CA2256761A1 - Modified factor vii - Google Patents

Abstract

The catalytic active site of Factor VII is modified to produce a compound which effectively interrupts the blood coagulation cascade. The modifications render Factor VIIa substantially unable to activate plasma Factors X or IX. The invention relates to novel methods of treatment and uses of modified Factor VII for treating preventing or treating myocardial injury associated with post-ischemic reperfusion, for improving regional myocardial blood flow during reperfusion, and maintaining or improving vascular patency in a patient, as well as topical application of modified Factor VII at vascular sites susceptible to thrombus formation.

Description

CA 022~676l l998-l2-Ol W O 97/47651 PCT~DK~7/00251 MODIFIED FACTOR Vll Field of the Invention The present invention relates to novel methods of treatment and novel uses of 5 modified forms of Factor Vll that inhibit thrombus formation, maintain or improve vascular patency, improve regional myocardial blood flow, and modulates myocardial injury during post-ischemic reperfusion.

Background Of The Invention Blood coagulation is a process consisting of a complex interaction of various blood components, or factors, which eventually gives rise to a fibrin clot. Generally, the blood components which participate in what has been referred to as the coagulation "c~scade" are proenzymes or zymogens, enzymatically inactive proteins which are converted to proteolytic enzymes by the action of an activator, itself an activated clotting factor. Coagulation factors that have undergone such a conversion and generally referred to as "active factors," and are designated by the addition of a lower case "a" suffix (e.g., Factor Vlla).
Activated Factor X ("Xa") is required to convert p~uthrolllbin to thrombin, which then converts fibrinogen to fibrin as a final stage in forming a fibrin clot. There are two systems, or pathways, that promote the activation of Factor X. The "i"ll insic pathway" refers to those reactions that lead to thrombin formation through utilization of factors present only in plasma.
A series of protease-mediated activations ultimately generates Factor IXa which, in conjunction with Factor Vllla, cleaves Factor X into Xa. An identical proteolysis is effected by Factor Vlla and its co-factor, tissue factor, in the "extrinsic pathway" of blood co~gul~tion.
Tissue factor is a membrane bound protein and does not normally circulate in plasma. Upon vessel disruption, however, it can complex with Factor Vlla to catalyze Factor X activation or Factor IX activation in the presence of Ca++ and phospholipid (Nemerson and Gentry, Biochem. 25:4020-4033 (1986)). While the relative importance of the two coagulation pathways in hemostasis is unclear, in recent years Factor Vll and tissue factor have been found to play a pivotal role in the regulation of blood coagulation.
- 30 Factor Vll is a trace plasma glycoprotein that circulates in blood as a single-chain zymogen. The zymogen is catalytically inactive (Williams et al., J. Biol. Chem. 264:7536-7543 (1989); Rao et al., Proc. Natl. Acad. Sci. USA. 85;6687-6691 (1988)). Single-chain CA 022~6761 1998-12-01 WO 97/47651 PCT/I)K971002~1 Factor Vll may be converted to two-chain Factor Vlla by Factor Xa, Factor Xlla, Factor IXa or thrombin in vitro. Factor Xa is bel.eved to be the major physiological activator of Factor Vll. Like several other plasma proteins involved in hemostasis, Factor Vll is depenclent on vitamin K for its activity, which is required for the g-carboxylation of multiple glutamic acid 5 residues that are clustered in the amino terminus of the protein. These g-carboxylated glutamic acids are required for the metal-associated interaction of Factor Vll with phospholipids.
The conversion of zymogen Factor Vll into the activated two-chain molecule occurs by cleavage of an internal peptide bond located approximately in the middle of the molecule.
10 In human Factor Vll, the activation cleavage site is at Arg,s2-lle,s3 (Hagen et al., Proc. Natl.
Acad. Sci. USA 83: 2412-2416 (1986); Thim et al., Biochem. 27:7785-7793 (1988) both of which are incorporated herein by references). Bovine factor Vll is activated by cleavage at the analogous Arg,s2-lle1s3 bond (Takeya et al., J. Biol. Chem. ;~: 14868-14877, 1988). In the presence of tissue factor, phospholipids and calcium ions, the two-chain Factor Vlla 15 rapidly activates Factor X or Factor IX by limited proteolysis.
It is often necessary to selectively block the co~gulation cascade in a patient.Antico~gul~nts such as heparin, coumarin, derivatives of coumarin, indandione derivatives, or other agents may be used, for exar"F'e, during kidney dialysis, or to treat deep vein thrombosis, disseminated intravascular coagulation (DIC), and a host of other medical 20 disorders. For example, heparin l,t:dl",enl or extracorporeal treatment with citrate ion (U.S.
Patent 4,500,309) may be used in dialysis to prevent coagulation during the course of treatment. Heparin is also used in preventing deep vein ll,rur"bosis in pdlients undergoing surgery.
Treatment with heparin and other anticoag~ nts may, however, have undesirable 25 side effects. Available antico~gul~nts generally act throughout the body, rather than acting specifically at a clot site. Heparin, for example, may cause heavy bleeding. Furthermore, with a half-life of approximately 80 minutes, heparin is rapidly cleared from the blood, necessit~tirlg frequent admi~ lldtion. Recause heparin acts as a cofactor for antithrombin lll (AT lll), and AT lll is rapidly depleted in DIC treatment, it is often difficult to maintain the 30 proper heparin dosage, necessitating continuous moniloring of AT lll and heparin levels.
Heparin is also ineffective if AT lll depletion is extreme. Further, prolonged use of heparin CA 022~6761 1998-12-01 W O 97/47651 PCTADX~7/00251 may also i"crease platelet agy,~gdlion and reduce platelet count, and has been imp'.G~ted in the dcv~lopr"ent of osteoporosis. Indandione derivatives may also have toxic side effects.
In addi;ion to the a"lico~g~ nts briefly desc,iLed above, several naturally occurring proteins have been found to have anlicoaglJl?~t activity. For example, Reutelingsperger (U.S. Patent No. 4,736,018) isolated anticoagulont proteins from bovine aorta and human umbilical vein arteries. Maki et al. (U.S.Patent No. 4,732,891) disclose human placenta-derived anticoagulant proteins. In addition, AT lll has been pr- posed as a therapeutic anticoag~'-nt (Schipper-et al., Lancet 1 (8069): 854-856 (1978); Jordan, U.S. Patent No.
4,386,025; Bock et al., U.S. Patent No. 4,517,294).
Proliferation of smooth muscle cells (SMCs) in the vessel wall is an important event in the formation of vascular lesions in atherosclerosis, after vascular reconstruction or in response to other vascular injury. For example, treatment of all,erosc'erosis frequently includes the clearing of blocked vessels by angioplasty, endarterectomy or reduc.tion atherectomy, or by bypass grafting, surgical procedures in which atherosclerotic plaques are cGn,prtssed or removed through c~tl.eleri~lion (ang;oplaaly), sl~i~,ped away from the arterial wall through an incision (endarterectomy) or bypassed with natural or synthetic grafts. These procedures remove the vascular endothelium, disturb the underlying intimal layer, and result in the death of medial SMCs. This injury is followed by medial SMC
proliferation and migration into the intima, which characleriatically occurs within the first few weeks and up to six months after injury and stops when the overlying endothelial layer is reest-~lished. In humans, these lesions are composed of about 20% cells and 80%
extracellular matrix.
In about 30% or more of palients treated by angioplasty, endai lerectomy or bypass grafts, thrombosis and/or SMC prol;~rdtion in the intima causes re-occlu~ion of the vessel and consequent failure of the reconstructive surgery. This closure of the vessel subsequent to surgery is known as reslenosis.
There is still a need in the art for improved cGr"posilions having anticoag~ nt ~ activity which can be adminialered at relatively low doses and do not produce the undesirable side effects associated with traditional anticoagul~nt compositions. The present 30 invention fulfills this need by providing anticoagulants that act specifically at sites of injury, and further provides other related advantages.

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 The modified Factor Vll molecu'es are particularly useful for ad~"i"i.,l,dlion to humans to treat a variety of conditions involving intravascular coag~lqtion. For example, although deep vein thrombosis and pulmonary embolism can be treated with conventional anticoagulants, the modified Factor Vll described herein may be used to prevent the occurrence of 5 thromboembolic complications in idenlified high risk pdlie~ , such as those undergoing surgery or those with congestive heart failure. In addition, modified Factor Vll may act as an antagonist for tissue factor-mediated induction of coagul~tion, thus blocking the production of thrombin and the sl~hsequent deposition of fibrin. As such, modified Factor Vll may be useful for inhibiting tissue factor activity resulting in, for exar"ple, the inhibition of blood 10 co~glllation, thrombosis or platelet deposition.
The modified Factor Vll molecules may be particularly useful in the treatment ofintimal hyperplasia or restenosis due to acute vascular injury. Acute vascular injuries are those which occur rapidly (i.e. over days to months), in contrast to chronic vascular injuries (e.g. atherosclerosis) which develop over a lifetime. Acute vascular injuries often result from 15 surgical procedures such as vascular reconstruction, wherein the techniques of angioplasty, endarterectomy, atherectomy, vascular graft emplacement or the like are e",,:l~yEd.
Hyperplasia may also occur as a delayed response in ,esponse to, e.g., graft emplacement or organ l,~nsplantalion. Since modified Factor Vll is more selective than heparin, generally binding only tissue factor which has been exposed at sites of injury, and because modified 20 Factor Vll does not destroy other coagulation proteins, it will be more effective and less likely to cause bleeding complications than heparin when used prophylactically for the prevention of deep vein thrombosis.
Recent advances in the treatment of coronary vascular disease include the use ofmechanical interventions to either remove or displace orrari.ling plaque material in order to 25 re-est~l sh adequate blood flow through the coronary arteries. Despite the use of multiple forms of mechanical interventions, including balloon angioplasty, reduction atherectomy, pla-cement of vascular stents, laser therapy, or rotoblalor, the effectiveness of these techniques remains limited by an approxi",al~ly 40% restenosis rate within 6 months after treatment.
Restenosis is thought to result from a complex interaction of biological processes 30 including platelet deposition and thrombus for"~dlion, release of chemotactic and mitogenic factors, and the migration and proliferation of vascular smooth muscle cells into the intima of the dilated arterial seg",ent.

CA 022~676l l998-l2-Ol W O97/47651 PCT~DK~7/00251 The inhibition of platelet accumulation at sites of mechanical injury can limit the rate of restenosis in human subjects. Therapeutic use of a monoclonal antibody to platelet Gpllb/llla is able to limit the level of reslenosis in human subjects (Califf et al., N. Engl. J.
~a~, 330:956-961 (1994)). The antibody is able to bind to the Gpllb/llla receptor on the 5 surfaces of pl*tolets and thereby inhibit platelet accumulation. This data suggests that inhibition of platelet accumulation at the site of mechani~' injury in human coronary arteries is beneficial for the ultimate healing response that occurs. While platelet accumulation occurs at sites of acute vascular injuries, the generation of thrombin at these sites may be responsible for the activation of the platelets and their subsequent accumulation.
As shown in the examples that follow, the modified Factor Vll of the present invention is able to bind to cell-surface tissue factor. For example, DEGR-Factor Vlla binds cell-surface tissue factor with an equivalent or higher affinity than wild-type Factor Vlla.
DEGR-Factor Vlla, however, has no enzymatic activity, yet it binds to tissue factor and acts as a competitive antagonist for wild-type Factor Vlla, thereby inhibiting the subsequent steps 1~ in the extrinsic pathway of coagulation leading to the generation of lh(ulllhin.
Modified Factor Vll molecules which maintain tissue factor binding inhibit platelet accumulation at the site of vascular injury by b'~ching the production of thrombin and the subsequent deposition of fibrin.
Due to the ability of DEGR-Factor Vll to block thrombin generation and limit platelet 20 deposition at sites of acute vascular injury, modified Factor Vll molecules which maintain tissue factor binding activity but lack Factor Vlla enzymatic activity can be used to inhibit vascular restenosis.

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 Compositions co",prisil-g modified Factor Vll are particularly useful in methods for l,t:aling patients when formulated into pharmaceutical compositions, where they may be given to individuals suffering from a variety of ~isease states to treat coagulation-related conditions. Such modified Factor Vll mol~cu'es, c~p~!e of binding tissue factor but having a substantially reduced ability to catalyze activation of other factors in the clotting cascade, may possess a longer plasma half-life and thus a correspondi"gly longer period of anticoagu'-tive activity when compared to other antiGo~gl ~-nts. Among the medical indications for the subject compositions are those commonly treated with antico~gul~nts, such as, for example, deep vein thrombosis, pulmonary embolism, stroke, disseminated 10 intravascular coagu~ation (DIC), fibrin deposition in lungs and kidneys associated with gram-negative endotoxemia, and myocardial infarction. The compositions can be used to inhibit vascular restenosis as occurs following mechanical vascular injury, such as injury caused by balloon angioplasty, encJallar~;tor"y, reductive atherectomy, stent placement, laser therapy or rotablation, or as occurs secondary to vascular grafts, stents, bypass grafts or organ 15 transplanls. The compositions can thus be used to inhibit platelet deposition and associated disorders. Thus, a method of inhibiting coagulation, vascular restenosis or platelet deposition, for example, comprises administering to a patient a composition comprising modified Factor Vll, such as that having at least one amino acid substitution in a catalytic triad of Ser344, Asp242 and His,93, in an amount sufficient to effectively inhibit coagulation, 20 vascular restenosis or platelet deposition. The methods also find use in the treatment of acute closure of a coronary artery in an individual (e.g. acute myocardial infarction), which comprises adminislering the modified Factor Vll, which includes DEGR-Factor Vll and FFR-Factor Vll, in conjunction with tissue plasminogen activator or streptokinase, and can accelerate tPA induced thrombolysis. The modified Factor Vll is given prior to, in conjunction 25 with, or shortly following ad",i"i;,l,dlion of a thrombolytic agent, such as tissue plasminogen activator.
International Application No. WO 92/15686 relates to modified Factor Vlla, polynucleic acid and mammalian cell lines for the production of modified Factor Vlla, and compositions comprising modified Factor Vlla for inhibiting blood co~gu~tion.
International App'~ tion No. WO 94/27631 relates to methods for inhibiting vascular restenosis, tissue factor activity, and platelet deposition.

CA 022~6761 1998-12-01 Inler"aliG"al A~plic~tion No. WO 96/1Z800 relates to a method for treatment of ~ acute closure of a coronary artery comprising to the individual a cGi"position which con,prises modified Factor Vlla in conjunction with tissue plasminogen activator or streptokinase.

Summary of the invention The present invention relates to a method for inhibiting thrombus ~r"lalion in apatient comprising acl~ "i. Ii~,lering topically to a vascular site susceptible to li ,ror"bus formation in the patient a therarelltically effective dose of a composition comprising Factor 10 Vll having at least one modification in its catalytic center, which modification sul,stanlially inhibits the ability of the modified Factor Vll to activate plasma Factor X or IX. The site of thrombus formation may be assoGi~ted with surgery, microsurgery, an~;o,~'ssty or trauma.
The invention further relates to a method for maintaining or improving vascular patency in a patient co",prising ad,llillislering locally to a vascular site susceptible to 1~ dec,eased patency a therapeutically effective dose of a composition comprising Factor Vll having at least one modification in its catalytic center, which modification suLslanlially inhibits the ability of the modified Factor Vll to activate plasma Factor X or IX. The site of thrombus formation may be associated with surgery, microsurgery, angioplasty or trauma.
The invention further relates to a method for preventing or minimizing myocardial 20 injury associated with post-ischemic reperfusion in an individual, comprising administering to the individual a composition which comprises a pharmacologically acce,~ le Factor Vll ha-ving at least one modiricdlion in its catalytic center, which modificalion s~ l,slarilially inhibits the ability of the modified Factor Vll to activate plasma Factor X or IX.
The invention further relates to a method for improving regional myocardial blood 25 flow during post-ischerllic reperfusion in an individual, comprising a.ll-li"islering to the indivi-dual a composition which co",prises a phallllacologically acceptable Factor Vll having at le-ast one modiricdlion in its catalytic center, which modiricdtion substantially inhibits the ability of the modified Factor Vll to activate plasma Factor X or IX.
In a preferred embodiment the modification of Factor Vll comprises reaction of the 30 Factor Vll with a serine protease inhibitor. In a more preferred aspect the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide. In an even more preferred aspect the protease inhibitor is a peptide halomethyl CA 022~676l l998-l2-Ol W O 97/47651 PCT~DK~7/00251 ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloro",ethyl ketone, Dansyl-Phe-Phe-Arg chloror"ell,yl ketone and Phe-Phe-Arg chloro",ethylketone, Phe-Phe-Arg chloron,ethylketone being the most prefer,ed.
The invention further relates to the use of Factor Vll having at least one modification 5 in its catalytic center, which modification substantially inhibits the ability of the modified Factor Vll to activate plasma Factor X or IX, for the manufacture of a composition for preventing or minimizing myocardial injury ~ssociated with post-ischemic reperfusion. The invention further relates to the use of Factor Vll having at least one modificdlion in its catalytic center, which modification substantially inhibits the ability of the modified Factor Vll 10 to activate plasma Factor X or IX, for the manufacture of a composition for improving regional myocardial blood flow during post-ischemic reperfusion.
The present invention provides methods and compositions to inhibit deleterious events associated with ischemic reperfusion. Severe ischemia to a tissue, organ or limb may be due to a decrease in blood flow and may be associated with trauma, surgical 15 manipulation, or lowered blood pressure. One of the cG",plicalions associated with severe ischemia is the up-regulation of tissue factor in the arterial system. This increased expression of tissue factor is kel eved to stimulate a procoagulant response, primarily in the capillary bed, thus initiating and/or sustaining intravascular thrombus formation.
Furthermore, the de novo synthesis of TF during reperfusion of post-ischemic hearts by 20 endothelial cells within the coronary vascl ~'atllre may lead to a decrease in coronary blood flow during reperfusion and thus influencing the fate of the ischemic myocardium that will ullin,~toly undergo necrosis. TF antigen and procoagulant activity is increased in atherectomy specimens obtained from palients with unstable angina, as compared to palier,l~ with stable angina. Thus, it is believed taht in these patients u" t ~le angina may be 25 preticipated by exposure of TF in the subendoll,c' I tissue of a large epicardial coronary artery as a result of piaque damage. This will eventually promote an intracoronary thrombus formation with a consequent abso' lte reduction in coronary flow.
TF may also effect coronary flow in a different way. Following reperfusion to the ischemic tissue, thrombi can be generaled which may be either occlusive or non-occlusive.
30 The generation of thrombi in the arterial bed, and the deposition of pldlal_l~ along the II,ror"busl lead to the secondary generation of ischemia to the tissue. The generation of the thrombi and the presence of plal31ets can then cause the generation and release of multiple CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/002Sl bioactive factors, including those generated from the coagulation pathway, such as thrombin and Factor X, as well as factors released from activated platelet:,. In turn, these factors may induce the generation of addilional factors by the underlying endoll,e' -' and smooth muscle cells, or by adjacent mononuclear celis, such as TNF-alpha and IL-1. These factors, in turn, 5 can then activate the endothe' ~l cells leading to the up-regulation of various adhesion molecules associated with monocyte and neutrophil binding. Normally, endothelial cells, being in contact with circulating blood, do not express signirican~ TF activity. Under certain circumstances endothelial cells may actively promote co~gulation by ex~.ressing TF-like procoagulant activity. In particular, both exogenous and endogenously generated oxygen 10 free radicals (OFRs) can stimulate endothelial cells within the coronary vasculature to synthesize and express sig"ificanl amounts of TF. OFRs are highly reactive molecular species that may attack various cell constituents. A burst of OFR generation follows reslordlion of flow after a period of ischemia, and these oxidant species might be responsible for a specific form of reperfusion-mediated tissue injury, secondary to lipid peroxidation and 15 other irreversible alterdlions of cell constituents. OFRs also dramatically decrease the activity of tissue factor pathway inhibitor (TFPI), a Kunitz-type protein synthesized by endothelial cells, which inhibits the extrinsic co~g~ ~ation pathway. This double effect of OFRs (TF expression by endothelial cells and decrease in TFPI activity) may shift the natural anticoagulant properties of the normal endothelium toward a procoagulant state, thus 20 favouring an unwanted intravascular activation of the coagulation. Thus, OFR-mediated TF
expression within the coronary circulation results in a significant reduction in coronary blood flow during post-ischemic reperfusion. This OFR-mediated expression of TF, with its attendant activation of the extrinsic co~gu~tion pathway, has important consequences, as this phenomenon impacts on the pathophysiology of post-ischemic reperfusion, particularly 25 in patients with acute myocardial i"rdr~;tion u"deryoing coronary thrombolysis.
The no-reflow phenomenon, that is, lack of uniform perfusion to the microvasculatu-re of a previously ischemic tissue has been described for the first time by Krug et al., (Circ.
Res. 1966; 19:57-62). The most important determinants that may influence the fate of ischemic myocardium are believed to be the amount of collateral flow during ischemia, the 30 size of the area at risk and the myocardial oxygen demand.
Over the past decade there has been intense interest in the concept of treating pa-tients with acute myocardial infarction with reperfusion strategies, including coronary throm-CA 022~676l l998-l2-Ol W O 97/47651 PCTADK~7/00251 bolysis, primary angioplastry, or both, However, nor all studies have demonsl,ated an impro-vement in left ventricular function after recanalization of the infarct-related artery. At the moment, a subslantial number of palienls exhibit a "low-flow" condition in the infarct-related coronary bed. This condition is related with an almost co",F'etc lack of benefits at least in 5 the term of mortality. These low-flow con-litions is believed to be caused, at least in part, by the inability of blood to re-enter all of the vasculature of the previously ischemic myocardium.
It has now surprisingly been shown that FVllai effects, and increases, the regional myocar-dial blood flow during reperfusion. It has also su, ~risi"gly been shown that FVllai results in a significant reduction in the area of no-reflow, The binding and l.dns",igralion of monocytes and neutrophils, the release of bioactive compounds by these cells, including the generation of free-oxygen radicals, can exacerbate the level of endothelial cell activation and damage. Ulti",ately, if the c~scade of events goes unchecked, this can lead to systemic comF' ,ations and the potential to stimulate multiple organ failure. By blocking tissue factor by administering a specific inhibitor for tissue factor/Factor Vll binding (e.g., FFR-FVlla), and thereby blocking the initiation of the extrinsic pathway of coag~ ~~tion, the initiation of the cascade of events may be prevented, thereby modulating the extent deleterious events associated with ischemia/reperfusion, such as, for example, eliminating, or minimizing the myocardial injury or necrosis.

Brief Description Of The Figures Fig. 1 illustrates the construction of an ek~ression vector for a Ser344~)Ala modified Factor Vll DNA sequence. Symbols used include 0-1, the 0-1 map unit sequence from adenovirus 5; E, the SV40 enhancer; MLP, the adenovirus 2 major late promotor; SS, a set of splice sites; and pA, the polyadenylation signal from SV40 in the late o~ienLa~ion.
Fig. 2 shows the effect of bolus injection of DEGR-Factor Vlla on thrombus formation (platelet deposition) on endarterectomized baboon aorta when compared to saline-treated con~,.,ls. The arteries were measured over 60 minutes. The DEGR-Factor Vlla significantly inhibited the dcvelopment of platelet rich ~h-olllbi in this primate model of acute vascular injury.
Fig. 3 shows results obtained when baboon smooth muscle cells were incubated with increasing concentrations of either FVlla (open box), or DEGR-FVlla in the presence of a conslant amount of FVlla (5 nM) (closed box). The level of FX activation was CA 022~6761 1998-12-01 s~ ~hsecluently determined using the chromogenic substrate S-2222. The data are presented as the amidolytic activity as a pe~centage of the activity generated in the pr~sence of 5 nM
FVlla alone.
Fig. 4 depicts the size of the intimal area of baboons fol'_~;ng carotid artery 5 endarterectomy and treatment with DEGR-Factor Vlla for 7 or 30 days, compared to control animals.
Fig. 5 illustrates the ratio of the intimal area to the intimal + media area of baboon femoral artery following balloon injury and t,eat")ent with DEGR-Factor Vlla, where the control group included 5 vessels, 7 day treatment examined 11 vessels, and 30 day 10 treatment examined 2 vessels (n= number of vessels examined).
Fig. 6 illustrates the experimental protocol for measuring infarct size (IS), no-reflow (NR), area at risk (AR), prothrombin time (PT) and activated partial thromboplastin time (aPTT).
Fig. 7 shows a plot of infarct size at the end of the reperfusion period (IS) expressed 15 as a percent of the area at risk of infarcting in the three treatment groups, the three treatment groups being animals treated with FFR-Factor Vlla, Factor Vlla and saline, respectively. Each bar represents the mean of eight animals + SD.
Fig. 8 shows a plot of the area of no-reflow (NR) at the end of the reperfusion period expressed as a percent of the area at risk of infarcting. (animals treated with FFR-Factor 20 Vlla, Factor Vlla and saline, respectively.) Each bar represents the mean of eight animals +
SD.
Fig. 9 shows the relationship between expected no-reflow c~lcl~lated (as a percent of left ventricle, LV) for each animal by the multiple linear reg,ession equation and the no-reflow actually observed (as percent of LV).
Fig. 10 shows a plot of regional myocardial blood flow (RMBF) for ischemic myocardium ~ssesed at 20 min of ischemia, and after 10 min and 2 hrs of reperfusion.
Fig. 11 shows the effect of FFR-Factor Vlla and Factor Vlla on prulhrur,,bin time ~ (PT) and activated partial thromboplaslin time (aPTT).

CA 022~6761 1998-12-01 W O 971476Sl PCT~DK97/00251 12 Description Of The Specific Frnbodiments Modified Factor Vll can be in the form of the zymogen (i.e., a single-chain molecule) or can be cleaved at its activation site. Thus, by "modified Factor Vll" is meant to include modified Factor Vll and modified Factor Vlla ",clec~'es that bind tissue factor and inhibit the 5 activation of Factor IX to IXa and Factor X to Xa. The Factor Vll sequence has at least one amino acid modification, where the modification is selected so as to substantially reduce the ability of activated Factor Vll to catalyze the activation of plasma Factors X or IX, and thus is caFV~'e of inhibiting clotting activity. The modified FactorVII has an active site modified by at least one amino acid suhstitution, and in its modified form is c~p- ~IE of binding tissue 10 factor. The modified Factor Vll compositions are typically in substantially pure form.
In preferred embodiments of human and bovine Factor Vll, the active site residueSer344 is modihed, replaced with Gly, Met, Thr, or more preferably, Ala. Such substitution could be made separalely or in combination with substitution(s) at other sites in the catalytic triad, which includes His,93 and Asp242.
Compositions of the modified Factor Vll are suitable for admir,i~l,dlion to a variety of mammals, particularly humans, to inhibit the coagulation c~sc~de. Modified Factor Vll may be adr"i"islered to a patient in conjunction with or in place of other antico~gul-nt compounds. Typically, for administration to humans the pharmaceutical compositions will comprise modified human Factor Vll protein and pharmaceutically-accept~'e carriers and buffers.
Factor Vll plays an important role in the coagulation cascade, particularly thatinvolving the extrinsic pathway. Present in the circulating plasma as an inactive single chain zymogen protein, once activated, Factor Vlla, in co",b.,,dlion with tissue factor and calcium ions, activates Factor X to Xa and activates Factor IX to IXa, with the eventual formation of a fibrin clot.
Factor Vll proteins have a catalytic site which is modified to decrease the catalytic activity of Factor Vlla, while the molecule retains the ability to bind to tissue factor. The modified Factor Vll moleu~'es co",pete with native Factor Vll and/or Vlla for binding to tissue factor. As a result, the activation of Factors X and IX is inhibited.
Modified Factor Vll may be encoded by a polynucleotide molecule comprising two operatively linked sequence coding regions encoding, respectively, a pre-pro peptide and a gla domain of a vitamin K-dependent plasma protein, and a gla domain-less Factor Vll CA 022~6761 1998-12-01 protein, wherein upon e)~ ression said polynucleotide encodes a modified Factor Vll molecule which does not significantly activate plasma Factors X or IX, and is capable of binding tissue factor. The modified Factor Vll molecule e~c~ ressed by this polynucleotide is a biologically active antico~gul?rlt, that is, it is c~pa~'e of inhibiting the coagulation cascade 5 and thus the formation of a fibrin deposit or clot. To express the modified Factor Vll the polynucleotide molecule is transfected into mammalian cell lines, such as, for exa"~r!e, BHK, BHK 570 or 293 cell lines.
The catalytic activity of Factor Vlla can be inhibited by cl-er"i~ -~ derivali~alion of the catalytic center, or triad. Derivdli~alion may be accorn~lis'-ed by reacting Factor Vll with an 10 irreversible inhibitor such as an organophosphor compound, a sulfonyl fluoride, a peptide halomethyl ketone or an azapeptide, or by acylation, for example. Preferred peptide halomethyl ketones include PPACK (D-Phe-Pro-Arg chloromethyl-ketone; (see U.S. Patent No. 4,318,904, incorporated herein by reference), D-Phe-Phe-Arg and Phe-Phe-Arg chloromethylketone (FFR-cmk); and DEGRck (dansyl-Glu-Gly-Arg chloromethylketone).
The catalytic activity of Factor Vlla can also be inhibited by substituting, inserting or deleting amino acids. In pr~:~r~ad embodiments amino acid substitutions are made in the amino acid sequence of the Factor Vll catalytic triad, defined herein as the regions which contain the amino acids which contribute to the Factor Vlla catalytic site. The suhstit-ltions, insertions or deletions in the catalytic triad are generally at or adjacent to the amino acids 20 which form the catalytic site. In the human and bovine Factor Vll proteins, the amino acids which form a catalytic"triad" are Ser344, Asp242, and His,93 (subscript numbering indicating position in the sequence). The catalytic sites in Factor Vll from other mammalian species may be determined using presently available techniques including, among others, protein isolation and amino acid sequence analysis. Catalytic sites may also be deter~"i,led by 25 aligning a sequence with the sequence of other serine proteases, particularly chymotrypsin, whose active site has been previously determined (Sigler et al., J. Mol. Biol., 35:143-164 (1968), incorporated herein by reference), and therer,onl determining from said alignment ~ the analogous active site re~idues.
The amino acid sl Ihstitutions, insertions or deletions are made so as to prevent or 30 otherwise inhibit activation by the Factor Vlla of Factors X and/or IX. The Factor Vll so modified should, however, also retain the ability to compete with authentic Factor Vll and/or Factor Vlla for binding to tissue factor in the coagulation cascade. Such competition may CA 022~6761 1998-12-01 readily be determined by means of, e.g., a cloUing assay as desc,il.ed herein, or a co",pelilion binding assay using, e.g., a cell line having cell-surface tissue factor, such as the human bladder car~ ,ol"a cell line J82 (Sakai et al. J. Biol. Chem. 264: g980-9988 (1989), incorporated by reference herein.) The amino acids which form the catalytic site in Factor Vll, such as Ser344, ASP242, and His,93 in human and bovine Factor Vll, may either be substituted or deleted. Within the present invention, it is preferled to change only a single amino acid, thus minimizing the likelihood of increasing the antigenicity of the molecule or inhibiting its ability to bind tissue factor, however two or more amino acid changes (s~ Ih5tjtutjons, additions or deletions) may 10 be made and combinalions of suhstitl~tion(s), addition(s) and deletion(s) may also be made.
In a preferred embodiment for human and bovine Factor Vll, Ser344 is preferably substituted with Ala, but Gly, Met, Thr or other amino acids can be su~stituted. It is preferred to replace Asp with Glu and to replace His with Lys or Arg. In general, sl~hstit-ltions are chosen to disrupt the tertiary protein structure as little as possible. The model of Dayhoff et al. (in Atlas 15 of Protein Structure 1978, Nat'l ~3iomed. Res. Found., Washington, D.C.), incorporated herein by reference, may be used as a guide in selecting other amino acid substitlltions.
One may introduce residue alterations as described above in the catalytic site of appropriate Factor Vll sequence of human, bovine or other species and test the resulting protein for a desired levei of inhibition of catalytic activity and resulting anticoagulant activity as described herein. For the modified Factor Vll the catalytic activity will be substantially inhibited, generally less than about 5% of the catalytic activity of wild-type Factor Vll of the corresponding speci s, more preferably less than about 1 %.
The modified Factor Vll may be produced through the use of recGr"~ .. ,anl DNA
techn.~les. In general, a cloned wild-type Factor Vll DNA sequence is modified to encode 25 the desired protein. This modified sequence is then inserted into an expression vector, which is in turn transformed or transfected into host cells. Higher eukaryotic cells, in particular cultured mammalian cells, are pret~r,t:d as host cells. The complete nucleotide and amino acid sequences for human Factor Vll are known. See U.S. Pat. No. 4,784,950, which is incorporated herein by reference, where the cloning and expression of recombinant 30 human Factor Vll is described. The bovine Factor Vll sequence is described in Takeya et al., J. Biol. Chem. 263:14868-14872 (1988), which is incorporated by reference herein.

CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/002Sl The amino acid sequence alterations may be accomplished by a variety of techniques. Modiric~lion of the DNA sequence may be by site-specific mutagenesis.
Techniques for site-speciric mulagenesis are well known in the art and are described by, for example, Zoller and Smith (DNA 3:479~88, 1984). Thus, using the nucl~Qtide and amino acid sequences of Factor Vll, one may introduce the alteration(s) of choice.
The Factor Vll modified accordingly includes those proteins that have the amino-terminal portion (gla domain) suhstituted with a gla domain of one of the vitamin K-dependent plasma protei,)s Factor IX, Factor X, proll,r~,"~bin, protein C, protein S or protein Z. The gla domains of the vitamin K-dependent plasma proteins are characterized by the 10 presence of gamma-carboxy glutamic acid residues and are generally from about 30 to about 40 amino acids in length with C-termini corresponding to the positions of exon-intron boundaries in the respective genes. Methods for producing Factor Vll with a heterologous gla domain are disclosed in U.S. Patent No. 4,784,950, incor~,oraled by ~e~crence herein.
DNA sequences for use in producing modified Factor Vll will typically encode a pre-15 pro peptide at the amino-terminus of the Factor Vll protein to obtain proper post-translational processing (e.g. gamma-carboxylation of glutamic acid residues) and secretion from the host cell. The pre-pro peptide may be that of Factor Vll or another vitamin K-dependent plasma protein, such as Factor IX, Factor X, proll,lombin, protein C or protein S. As will be appreciated by those skilled in the art, additional modif,cdlions can be made in the amino 20 acid sequence of the modified Factor Vll where those modifications do not significantly impair the ability of the protein to act as an anticoagu'-nt. For example, the Factor Vll modified in the catalytic triad can also be modified in the activation cleavage site to inhibit the conversion of zymogen Factor Vll into its activated two-chain form, as generally described in U.S. Patent 5,288,629, incor~ordled herein by reference.
Ex~.(ecsion vectors for use in expressing modified Factor Vlla will comprise a promoter c:~p-~'e of directing the transcription of a cloned gene or cDNA. Preferred promoters for use in cultured ",ai."nalian cells include viral promoters and cellu!ar p,omoters. Viral promoters include the SV40 promoter (Subramani et al., Mol. Cell. Biol.
1 :854-864, 1981) and the CMV promoter (Boshart et al., Ç~ll 41 :521-530, 1985). A
30 particularly preferred viral promoter is the major late promoter from adenovirus 2 (Kaufman and Sharp, Mol. Cell. Biol. 2:1304-1319, 1982). Cellular promoters include the mouse kappa gene promoter (Bergman et al., Proc. Natl. Acad. Sci. USA 81 :7041 -7045, 1983) and the CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 mouse VH promoter (Loh et al., ~1133:85-93, 1983). A particularly prafe"~d cellular promoter is the mouse metalloll ,ioneL ,-l promoter (Palmiter et al., Science 222:809-814, 1983). Expression vectors may also contain a set of RNA splice sites located downstream from the promoter and u~,sl,aar" from the insertion site for the Factor Vll sequence itself.
5 Preferred RNA splice sites may be obtained from adenovirus and/or immunoglobulin genes.
Also contained in the ex~,lession vectors is a polyadenylation signal located downstream of the insertion site. Particularly preferred polyadenylation signals include the early or late polyadenylation signal from SV40 (Kaufman and Sharp, ibid.), the polyadenylation signal from the adenovirus 5 Elb region, the human growth hormone gene terminator (DeNoto et al.
Nuc. Acids Res. 9:3719-3730, 1981) or the polyadenylation signal from the human Factor Vll gene or the bovine Factor Vll gene. The expression vectors may also include a noncoding viral leader sequence, such as the adenovirus 2 tripartite leader, located between the promoter and the RNA splice sites; and enhancer sequences, such as the SV40 enhancer.
Cloned DNA sequences are introduced into cultured mammalian cells by, for example, calcium phosphate-mediated transfection (Wigler et al., Cell 14:725-732,1978;
Corsaro and Pearson, Somatic Cell Genetics 7:603-616, 1981; Graham and Van der Eb, Virology 52d:456-467, 1973) or elect,opordlion (Neumann et al., EMBO J. 1 :841 -845,1982).
To identify and select cells that express the exogenous DNA, a gene that confers a selectable phenotype (a selectable marker) is generally introduced into cells along with the 20 gene or cDNA of interest. Preferred selectable markers include genes that confem~sislance to drugs such as neomycin, hygromycin, and methotrexate. The sele~ !e marker may be an amplifiable selectable marker. A prarer,ad amplifiable selec~able marker is adihydrofolate reductase (DHFR) sequence. Selectable markers are reviewed by Thilly (Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA, i"co".or~lad herein 25 by reference). The choice of select~h'e markers is well within the level of ordinary skill in the art.
Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plas,-,id. If on the same plasn,id, the selectable marker and the gene of interest may be under the control of 30 different promoters or the same promoter, the latter arrangement producing a dicistronic mess~ge. Constructs of this type are known in the art (for example, Levinson and CA 022~676l l998-l2-Ol W O 97/47651 PCT~DK~7/00251 Simonsen, U.S. Patent 4,713,339). It may also be advanlageous to add additional DNA, known as "carrier DNA," to the mixture that is introduced into the cells.
After the cells have taken up the DNA, they are grown in an appropriate growth medium, typically 1-2 days, to begin e~,u,essing the gene of interest. As used herein the term "appropriate growth medium" means a medium containing nutrients and other components required for the growth of cells and the ex~,ression of the modified ~actor Vll gene. Media generally include a carbon source, a nit,~gen source, essential amino acids, essential sugars, vitamins, salts, phospholipids, protein and growth factors. For production of gamma-carboxylated modified Factor Vll, the medium will contain vitamin K, prefelably at a concentration of about 0.1 mg/ml to about 5 mg/ml. Drug selection is then applied to select for the growth of cells that are expressing the sele~t .-'- !e marker in a stable fashion.
For cells that have been transfected with an amplifiable sele~ta~!e marker the drug concentration may be increased to select for an increased copy number of the cloned sequences, thereby increasing expression levels. Clones of stably transfected cells are then screened for expression of modified Factor Vll.
Preferred mammalian cell lines include the COS-1 (ATCC CRL 1650), baby hamster kidney (BHK) and 293 (ATCC CRL 1573; Graham et al., J. Gen. Virol. 36:59-72, 1977) cell lines. A preferred BHK cell line is the tk- ts13 BHK cell line (Waechter and Baserga, Proc.
Natl. Acad. Sci. USA 79:1106-1110,1982, incorporated herein by reference), hereinafter referred to as BHK 570 cells. The BHK 570 cell line has been deposited with the American Type Culture Collection, 12301 Parklawn Dr., Rockville, MD 20852, underATCC accession number CRL 10314. A tk- ts13 BHK cell line is also available from the ATCC underaccession number CRL 1632. In addition, a number of other cell lines may be used, including Rat Hep I (Rat hepatoma; ATCC CRL 1600), Rat Hep ll (Rat hepatoma; ATCC

2~ CRL 1548), TCMK (ATCC CCL 139), Human lung (ATCC HB 8065), NCTC 1469 (ATCC
CCL 9.1), CHO (ATCC CCL 61) and DUK~( cells (Urlaub and Chasin, Proc. Natl. Acad. Sci.
USA77:4216-4220, 1980).
~ Transgenic animal technology may be employed to produce modified Factor Vll. It is preferred to produce the proteins within the mammary glands of a host female mammal.
Ex~ ression in the mammary gland and subsequent secretion of the protein of interest into the milk overcomes many difficulties encountered in isolating protei.ls from other sources.
Milk is readily collectect, available in large quantities, and well cha~acLerized biochemically.

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 18 Fu,li,er")or~:, the major milk prutei~ls are present in milk at high conc~nl,dlions (typically from about 1 to 15 g/l).
From a commercial point of view, it is clearly preferable to use as the host a species that has a large milk yield. While smaller animals such as mice and rats can be used (and 5 are plefer,t:d at the proof of principle stage), it is pr~fer,ed to use livestock mammals including, but not limited to, pigs, goats, sheep and cattle. Sheep are particularly preferred due to such factors as the previous history of transgenesis in this species, milk yield, cost and the ready availability of equipment for co'lev~i,,g sheep milk. See WIPO Publication WO
88/00239 for a comparison of factors influencing the choice of host species. It is generally 10 desirable to select a breed of host animal that has been bred for dairy use, such as East Friesland sheep, or to introduce dairy stock by breeding of the l,dnsgenic line at a later date.
In any event, animals of known, good health status should be used.
To obtain ex~.ressiol, in the mammary gland, a transcription promoter from a milk protein gene is used. Milk protein genes include those genes encoding casei~ ,s (see U.S.
Patent No. 5,304,489, incorporated herein by reference), beta-lactoglQbulin, a-lactalbumin, and whey acidic protein. The beta-lactoglobulin (BLG) promoter is preferred. In the case of the ovine beta-lactoglobulin gene, a region of at least the proximal 406 bp of 5' rlar,ki"g sequence of the gene will generally be used, although larger portions of the 5' flanking sequence, up to about 5 kbp, are prer~r,ed, such as a ~4.25 kbp DNA segment 20 encompassing the 5' flanking promoter and non-coding portion of the beta-lactoglobulin gene. See Whitelaw et al., Biochem J. 286: 31-39 (1992). Similar fragments of promoter DNA from other species are also suitable.
Other regions of the beta-lactoglobulin gene may also be incorporated in constructs, as may genomic regions of the gene to be expressed. It is generally accepted in the art that 25 constructs lacking introns, for example, express poorly in comparison with those that contain such DNA sequences (see Brinster et al., Proc. Natl. Acad. Sci. USA 85: 836-840 (1988);
Palmiter et al., Proc. Natl. Acad. Sci. USA 88: 478-482 (1991); Whitelaw et al., Transgenic Res. 1: 3-13 (1991); WO 89/01343; and WO 91102318, each of which is incorporated herein by reference). In this regard, it is generally preferred, where possible, to use genomic 30 sequences containing all or some of the native introns of a gene encoding the protein or polypeptide of interest, thus the further inclusion of at least some introns from, e.g, the beta-lactoglobulin gene, is p,efer,~:d. One such region is a DNA segment which provides for CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/00251 19 intron splicing and RNA polyadenylation from the 3' non-coding region of the ovine beta-lactoglobulin gene. When suhstitut~sd for the natural 3' non-coding sequences of a gene, this ovine beta-lactoglobulin segment can both enhance and sl~hi';~e ex~,ression levels of the protein or polypeptide of interest. Within other embodiments, the region 5 surrounding the i"itialion ATG of the modified Factor Vll sequence is replaced with corresponding sequences from a milk specific protein gene. Such replacement provides a putative tissue-specific initiation environment to enhance ex~.ression. It is convenient to replace the entire ",odif,ed Factor Vll pre-pro and 5' non-coding sequences with those of, for example, the BLG gene, although smaller regions may be replaced.
For expression of modified Factor Vll in l,ansgenic animals, a DNA segment encoding modified Factor Vll is operably linked to additional DNA segments required for its expression to produce e~Jr~ssion units. Such ad.litional segments include the above-mentioned promoter, as well as sequences which provide for termination of transcription and polyadenylation of mRNA. The expression units will further include a DNA
segment encoding a secretory signal sequence operably linked to the seg",enl encoding modified Factor Vll. The secretory signal sequence may be a native Factor Vll secretory signal sequence or may be that of another protein, such as a milk protein. See, for example, von Heinje, Nuc. Acids Res. 14: 46834690 (1986); and Meade et al., U.S. Patent No.
4,873,316, which are incGr~,orated herein by reference.
Construction of ex,uression units for use in transgenic animals is conveniently carried out by inserting a modified Factor Vll sequence into a plasmid or phage vector containing the additional DNA segments, although the ex~,rt:ssion unit may be constructed by essentially any sequence of ligations. It is particularly convenient to provide a vector containing a DNA segment encoding a milk protein and to replace the coding sequence for the milk protein with that of a modified Factor Vll polypeptide, thereby creating a gene fusion that includes the expression control sequences of the milk protein gene. In any event, cloning of the ek~ression units in plasm.~s or other vectors facilitates the ai"F';ricaLion of the modified Factor Vll sequence. An,F!irication is conveniently carried out in bacterial (e.g. E.
~Q!i) host cells, thus the vectors will typically include an origin of replication and a selectable marker functional in bacterial host cells.
The ex~,ression unit is then introduced into fertilized eggs (including early-stage embryos) of the chosen host species. Introduction of heterologous DNA can be CA 022~676l l998-l2-Ol W O 97/47651 PCT~DK~7/00251 accGr,.r' shed by one of several routes, including microinjection (e.g. U.S. Patent No.
4,873,191), retroviral infection (Jaenisch, Science 240: 1468-1474 (1988)) or site-directed i~teyldtion using embryonic stem (ES) cells (reviewed by Bradley et al., Bio/Technology 10:
534-539 (1992)). The eggs are then implanted into the oviducts or uteri of pseudopregnant 5 females and allowed to develop to term. orr~p, ing carrying the introduced DNA in their germ line can pass the DNA on to their progeny in the normal, Mendelian fashion, allowing the development of transgenic herds.
General procedures for producing transgenic animals are known in the art. See, for example, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory, 1986; Simons et al., Bio/Technology 6: 179-183 (1988); Wall et al., Biol.
Reprod. 32: 645-651 (1985); Buhler et al., Bio/Techl-.aloyy 8: 140-143 (1990); Ebert et al., Bio/Technology 9: 835-838 (1991); K,i",penrort et al., Bio/Technology 9: 844-847 (1991);
Wall et al., J. Cell. Biochem. 49: 113-120 (1992); U.S. Patent Nos. 4,873,191 and 4,873,316;
WIPO public~tions WO 88/00239, WO 90/05188, WO 92/11757; and GB 87100458, which are incorporated herein by reference. Techniques for introducing foreign DNA sequences into mammals and their germ cells were originally developed in the mouse. See, e.g., Gordon et al., Proc. Natl. Acad. Sci. USA 77: 7380-7384 (1980); Gordon and Ruddle, Science 214: 1244-1246 (1981); Palmiter and Brinster, S~!141: 343-345 (1985); Brinster et al., Proc. Natl. Acad. Sci. USA 82: 4438-4442 (1985); and Hogan et al. (ibid.). These techniques were subsequently adapted for use with larger animals, including livestock species (see e.g., WIPO p~lic~tions WO 88/00239, WO 90/05188, and WO 92/11757; and Simons et al., Bio/Technology 6: 179-183 (1988). To summarize, in the most efricient route used to date in the generation of transgenic mice or livestock, several hundred linear molec~'es of the DNA of interest are in,erted into one of the pro-nuclei of a fertilized egg according to est~k'i~ hed techniques. Injection of DNA into the cytoplasm of a zygote can also be employed.
Production in l,dnsgenic plants may also be employed. Expression may be generalized or directed to a particular organ, such as a tuber. See, Hiatt, Nature 344:469-479 (1990); Edelbaum et al., J. Interferon Res.12:449-453 (1992); Sijmons et al., Bio/Technology 8:217-221 (1990); and European Patent Office Publication EP 255,378.
Modified Factor Vll may be purified by affinity chromatography on an anti-Factor Vll antibody column. The use of calcium-dependent monoclonal antibodies, as described by CA 022~6761 1998-12-01 Wakabayashi et al., J. Biol. Chem. 261 :11097-11108, (1986) and Thim et al., Biochem. 27:
7785-7793, (1988), incor,uorated by reference herein, is particularly prefer,~d. Additional purification may be achieved by conventional che",i--' pu,irication means, such as high performance liquid chromatography. Other methods of purification, including barium citrate p,eci,l~italion, are known in the art, and may be applied to the purification of the novel modified FactorVII desc,iled herein (see, ~enerally, Scopes, R., Protein Pu,iricalion.
Springer-Verlag, N.Y., 1982). Sul)~tanlially pure modified Factor Vll of at least about 90 to 95% homogeneity is pre~fe"ed, and 98 to 99% or more homoge"eity most pr~fer,ed, for pharmaceutical uses. Once purified, partially or to homogeneity as desired, the modified 10 Factor Vll may then be used therapeutically.
The modified Factor Vll is cleaved at its activation site to convert it to its two-chain form. Activation may be carried out according to procedures known in the art, such as those disclosed by Osterud, et al., Biochemistry 11:2853-2857 (1972); Thomas, U.S. Patent No.
4,456,591; Hednerand Kisiel, J. Clin. Invest. 71:1836-1841 (1983); or Kisiel and Fujikawa, 15 Behring Inst. Mitt. 73:29-42 (1983), which are incorporated herein by reference. The resulting molecule is then formulated and administered as described below.

Compositions The compounds will typically be administered within about 24 hours prior to pe, ror"~ing the 20 intervention, and for as much as 7 days or more thereafter. Admi"i~,~, alion for preventing or minimizing myocardial injury can be by a variety of routes as further described herein. The compounds can also be administered locally at vascular sites susceptihle of thrombus formation, for example, at sites of anastomosis, or locally at vascular sites susceptible to decreased patency.
In the prevention of or treatment of myocardial injury, the dose of modified Factor Vll ranges from about 50 mg to 500 mg/day, more typically 1 mg to 200 mg/day, and more preferably 10 mg to about 175 mg/day for a 70 kg patient as loading and mainlenance doses, depending on the weight of the patient and the severity of the condition.The pharmaceutical compositions for treatment of myocardial injuries are intended 30 for parenteral administration for prophylactic and/or therapeutic treatment. Preferably, the pharmaceutical compositions are administered parenterally, i.e., intravenously, subcutaneously, or intramuscularly. The compositions for parenteral adminisl, dlion CA 022~6761 1998-12-01 W O 97t47651 PCT~DK97/00251 comprise a solution of the modified Factor Vll molecules dissolved in an acce~table carrier, pr~ferably an aqueous carrier. A variety of aqueous carriers may be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. The modified FactorVII mo'ecl~'es can also be formulated into liposome preparalions for delivery or targeting to sites of injury.
Liposome prepardlions are generally desc, ibed in, e.g., U.S. 4,837,028, U.S. 4,501,728, and U.S. 4,975,282, inco" oraled herein by ~~fer~nce. The co,npositions may be sterilized by conventional, well known sterilization techniques. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and Iyophilized, the Iyoph ' ~çd preparation being combined with a sterile aqueous solution prior to administration. The 10 compositions may contain pharmaceutically accept ~'e auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents and the like, for example, sodium acetate, sodium lactate, sodium ch'o. ide, potassium chloride, calcium chloride, etc. The concentration of modified Factor Vll in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1 %
1~ to as much as 15 or 20% by weight and will be selected primarily by fluid volumes, viscosities, etc., in accordance with the particular mode of ad",inisl,dlion selected.
Thus, a typical pharmaceutical composition for intravenous infusion could be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of modified Factor Vl l . Actual methods for preparing parenterally administrable compounds will be known or apparent to 20 those skilled in the art and are described in more detail in for example, Remington's Pharmaceutical Science, 16th ed., Mack Publishing Company, Easton, PA (1982), which is incorporated herein by reference.
The compositions containing the modified Factor Vll molecules can be acln, nistert:d for prophylactic andlor therapeutic treatments. In therapeutic app'.c~tions, compositions are 25 administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially arrest the disease and its comrl c~tions. An amount ~dequat~s to accomplish this !s defined as "therapeutically effective dose." Amounts effective for this use will depend on the severity of the di~.ea3e or injury and the weight and general state of the patient, but generally range from about 0.05 mg up to about 500 mg of modified 30 Factor Vll per day for a 70 kg patient, with dos~ges of from about 1.0 mg to about 200 mg of modified Factor Vll per day being more commonly used. It must be kept in mind that the materials of the present invention may generally be employed in serious dj-~E~5e or injury CA 022~6761 1998-12-01 W O 97/47651 PCTADX97/002Sl states~ that is, life-threatening or potentially life threatening situations. In such cases, in view of the minimization of extraneous substances and general lack of immunogenicity of modified human Factor Vll in humans, it is possible and may be felt desirable by the treating physician to ad",i~ ter substantial excesses of these modified Factor Vll compositions.
In prophylactic applications, cGmpositions containing the modified Factor Vll are administered to a patient susceptihle to or Gll,el~J ise at risk of a ~iee~se state or injury to enhance the patient's own anticoagu'ative capabilities. Such an amount is defined to be a "prophylactis;"y effective dose." In this use, the precise amounts again depend on the patient's state of health and weight, but generally range from about 0.05 mg to about 500 mg 10 per 70 kiloy,ar" patient, more commonly from about 1.0 mg to about 200 mg per 70 kg of body weight.
Single or multiple adlnini;~lldliGns of the co",positions can be carried out with dose levels and pattern being selected by the tl~dling physician. For ambulatory palierll~
requiring daily maintenance levels, the modified Factor Vll may be administered by 15 continuous infusion using a po, i lt le pump system, for example.
Local delivery of the ",odir,ed Factor Vll such as, for example topical ~pplicPIion of modified Factor Vll at vascular sites susceptible to ll,n.,nbus formation, (e.g. sites of anasto",osis) or at vascular sites susceptible to decreased patency may be carried out, for example, by way of spray, perfusion, double balloon call,eler:j, stent, incorporated into 20 vascular grafts or stents, hydrogels used to coat balloon catheters, or other well established methods. In any event, the pharmaceutical formulations should provide a quantity of modified Factor Vll of this invention sufficient to effectively treat the patient.

The fo'low;"g examples are offered by way of illusl(dtion, not by way of limitation.

,,, , ....... .... . ~ . , .

CA 022~6761 1998-12-01 W O 97/476Sl PCTADK~7/00251 EXAMPLES

FXAMPI F I
F~ression of Ser~11~Ala;~ Factor Vll To generate the Ser344~3)Ala Factor Vll active site mutant, plasmid FVI1(565 1 2463)/
pDX (U.S. Patent No. 4,784,950 incorporated herein by ,~rer~nce; deposited with the American Type Culture Collection under accession number 40205) was digested with Xba I
and Kpn 1, and the resulting 0.6 kb r,ay"~en~, cG",pri~i"g the coding region for serine 344, was recovered. This t~aylllent was cloned into Xba 1, Kpn l-digested M13mp19 as shown in the Figure. This manipulation and subsequent steps described below were generally pe, rurmed according to slandard protocols (as described, for example, by Maniatis et al., Molecular Cloning. A I ~h~ratory Manual. Cold Spring Harbor Labora~ory Press, Cold Spring Harbor, N.Y. (1982) incorporated herein by reference).
Mutagenesis was carried out on the M13 template according to the methods of Zoller and Smith, supra, using the mutagenic oligonucleotide ZC1656 (5' TGG GCC TCC
GGC GTC CCC CTT 3') and the "universal" second primer ZC87 (5' TCC CAG TCA CGA
CGT 3'). ~eaction products were screened using kinased ZC1656. Positive plaques were picked, and template DNA was prepared and sequenced from the Pst I site at 1077 to the Kpn I site at 1213. Sequence analysis confirmed the presence of the desired mutation. The mutant clone was designated 1656.
An expression vector was then constructed using the 1656 clone. The mutagenized sequence was isolated from the M13 vector as a ~0.14 kb Pst l-Kpn I fragment. This fragment was ligated to the 1.7 kb Hind Ill-Xba I fragment from FVI1(565+2463)/pDX, the 0.5 kb Xba l-Pst I r,agmenl from FVI1(565+2463)/pDX, and the 4.3 kb Kpn l-Hind lll fragment from FVI1(565+2463)/pDX, as shown in the Figure. The presence of the desired mutant sequence was confirmed by digesting mutant and wild-type clones with Pst 1, and a mutant Factor Vll insert in M13 with Kpn I and Xba 1, preparing Southern blots of the digested DNA, and probing the blots with radiolabeled ZC1656.
The baby hamster kidney cell line BHK 570 (deposited with the American Type Culture Collection under accession number 10314) was transfected with two isolates (designated #544 and #545) of the 1656 expression vector. The cells were prepared by diluting a confluent 10 cm plate of BHK 570 cells 1:10 into five 10 cm plates in non-selective CA 022~676l l998-l2-Ol W O 97/47651 PCTnDK~7/00251 medium (Dulbecco's "~odiried Eagle's medium [DMEM] containing 10% fetal bovine serum and 1 % PSN antibiotic mix lGIBCO Life Technologies, Gaithersburg, MD]). After 24 hours, when the cells had reached 20-30% confluency, they were co-transfected with one isolate of the ex~,ression vector encoding the 1656 mutation, plasmid p486 (comprising the Adenovirus 5 ori, SV40 enhancer, Adenovirus 2 major late promotor, Adenovirus 2 ll ipa, lil~ leader, 5' and 3' splice sites, the DHFR' cDNA and SV40 polyadenylation signal in pML-1 (Lusky and Botchan, Nature 293: 79-81, (1981)) and 10 mg of carrier DNA (sonicated salmon sperm DNA) as shown in Table 1. The DNA was added to a 15 ml tube, then 0.5 ml of 2X Hepes (25 9 Hepes, 40 9 NaCI, 1.8 9 KCI, 0.75 9 Na2HPO42H20, 5 9 dextrose diluted to 2.51 with 10 distilled water and pH adjusted to pH 6.95-7.0) was added and the tubes were mixed. The DNA in each tube was prec;pit~ted by the addition of 0.5 ml of 0.25 M CaCI2 while air was bubbled through the DNA/Hepes solution with a pasteur pipet. The tubes were thenvortexed, incubated at room temperature for 15 minutes, and vortexed again. The DNA
mixtures were then added d,opwise onto the plates of cells with a pipette. The plates were 15 swirled and incubated at 37~C for 4-6 hours. After inc~ ~b~tion, 2 ml of 20% glycerol diluted in Tris-saline (0.375 9 KCI, 0.71 9 Na2HPO4, 8.1 g NaCI, 3.0 g Tris-HCI, 0.5 9 sucrose, diluted in a total of 1 liter and pH adjusted to pH 7.9) was then added to each plate. The plates were swirled and left at room temperature for two minutes. The medium was then removed from the plates and repl~ced with 2 ml of Tris-saline. The plates were left at room temperature for 2 minutes, then the Tris-saline was removed and replaced with 10 ml of non-selective medium. The plates were incubated at 37~C for two days.

. .

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 Table 1 Transfection*

544 545 544 Control 545 Control Plasmid Name Clone 544 15 mml --- 15 ml ---Clone 545 --- 30 ml --- 30 ml p486 1.5 ml 1.5 ml --- ---Carrier DNA 1.6 ml 1.6 ml 1.6 ml 1.6 ml ~ DNA concen~,dlions used were: clone 544: 0.7 mg/ml; clone 545: 0.3 mg/ml; p486: 1.49 mg/ml.

After the two day incubation, the cells were diluted in selection medium (DMEM
containing 10% dialyzed fetal bovine serum, 1% PSN antibiotic mix and 150 nM
methotrexate) and plated at dilutions of 1:100, 1:250 and 1:500 in maxi plates. The plates were incubated at 37~C for one week. After one week, the medium was changed and replaced with selection medium, and the plates were checked for colony for"~ation.
Eight days later, after colony formation, twelve colonies were randomly chosen from the 1 :500 dilution plates of the #544 and #545 transfection plates. Each clone was plated into one well of a 6-well plate and grown in selection medium. After seven days, the plates were confluent, and the clones were each split into 10 cm plates in selection medium.
The clones described above and control cells lrdn~ected to express wild-type factor Vll were metabolically labeled with 35S-Methionine-Cysteine Protein Labeling Mix (NEN
DuPont Biotechnology Systems, Wilmington, DE). The clones were grown and prepared for a pulse label experiment in selective medium. The cells were rinsed with phosphate buffered saline (Sigma, St. Louis, MO) and pulsed for four hours in 20 mCi/ml 35S-Cys-35S-Met. After four hours, supernatants and cells were harvested. The cells were Iysed essentially as described by Lenk and Penman (Cell 16: 289-302, (1979)) and 400 ml of each Iysate and precleared with 50 ml of staph A (Sigma, St. Louis, MO).

CA 022~6761 1998-12-01 PCT~DK~7/00251 Samples from the met~h~ lly labeled cells were radioimmunoprecipitated (RIP) - by first incubating the samples with 6 ml of anti-Factor Vll polyclonal antisera for four hours.
Sixty microliters of washed staphylococc~l protein A was added to each sample, and the samples were rocked for 1.5 hours at 4~C. The samples were centrifuged, and the 5 super"atanl was removed. The pellets were washed twice in 0.7 M RIPA buffer (10 mM
Tris, pH 7.4, 1% deoxycholic acid [Calbiochem Corp., La Jolla, CA],1 % Triton X-100, 0.1%
SDS, 5 mM EDTA, 0.7 M NaCI) and once in 0.15 M RIPA buffer (10 mM Tris, pH 7.4, 1%
deoxycholic acid [Calbiochem Gorp., La Jolla, CA],1 % Triton X-100, 0.1 SDS, 5 mM EDTA, 0.15 M NaCI). One hundred microliters of 1x SDS dye (50 mM Tris-HCI, pH 6.8, 100 mM
dithiothreitol, 2% SDS, 0.1% bromphenol blue, 10% glycerol) was added to each sample, and the sai"F!es were boiled for 5 minutes followed by centrifugation to remove the protein A. Fifty micrc!i'e:r~ of each sample was run on a 10% polyacrylamide gel. Results showed that 9 of 10 clones secreted modified Factor Vll.

15 FXAMPLE ll ANTICOAGUI ~NT ACTIVITY OF MODlFl~n FACTOR Vll The ability of the modified Factor Vll protein to inhibit clotting was measured in a one-stage clotting assay using wild-type Factor Vll as a control. Recombinant p/~tei"s were prepared essentially as described above from cells cultured in media co,)l~i.,ing 5mg/ml 20 vitamin K. Varying amounts of the modified Factor Vll (from clone 544) or recombinant wild-type Factor Vll were diluted in 50 mM Tris pH 7.5, 0.1% BSA to 100 ml. The mixtures were inc~lhated with 100 ml of Factor Vll-deficient plasma (George King Bio-Medical Inc., Overland Park, KS) and 200 ml of thrombopla~l;" C (Dade, Miami, FL; contains rabbit brain thromboplastin and 11.8 mM Ca~). The clotting assay was performed in an automatic 25 coag~ ~'ation timer (MLA Electra 800, Medical Laboratory Automation Inc., Pleasantville, NY), and clotting times were converted to units of Factor Vll activity using a standard curve constructed with 1:5 to 1:640 dilutions of normal pooled human plasma (assumed to contain one unit per ml Factor Vll activity; prepared by pooling citrated serum from healthy donors).
Using this assay the pleparalions of modified Factor Vll exhibited no detect~hlE coagulant 30 activity. Table 2 shows results of the assay in terms of clotting times for control (unl,dn~ected) BHK cell-conditioned media (+/- vitamin K), wild-type Factor VII and two CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 28 iso!qtes of cells ex~r~ssi-,g the modified Factor Vll. Factor Vll activity is seen as a reduction in clotting time over control sar ,p es.

Table 2 Sample Dilution Clotting Time (sec.) Control ~K 1:5 33.1 1:10 33-4 Control-K 1:~ 34.3 1:10 33.2 Wild-type FactorVII 1:20 19.0 1.40 21.5 1 :80 23.3 Modified FactorVII (#6) 1:1 33.5 Modified FactorVII (#10) 1:1 32.5 To determine the effect of the modified Factor Vll on plasma factor substrates preparations of modified Factor Vll and recombinant wild-type or native Factor Vll are incubated with either Factor X or Factor IX and the activation thereof monitored by clotting assays or polyacrylamide gel electrophoresis.

FXAMPLE lll Ability of Modified Factor Vll to Bind Tissue Factor The ability of the modified Factor Vll to co",pete with wild-type Factor Vll for tissue 15 factor and inhibit its clotting activity was assessed in a one-step clotting assay in the presence of a limiting amount of tissue factor (thromboplastin).
Clotting times were determined in a one-step assay similar to that described in Example ll. A limited amount of tissue factor a constant amount of wild type Factor Vll and increasing amounts of variant Factor Vll were used in the mixing experiments. An inhibition 20 of Factor VllNlla procoagulant activity would be seen as an increase in clotting time in assays containing increasing amounts of variant Factor Vll.

CA 022~6761 1998-12-01 W O97/47651 PCT~DK97/00251 The amount of Factor Vll activity in the test samples was c~'sul~terl as a per~enlage of a slandard curve that measured Factor Vll activity in normal pooled plasma.
The standard curve for Factor Vll activity was generated using serial dilutions of normal pooled plasma in phosphate buffered solution (PBS) that ranged from 1:5 to 1:640. For this 5 purpose it was assumed that normal plasma contains approximately 500 ng/ml of Factor Vll and this was considered to be one unit of activity. A mixture of 100 ml Factor Vll-deficient plasma, 100 ml plasma dilution and 200 ml of thromboplastin-C (Dade, Miami, FL.) was used to measure clotting time on a MLA Electra 800 autGu~alic timer. To e~ l sh the slandard curve, the results were graphed as percentage of activity (1:5 = 100% activity) versus 10 clotting time in seconds.
The assay re~uired that the medium containing the wild type and variant Factor Vll be composed of less than one percent serum. The dilutions were made in PBS so that clotting times would fall along the standard curve. A minimum dilution of 1:2 was typical.
The final volume was 100 ml. Two different human Factor Vll Ser344 ~ Ala variants, 15 designated clones "#10" and "#6" were tested in the experiments. The results, set forth in the Table below, show that as the amount of Factor Vll variant increased, the percent of Factor Vlla activity decreased.

CA 022~676l l998-l2-Ol Table 3:
Results of mixing assay with Ser344 -~ Ala Variants (B4A1 (wild type~ medium was used as 100% activity at 10 )ll/reaction) Ser344-~Ala Variantmedium B4A1 medium BHKcontrol* PercentFVllaClone No. amount amount Activity #10 10 ,ul 10 ~11 0 70 #10 20 ,u1 10 ~11 0 51 #10 30 ~11 10 ~11 0 43 #10 40 ~11 10 ~l 0 34 #10 50 ~11 10 1ll 0 28 #10 (-K) 20 ~LI 10 ~l 0 78 #6 10 ~l 10 ~l 0 74 #6 20 ~l 10 1ll 0 56 #6 30 1ll 10 ~11 0 46 #6 40 1ll 10 ~l 0 41 #6 50 ~l 10 ~l 0 32 #6 20 ~l 10 1ll 0 85 BHK control 0 10 1ll 20 ~l 91 BHK control (-K) 0 10 ~l 20 1ll 107 Untransfected conditioned medium 5 For expression of the Factor Vll variant, cells were grown in the presence of vitamin K, except where noted "(-K)".
These experiments showed that variants of Factor Vll having a Ser344 ~ Ala substitution competed with native Factor Vll in a dose dependent fashion and inhibited the procoagulant activity of native Factor VllNlla. It can thus be concluded that Ser344 tg) Ala variant human Factor Vll competes with native human Factor Vlla and consequently inhibits 15 activation of Factor X and/or IX in human plasma.

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CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/00251 FXAMPI F IV
Reaction of Factor Vll with PPACK
Recombinant Factor Vll was produced in transfected baby har,lsler kidney cells.
The protein was purified and activated as ~Ecclosed by Thim et al. (Bioche",isl,y 27: 7785-7793, 1988), Brinkous et al. (Proc. Natl.Acad. Sci. USA 86: 1382-1386, 1989) and Bjoern and Thim (Res. Discl. No. ~9, 564, 1986), which are incor,uoraled herein by reference. The cell culture medium was recovered, filtered and diluted to reduce salt concentration. The diluted medium was then fractionated by anion exchange chromalog,dphy using an elution buffer containing CaCI2. The Factor Vll fraction was recovered and further purified by immunochromalog,aphy using a calcium-dependent anti-Factor Vll monoclonal antibody.
Additional purification was carried out using two anion exchange chrol"alog,dphy steps wherein Factor Vll was eluted using CaCI2 and NaCI, respectively. Factor Vlla was recovered in the final eluate.
Recombinant Factor Vlla (1 mM) in 50 mM Tris-HCI, 100 mM NaCI, 5 mM CaCI2, ph 7.4 was incuhated with 20 mM PPack (D-Phenylalanyl P~,lyl Arginyl Chloromethyl Ketone;
Calbiochem, La Jolla, CA) for 5, 20 and 60 minutes. Buffer containing the chromogenic substrate S2288 (H-D-lsoleucine-L-Prolyl-L-Arginine p-nitroanilide; Kabi Vitrum AB, Molndal, Sweden) was then added to obtain a 2.5 fold dilution and a final concentration of 0.3 mM
S2288. The generation of p-nitroaniline was measured and compared to results using untreated Factor Vlla as a control. The results indicated that Factor Vlla is fully inactivated after about 60 minutes under these reaction condilions.

FxAMpl F V
Generation of DFGR-Factor Vlla Recombinant human Factor Vlla was prepared as descl ibed in Example IV.
Recombinant human FactorVlla, in 10 mM glycine buffer, pH 8.0, 10 mM CaCI2, 50 mM
NaCI, was diluted to a concer,lldlion of 1.5 mg/ml. A 10-fold molar excess of Dansyl-L-Glu-Gly-Arg-Chloromethyl Ketone, DEGRck, (Calbiochem, La Jolla, CA 92037) which had been dissolved with distilled H2O was added to the Factor Vlla. After a 2 hr inc~lhation at 37~C, a ~ , CA 022~6761 1998-12-01 W O 97/47651 PCTnDK97/00251 second 1 0-fold molar excess of DEGRck was added to the mixture and incub~ted for an additional 2 hr at 37~C. A third 1 0-fold molar excess of DEGRck was added to the Factor Vlla and incubated for approxi",alely 16 hours at 4~C. The DEGR-Factor Vlla sample was then extensively dialyzed at 4~C against Tris buffered saline (0.05 M Tris-HCI 0.1 M NaCI
pH 7.5) to remove any free DEGRck.
The final DEGR-Factor Vlla mixture was tested for the presence of free DEGRck ina Factor Xa chrc,r"ogenic substrate assay. The DEGR-Factor Vlla mixture was added to purified human Factor Xa along with the chromogenic substrate S-2222. This substrate is cleaved specifically by Factor Xa and not by Factor Vlla. Unbound DEGRck in the mixture is 10 able to bind to the Factor Xa and there by inhibit the chromogenic activity of the Factor Xa.
Spiking free DEGR-ck into a Factor Xa mixture generated a standard curve to measure the level of free DEGRck in solution versus the inhibition of Factor Xa chromogenic activity.
Analysis of the DEGR-Factor Vlla mixture showed that the ratio of free DEGRck:DEGR-Factor Vlla was less than 0.5% following extensive dialysis thereby ensuring that the 15 inhibition observed by DEGR-Factor Vlla in the various assay systems described below was not due to the presence of free DEGRck.

EXAMPLE Vl Factor Xa Generation on Rat Smooth Muscle Cells Vascular smooth muscle cells were analyzed for the presence of cell-surface tissue factor by measuring the ability of the cells to stimulate the conversion of Factor X to Factor Xa using a chromogenic substrate that is specific for Factor Xa.
Rat vascular smooth muscle cells (Clowes et al. J. Clin. Invest. 93:644-651 (1994)) were plated into 96-well culture dishes (American Scientific Products Chicago Il.) at 8 000 25 cells per well in growth media (Table 4).

. . . ~ , .

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97100251 Table 4 500 ml Dulbecco's Modified Eagle's Medium (DMEM) (GIBCO-BRL, Gaill,er~burg, MD.)10% fetal calf serum (Hyclone, Logan, UT.) 5 1mM sodium pyruvate (Irvine, Santa Ana, CA.) 0.29 mg/ml L-glutamine (Hazelton, Lenexa, KS.) 1x PSN; (100X is 5 mg/ml penicillin, 5 mg/ml streptomycin, 10 mg/ml neomycin) (GIBCO-BRL, Gaill,er~burg, MD.) After a 48 hour incubation at 37~C the medium was changed to serum free medium (Table 5).

Table 5 250 ml Dl~lbecco's Modified Eagle's Medium (DMEM) 250 ml Ham's F-12 Medium (Fred Hutchinson Cancer Research Center, Seattle, WA) 1mM sodium pyruvate .29 mg/ml L-glutamine 20 mM transferrin (JRH, Lenexa, KS.) 5 mM insulin (GIBCO-BRL) 16 ng selenium (Aldrich, Milwaukee, Wl.) 1 mg/ml bovine serum albumin (Sigma, St. Louis, MO) The cells were inc~ ~hated 72 hours at 37~C. After incubation, either PDGF-BB
(10 ng/ml) or 10% fetal calf serum was added to the cells to stimulate tissue factor expression (Taubman et al., J. Clin. Invest. 91:547-552, 1993). A parallel set of cells received neither PDGF nor serum to monitor for intrinsic activity of unstimulated cells. After a 6 hour incubation, recombinant human Factor Vlla was added to the cells at a final concentration of 10 nM. One set of cells did not have Factor Vlla added as a negative control. The cells were incubated for 2 hours at 37~C and washed with HEPES buffer (10 mM HEPES, 137 mM NaCI, 4 mM KCI, 5 mM CaCI2, 11 mM glucose, 0.1% BSA). After washing, cells were incubated for 5 min with 50 ml per well of 200 nM plasma-purified human Factor X in a Tris-buffered saline supplemented with 5 mM CaCI2. Twenty-five . , ... .. , _ .. , CA 022~6761 1998-12-01 WO 97/476~1 PCT~DK97/00251 microliters of 0.5 M EDTA and 25 ml of an 800 mM solution of S-2222 chromogenic substrate (Kabi Pl,ar",acia, Franklin, OH) were added to each well. The plates were inc~ ~h~ted for 40 min at room temperature, then analyzed at 405 nm using a THERMOMAX
",icroplale reader (Molecular Devices, Menlo Park, CA).
Table 6 shows an increase in absorl.ance for the Factor Vlla treated wells as compared to the control wells (no Factor Vlla added). The increase in abso,Lance is a direct measurement of the level of Factor Xa generated in the wells and its subsequent cleavage of the chromogenic substrate, releasing the chromophore. The data also demonsl, dle that the level of chromogenic activity in cells pretreated with either PDGF-BB or 10% fetal calf serum 10 was higherthan unstimulated cells.

Table 6 Test Sample OD40s Control 0.043 Intrinsic 0.247 PDGF-BB 0.360 10% FCS 0.342 These results clearly show there is a Factor Vlla-dependent activation of Factor X to Factor Xa on the cell surface of rat vascular smooth muscle cells.

EXAMPLE Vll Inhibition of Cell-Surface ChromogenicActivity By DFGR-Factor Vlla Rat vascular smooth muscle cells were plated into 96-well culture dishes as described above. The cells were cultured for 72 hours in serum free media as described above and treated with the addition of 10% fetal calf serum for 6 hours to stimulate tissue factor expression. After stimulation, buffer only (control), 10 nM Factor Vlla, or 10 nM Factor Vlla + 100 nM DEGR-Factor Vlla was added to each well. The cells were incubated for 2 hours at 37~C, then washed with HEPES buffer. After washing, the cells were incubated for 5 minutes with 50 ml per well of 200 nM Factor X in Tris-buffered saline supplemented with 5 ~ ., CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/00251 mM CaCI2. Twenty-five microliters of 0.5 M EDTA and 25 ml of S-2222 (800 mM) chromogenic substrate (Kabi Pharmacia) were added to each well. The cells were incuh~ted at room temperature for 40 minutes. Chrumogenic activity was analyzed at 405 nm as described above.
Table 7 shows stimulation of ch~o",oyen c activity in the wells treated with Factor Vlla only, and inhibition of stimulation when DEGR-Factor Vlla was co-incubated with the Factor Vlla. These results demonsl, ale that DEGR-Factor Vlla acts as a competitive antagoni4t for Factor Vlla binding, thereby inhibiting the activation of Factor X to Factor Xa and the subsequent cleavage of the S-2222 chromogen.
Table 7 Test Sample OD40s Control 0 035 Factor Vlla 0.342 Factor Vlla + 0.073 DEGR-Factor Vlla .073 EXAMPLE Vlll 15 Dose Dependent Inhibition by DFGR-Factor Vlla of Cell Surface Chromo~enic Activity on Rat Smooth Muscle Cells Rat vascular smooth muscle cells were plated into 96-well culture dishes at 4,000 cells perwell in growth medium sl~rF!er"ented with 1% fetal calf serum (as in Table 4 without 10% fetal calf serum). After 5 days the medium was removed, and either increasing 20 concentrations of Factor Vlla alone or 10 nM Factor Vlla with increasing concentrations of DEGR-Factor Vlla were added to the cells. The cells were incuhated with the Factor Vll mixtures for 2 hours at 37~C. After incubation, the cells were washed and incubated with 50 ml of 200 nM Factor X in tris buffered saline for 5 minutes at room temperature. Each well had 25 ml of 0.5 M EDTA and 25 ml of 800 mM S-2222 (Kabi Pharmacia) added to it, and 25 the plates were incubated for 40 minutes at room temperature. Chromogenic activity was analyzed at 405 nm in a microplate reader as described above.

.. .. . . ..

CA 022~676l l998-l2-Ol W 0 97/47651 PCT~DK~7/00251 Table 8 shows a dose-depender)l increase in chromogenic activity with incleas" ,g amounts of Factor Vlla added to the wells. When the mixture of DEGR-Factor Vlla with 100 nM Factor Vlla was added to the cells (Table 9) there was a dose dependent inhibition in chromogenic activity. A 1:1 molar ratio of DEGR-Factor Vlla:Factor Vlla inhibited 5 approximately 95% of the chromogenic activity. These data suggest that in thisexpe,i",enlal design DEGR-Factor Vlla has a significantly higher affinity for cell-surface tissue factor than native Factor Vlla on smooth muscle cells in culture. If DEGR-Factor Vlla and Factor Vlla had equal affinity for binding tissue factor then the level of inhibition observed when the two molecules were added to the cells in an equal molar ratio would not 10 have been as high.

Table 8 Factor Vlla Conc. OD40s (nM) 0.10 0.005 0.39 0.025 1.56 0.058 6.25 0.111 25.00 0. 154 1 00.00 0.208 Table 9 shows the dose dependent inhibition of Factor Xa chromogenic activity on15 rat smooth muscle cells by DEGR-FactorVlla. Increasing concenl,dlions of DEGR-Factor Vlla were co-incubated with 100 nM Factor Vlla and the Factor Xa chromogenic activity determined using chromogenic substrate S-2222.

.

CA 022~6761 1998-12-01 Table 9 DEGR-Factor Vlla OD40s Conc. (nM) 0.10 0.208 0.39 0.176 1.56 0.116 6.25 0.073 25.00 0.026 100.00 0.014 Inhibition of Factor Xa generation by DEGR-Factor Vlla in a soluble tissue factor assay.
The conversion of Factor X to Factor Xa using purified recombinant soluble tissue factor was est?'- !is hed using a chromogenic assay. Tissue factor was expressed and purified from Saccharomyces cerevisiae (Shigematsu et al., J. Biol. Chem. 267:21329-21337, 1992). Soluble tissue factor was purified and characterized by Dr. W. Kisiel (University of New Mexico). A reaction mixture containing 65.9 ml of soluble tissue factor (2.2 mM), 29.0 ml of PCPS (1 mM, Sigma, St. Louis, MO), 29.5 ml human Factor X (4.1 mM), 2.77 ml Hank's buffer (25 mM Tris, pH 7.4, 150 mM NaCI, 2.7 mM KCI, 5 mM CaCI2, 0.1 % BSA) was prepared. Forty microliter of tissue factor/Factor X mixture, 25 ml Factor 15 Vlla diluted with TBS and 25 ml of DEGR-Factor Vlla diluted with TBS were added to each well of a 96-well microtiter plate. A control using 40 ml of tissue factorlFactor X mixture; 25 ml Factor Vlla diluted with TBS, and 25 ml of TBS only was included. Ten microliters of S-2222 (4 mM) chromogenic substrate was added to the reaction mixture in the wells and incubated at room temperature for 2-10 minutes. Results were analyzed at 405 nm in a 20 microplate reader as described above.
Determination of a slanda,d curve for Factor Vlla activation of Factor X was made using increasing concentrations of Factor Vlla added in the absence of DEGR-Factor Vlla.
The results, presented in Table 10, show that there is a dose-dependent increase in , . ~ , ... ..

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 chromogenic activity with increasing amounts of Factor Vlla added to the reaction mixture.
The simultaneous addition of varying amounts of DEGR-Factor Vlla and 100 nM Factor Vlla led to a dose dependent decrease in chromogenic activity (Table 11). These data demonstrate that DEGR-Factor Vlla acts as a competitive antagonist for native Factor Vlla 5 binding to soluble tissue factor, and thereby inhibits the generation of Factor Xa as measured by the decrease in chromogenic activity towards the chromogenic substrate S-2222.

Table 10 10 Stimulation of Factor Xa chromogenic activity with increasing concentrations of Factor Vlla added to soluble tissue factor. Changes in optical density were measured using chromogenic substrate S-2222.

Factor Vlla Conc (nM) OD40s 0.78 0.168 1.56 0.288 3.12 0.478 6.25 0.694 12.50 0.764 25.00 0.790 50.00 0.738 100.00 0.770 CA 022~6761 1998-12-01 Table 11 Inhibition of Factor Xa chromogenic activity by the addition of DEGR-Factor Vlla to soluble tissue factor in the presence of native Factor Vlla is measured. Changes in optical density 5 were measured using the .;I,romogen.c substrate S-2222.

DEGR-Factor Vlla OD40s Conc. (nM) - ~
0 0.810 50 0.750 100 0.609 200 0.296 400 0.167 800 0.083 1600 0.055 EXAMpl F X
10 Inhibition of Coagulation by DEGR-Factor Vlla Standard clotting assays to monitor the effect of DEGR-Factor Vlla on clotting time were prepared as follows: 100 ml of normal baboon plasma, collected with sodium citrate as anticoagul?nt, was added to 100 ml of varying concenl,alions of DEGR-Factor Vlla diluted in TBS (20 mM Tris, pH 7.4,150 mM NaCI). The samples were mixed and briefly inc~bated at 37~C. The samples were added to an Electra 800 Automatic Goagu'~tion Timer (Medical Laboratories Automation, Pleasantville, NY). After incubation, 200 ml of a tissue factor p,epardlion containing 25 mM CaCI2was added to the DEGR-Factor Vlla prepa,~lions. A
tissue factor preparation was made as a saline extract of baboon brain from freshly frozen brain tissue and cha,dcteri~ed for its ability to initiate coagulation in baboon plasma~ A
20 concentration of tissue factor that gave a clotting time of about 40 seconds was selected.

... ... .... . ..

CA 022~6761 1998-12-01 W O 97/47651 PCT~DX~7/00251 The data presented in Table 12 der"or,sl,dles a dose-dependent increase in clotting time due to the addition of DEGR-Factor Vlla. A dose as low as 1 mglml of DEGR-Factor Vlla in plasma resulted in a significant increase in clotting time.

Table 12 Dose dependent increase in clotting time due to DEGR-Factor Vlla.

DEGR-Factor Clotting Time Vlla (seconds) (~lg/ml plasma) 0 40.7 0.~ 46.2 1.0 50.8 2.5 64.5 5.0 108.1 10.0 158.4 10 EXAMPI F Xl Inhibition of Platelet Accumulation With DEGR-Factor Vlla DEGR-Factor Vlla was analyzed for its ability to inhibit platelet accumulation at sites of arterial thrombosis due to mechanical injury in non-human pri",ales. A model of aortic enda, lerectomy was utilized in baboons essentially as described by Lumsden et al. (Blood 81:1762-1770 (1993)). A section of baboon aorta 1-2 cm in length was removed inverted and scraped to remove the intima of the artery and approximately 50% of the media. The artery was reverted back to its correct orientation cannulated on both ends and placed into an extracorporeal shunt in a baboon thereby exposing the mechanically injured artery to baboon blood via the shunt. Just prior to opening of the shunt to the circulating blood 1l'1n-20 labeled autologous pl~teletc were i",ected intravenously into the animal. The level of platelet accumulation at the site of the injured artery was determined by real-time gamma camera imaging.

CA 022',6761 1998-12-01 Evaluation of DEGR-Factor Vlla for inhibition of platelet accumulation was done using bolus injections of DEGR-Factor Vlla or saline control and were given just prior to the opening of the shunt. The injured arteries were measured continuously for 60 minutes. A
dose of 0.005 mg/kg of DEGR-Factor Vlla inhibited platelet accumulation. At a 1.0 mglkg bolus injection, approximately 90% of platelet accumulation was inhibited at 1 hour post drug all"ini~l,dlion. These results are shown in Fig. 2.
These data show that inhibition of tissue factor with DEGR-Factor Vlla can significantiy inhibit the dcvelopn,ent of platelet-rich lh,ol"bi in a nonhuman primate model of acute vascular injury.

FXAMPI F Xll DFGR-FVlla Inhibits Vascular Reslenosis Fcl'.~ i,lg Balloon Any;opla;~ly in Atherosclerotic 1 5 Rabbits DEGR-FVlla was evaluated for its ability to modulate lesion development following balloon angioplasty in New Zealand White (NZ'\/V) atherosclerotic rabbits. This animal model has been well characterized and has proven to be a good model for evaluating anti-thrombotic compounds on vascular lesion development (Gimple et al., Circulation 86:1536-1546 (1992), and Rogosta et al., Circulation 89:1262-1271 (1994)). The animal model used to evaluate DEGR-FVlla is essenliG' y as described by Ragosta, ibid.
Anesthesia was induced in rabbits with 5 mg/kg xylazine and 35 mg/kg ketamine byintramuscular injection. The proximal femoral arteries were exposed by cutdown below the inguinal ligament with proximal and distal ligatures. The isolated segmer,l~ were cannulated with 27 gauge needles. A vent was created by needle puncture. The isol ~lec! seg",enLs were flushed with saline to clear residual blood, and desicca,Lsd by air infused at a rate of 80 ml/min for 8 minutes. Following air-drying, the isolated segments were again flushed with saline and the ligatures removed. Hemostasis was maintained with non-occlusive local pressure. The segments were demarcated with metal clips. Local spasm was treated with Xylocaine 1% locally. The day following surgery, the animals were placed on 1% cholesterol and 6% peanut oil diet for one month until balloon angioplasly. Tylenol 10 mg/kg orally was CA 022~6761 1998-12-01 WO 97/47651 PCT~DK~7/00251 given for postoperdlive pain relief for 3-5 days. Ambipen 1cc was given after the surgical procedure during postoperative days 3 to 5.
The test drug delivery for the animals consisted of an initial bolus injection immediately prior to balloon angioplasty, followed by a continuous systemic infusion by 5 osmotic pump via the internal jugular vein. The duration of the drug infusion was 3 days.
The control animals received heparin, 150 Ulkg IV bolus, prior to balloon angioplasty rol'ovled by saline infusion. The DEGR-FVlla treated animals received a 1 mg/kg bolus i",e t cn followed by 50 mglkglhr infusion.
For the placement of the osmotic pumps for continuous systemic infusion, 10 anesthesia was induced in the animals as described above, and maintained throughout the procedure with additional IM injections of ketamine and Xylazine. Through a midline neck incision, the right internal jugular vein was isolated by blunt dissection and the distal end ligated. A silastic tube (PE-160) was introduced into the right internal jugular vein. A
subcutaneous tunnel was created to pass the silastic tube. This tube was connected with 15 the osmotic pump. The osmotic pump was implanted subcutaneously in the back of the rabbit. The right common carotid artery was isolated by blunt dissection and the distal end ligated. Via an arteriotomy, a 5F introducer was placed and advanced to the junction of the aortic arch. Blood was drawn for determination of hemostatic parameters, drugs and choleslerol levels. Twenty milligrams of xylocain was injected ir,l,da,lerially. A control 20 aortoiliofemoral angiogram was performed via a 5F Berman cdtl,eter positioned above the aortic bifurcation using 3~ ml renographin injected over 3 seconds by hand.
After removal of the Berman cdlheter, a 0.014-inch guidewire was introduced in the descending aorta and positioned above the aortic bifurcation. Under fluoroscopic guidance, an appropriately sized balloon angioplasty c~tl,eter of 2.0 to 2.5 mm was introduced and 25 advanced over the gu~de~;,e and positioned across the stenosis. The balloon was inflated to 6 atmospheres for 60 seconds with a hand inflator. Three i"fldlions were performed with 60 second intervals ~etvJecn inrldliGns. This procedure was pe,r~r",ed in both femoral arteries in each animal.
Following balloon dilatation, the angioplasty catheter was withdrawn and the Berman 30 catheter reintroduced to a position 3 cm above the aortic bifurcation. To minimize spasm 20 mg of lidocaine was given intraarterially. A post procedure an~;oy,dl" was perFormed as described above. A 1 cm grid was positioned at the level of the femoral artery to calculate , ... . . .. .

CA 022~6761 1998-12-01 the actual diarl ,eter. The Odtlleter was then removed. The right carotid artery was ligated with 3-0 silk and the wound sutured by layers. Ambipen and aceta,l. .lophen were given as above.
Prothrombin time and concentldlion of DEGR-FVlla in the blood were deterll,;ned at 5 immediately pre-bolus in, ';on of the test compound, 1 hr post bolus ill,ec'icn, and at 3 days at the end of continuous infusion. One to two mls of citrated plasma was obtained and the ploltlrorllbin times and antigen levels determined.
A slandar-l clotting assay was used to monitor the protllro-llt..) time in the control and DEGR-FVlla-treated animals as follows. Twenty-five microliters of test rabbit plasma, collected with sodium citrate as anticoag~ nt, was added to 150 ml of TBS (20 mM Tris, pH
7.4, 150 mM NaCI). The samples were mixed and added to an Electra 800 Automated Co~gu~ation Timer (Medical Laboratories Automation, Pleasantville, NY). After incubation, 200 ml of thrombop'astirl prepardlion (Sigma Chemical) containing 25 mM CaCI2 was added to the plasma prepar;3~ions. A concelltldlion of thrornhopl~slin that gave a clotting time of approximately 20 seconds in the control rabbit plasma was selected.
An ELISA assay was used to determine the concentration of DEGR-FVlla in plasma samFles from the control and DEGR-FVlla treated rabbits. The assay involved first diluting an anti-human FVII monoclonal antibody (Dr. W. Kisiel, U. of New Mexico) to 2.0 mg/ml in 0.1 M carbonate buffer pH 9.6, and adding 100 ml/well to 96-well plates. The plates were then incubated at 4~C overnight and subsequently washed two times using wash buffer (PBS, pH 7.4, containing 0.05% Tween 20). Blocking of nonspecific binding sites was achieved with 200 ml of blocking buffer per well (PBS, pH 7.4, containing 0.05% Tween 20 and 1% BSA) incut)~ted at 37~C for 2 hr, f.'lD~ ecJ by a wash using the wash buffer.
After blocl;ing, a ~landald dilution series of DEGR-FVlla ranging from 20-0.027 ng/ml was added, along with a dilution series of the test rabbit plasma (1:100 to 1:4000 in b'o~'~ing buffer) applied at 100 ml/well. Non-immune rabbit plasma was used as a negative control. Plates were then incubated for 1 hr at 37~C follo~Jed by four washes with wash buffer.
DEGR-FVlla was detected by adding 100 ml/well of a 1:1,000 dilution of rabbit anti-human FVII polyclonal antibody (Dr. Kisiel, U. New Mexico) in k'o hil,g buffer. Plates were inc~lbated for 1 hr at 37~C, followed by five washes with wash buffer. Specific antibody binding was detected using 100 ml/ well of a 1:2,000 dilution of goat anti-rabbit IgG antibody-, . . .

CA 022~6761 1998-12-01 peroxida~e con ug~te (Tago Inc.). Plates were inu~hated for 1 hr at 37~C and washed six times with wash buffer. Finally 100 ml of substrate solution was added (0.42 mg/ml of o-phenylenediamine dihydrochloride [OPDl in 0.2 M citrate buffer pH 5.0 containing 0.3%
H2O2). After 1-3 min at room temp. the color reaction was stopped by adding 100 ml/well of 5 1 N HzSO4 and the plates were read at 490nm on a Micropldle spect,ophotometer. The concentration of DEGR-FVlla in the plasma sal"F es was determined by comparing the A490 values of the unknown to those of the DEGR-FVlla standard curve.
Analysis of plasma samples for pr~tl,ror"bin times and DEGR-FVlla antigen levels is shown in Table 13 and Table 14 respectively. The data are p(esenled for each individual 10 animal. Table 15 shows a summary of the mean clotting times. In all cases theDEGR-FVlla treated animals had elevated proll,r~r"b.n times at the 1 hr post-bolus injection time point which returned to near pre-treatment levels at the 3-day time point. Analysis of the DEGR-FVlla antigen levels also showed a high level of DEGR-FVlla in the plasma at the 1 hr time point ranging between 2-6 mg/ml in the plasma with much lower circulating levels 15 at the 3 day time point. The levels of DEGR-FVlla measured at the 1 hr time period cGr,espond with a predicted increase in prothrombin time as determined by spiking normal rabbit plasma with DEGR-FVlla in vitro and determining proll,lor"~ ., times in a standard dilute thromhoplastin assay.

CA 022~6761 1998-12-01 Table 13 MEASUREMENT OF PROTHROMBIN TIMES

Clotting Time (seconds) Animal Number Treatment D,~t,t:al",ent 1 hour 3days 73 Control 24.8 22.3 17.8 74 Control 24.8 27.9 18.6 Control 24.6 N/D 20.5 76 Control 22 N/D 17.9 169 Control 21.2 22.9 22 170 Control 24.9 23.5 18.6 173 Control 25.9 21 20.8 174 Control 25 29.4 20.1 77 DEGR-FVlla 22.5 40.1 18.3 78 DEGR-FVlla 24.3 34 20.9 DEGR-FVlla 24.7 50 21.7 96 DEGR-FVlla N/A N/A 21 97 DEGR-FVlla 23.6 33.3 21.2 171 DEGR-FVlla 20.6 45.8 21.9 172 DEGR-FVlla 23.5 41.6 22.4 ~IA = Data Not Available , . ....

CA 0225676l l998-l2-Ol Table 14 ELISA TO DETECT DEGR-FVlla IN RABBIT PLASMA

FVlla ELISA (ng/ml) Animal Number Treatment P~ at,-)enl 1 hour3days 73 Control 0 13 0 74 Control 36 14 4 Control 0 N/A 9 76 Control 0 N/A 14 169 Control 0 0 170 Control 0 0 0 173 Control 36 31 0 174 Control 87 86 160 77 DEGR-FVlla 0 3,210 102 78 DEGR-FVlla 1 4,950 7 DEGR-FVlla 13 4,543 661 96 DEGR-FVlla 65 4,900 117 97 DEGR-FVlla 4 4,600 502 171 DEGR-FVlla 13 2,145 212 172 DEGR-FVlla 9 2,830 228 ~/A = Data Not Available ... ..

5 Table 15. Stdli:,lical Summary of Plasma Clotting Times.

Unpaired t-Test X PPF-BI FFn DF: UnpairedtValue: Prob. (2-tail):
12 1.12 0.2852 Group: Count: Mean: Std. Dev.: Std. Error:
Control 8 24.15 1.64 0.58 DEGR-Vlla 6 23.2 1.48 0.60 Unpaired t-Test X 1 Hr POST ANGIO
DF: Unpaired t Value: Prob. (2-tail):
-5.44 0.0003 Group: Count: Mean: Std. Dev.: Std. Error:
Control 6 24.5 3.35 1.37 DEGR-Vlla 6 40.8 6.53 2.67 Unpaired t-Test X 3 Days POST ANGIO
DF: Unpaired t Value: Prob. (2-tail):
13 -2.04 0.0622 Group: Count: Mean: Std. Dev.: Std. Error:
Control 8 19.54 1.53 0.54 DEGR-Vlla 7 21.06 1.33 0.50 CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 48 Three weeks post-angioplasty a follow-up angiogram was repeated as described above via the left carotid artery immediately prior to sacrifice. Through a vertical lower abdominal incision, the distal aorta was isolated, tied off proximally, and the perfusion 5 cannula inserted above the aortic bifurcation. The distal aorta was flushed with 50 ml of saline followed by ~n vivo fixation with 500 ml of Histochoice (AMRESCO, Solon, OH) solution infused over 15 mins at 120 mmHg. Once perfusion was started, the animals were sacrificed with an overdose of nembutal (3 ml sodium penluba-l,ital IV, 65 mg/ml). A 5 cm segment of femoral artery was excised bilaterally. The tissue was preserved in Histochoice 10 solution for light microscopy.
To dett:r",;.,e intimal lesion dc~elop,nent at the site of balloon angicp'~ ~y, the excised femoral a, leries were cut in serial 3 mm sections, embedded in paraffin, and sections cut from multiple regions of each artery. The sections were mounted onto glass slides and the slides stained with hematoxylin and eosin, and Van Giemson stains.
15 Morphometric analysis was performed with Bioquant Program to obtain area measurements for the lumen, the intima and the media. Morphometric analysis of tissue sections from the injured arteries were done measuring the total luminal area; the area of the intima, determined by measuring the area within the internal elastic lamina and subtracting the corresponding luminal area from each tissue section; and the area of the media, determined 20 by measuring the area inside the external elastic lamina and subtracting the area inside the internal elastic lamina. Measurements for intimal lesions in the femoral arteries in control and DEGR-FVlla treated animals showed that there was a s4niricalll decrease in the size of the intima in the DEGR-FVlla treated animals (Table 16). In contrast, measurement of the medial area showed no significant difference between the two groups.

.. . . . .... .. .. .... . . .

CA 022~676l l998-l2-Ol W 0 97/47651 PCT~DK~7/00251 Table 16. MEASUREMENTS OF THE INTIMA AND MEDIA IN
BALLOON ANGIOPLASTY TREATED RABBITS
Group N Intima (mm2) Std. Dev. Prob. (2-tail) Control 13 0.819 0.414 0.0138 DEGR-FVlla 10 0.438 0.192 Group N Media (mm2) Std. Dev. Prob. (2-tail) Control 13 0.389 0.098 0.172 DEGR-FVlla 10 0.329 0.105 The data from the angiographic measu,t:",er,ls are presented in Table 17 as the 5 Mean Luminal Diameter (MLD) +/- standard deviation for the control and DEGR-FVlla treated animal for all three time points: i",r"e.liately pre-angioplasty immediately post-angioplasty and 21 days post-angioplasty. There was no significant difference in the MLD between the control and DEGR-FVlla treated animals at either the pre- or immediately post-angioplasty measurements. A significant i"c,~ase in MLD was obseNed however in 10 the DEGR-FVlla treated animals at the 21 day post-angioplasty measurement.

. . ~. ~

CA 022~6761 1998-12-01 PCT~DK97/00251 Table 17. MEASUREMENT OF MINIMAL LUMINAL DIAMETER (MLD) Pre-PTCA Measurement of MLD
Group N Mean MLD Std. Dev. Prob. (2-tail) Control 13 1.202 0.24 0.3883 DEGR-FVlla 10 1.283 0.19 Post-PTCA Measurement of MLD
Group N Meal MLD Std. Dev. Prob. (2-tail) Control 13 1.492 0.551 0.5326 DEGR-FVlla 10 1.323 0.725 21 Day Measurement of MLD
Group N Mean MLD Std. Dev. Prob. (2-tail) Control 13 0.889 0.228 0.0001 DEGR-FVlla 10 1.393 0.242 5 EXAMPLE Xlll Inhibition of Cell-Surface Factor Xa Generation on Baboon SMCs by DEGR-FVlla A cell-surface chromogenic assay was developed, essentially as described in 10 Example Vlll above, to measure the efficacy of DEGR-FVlla to block FVlla binding to CA 022~676l l998-l2-Ol W O 97/476Sl PCTADKg7/00251 cell-surface tissue factor and the subsequent conversion of Factor X to Factor Xa on monolayers of baboon smooth muscle cells (SMCs). This method is a mo,~i~icalion of those described by Sakai et al., J. BjQ. Chem. 264:9980-9988 (1989) and Wildgoose et al., Proc.
Natl. Acad. Sci. USA. 87:7290-7294 (1990). Baboon SMCs were obtained from the University of Washington, Seattle, WA, and were cultured from aortic explants. The baboon SMCs were plated into 96-well culture dishes at a concentration of 8,000 cells/well in 200 ml/well DMEM culture media supplemented with 10% fetal calf serum, and mail)lained in this media for 4 days at 37~C in 5% CO2. At the time of assay 110 ml of culture media was removed, and increasing concentrations of FVlla or FVlla in combination with DEGR-FVlla were added to wells. A standard curve for FVlla concenl,ation was generated, ranging from 5 nM to 0.04 nM. To measure the inhibitory activity of DEGR-FVlla on FVlla activity, in~ asi"g concenl,dlions of DEGR-FVlla were added to test wells in the presence of a constant amount of FVlla (5 nM). Both FVlla and DEGR-FVlla were diluted with HEPES
buffer(10 mM HEPES, 137 mM NaCI, 4 mM KCI, 5 mM CaCI2, 11 mM glucose, 0.1% BSA) and 10 ml of 10x stock solutions added to the cells. The cells were incubated with the test compounds for 2 hr at 37~C, then washed 3 times with HEPES buffer. Fifty micr~l:ler~ of a 200 nM solution of Factor X in tris buffer (25 mM Tris, pH 7.4, 150 mM NaCI, 2.7 mM KCI, 5 mM CaCI2, 0.1 % BSA) was then added to each well. After 4 mins at room temp., 25 ml of 0.5 M EDTA was added to stop the Factor X to Xa conversion. Twenty-five microliters per well of 0.8 mM S-2222, a factor Xa-specific chromogenic substrate, in Tris buffer was added and the absorbance at 405 nM read after 60 mins in a Thermomax mic,oplale reader(Molecular Devices Corp., Menlo Park, CA).
The results, shown in Fig. 3, demonstrate a dose dependent increase in amidolytic activity for the FVlla treated wells (open boxes). The increase in absorbance is a direct measure of the level of Factor Xa generated in the wells and its subsequent cleavage of the chromogenic substrate. The addition of increasing amounts of DEGR-FVlla with a constant amount of FVlla (5 nM) showed a dose dependent decrease in amidolytic activity with increasing levels of DEGR-FVlla (closed boxes). An equal molar ratio of DEGR-FVlla to FVlla was able to inhibit ~90% of the chromogenic activity. Even at a 10-fold lower level of DEGR-FVlla, there was still a 40% inhibition in the generalion of Factor Xa chromogenic activity. These results support the conclusion that DEGR-FVlla is an extremely potent .. . . .. .

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 52 antagonist of the activation of Factor X to Xa by FVlla on the surface of intact cell monolayers of SMCs.

Fffect of DFGR-Factor Vlla on Vascular Thrombosis Formation and Vascular Lesion For",ation in Baboons ..
Human DEGR-Factor Vlla was tested for the ability to inhibit tissue factor (TF) and 10 activated Factor Vll (FVlla) mediation of vascular lesion formation (VLF) induced by mechanical vascular injury in nonhuman pdmales.
Beginning i-"me-Jialely prior to creating mechani~, ' vascular injury in baboons, DEGR-Factor Vlla was infused intravenously for 7 days (5 animals) or 30 days (1 animal).
Measurements were performed for vascular lesion formation on day 30. The results in 5 15 treated animals were compared with the findings in 5 concurrent vehicle buffer-infused conl, uls.
Baseline measurements were obtained on study animals for: a) platelet counts, neutrophil counts, monocyte counts and red cell counts; b) plasma rll,ri"ogen level; c) activity levels of plasma co~gu~~tion factors Vll, Vlla, X and V, together with the antigenic 20 levels of FVII; and d) baseline plasma sample for anti-Factor Vlla anlibody level.
Under h-'-ll,ane anesll,esia and sterile operating conditions, animals labeled with autologous "'In-platelets received intravenous infusions of DEGR-FVlla using the tether system for continuous intravenous administration (initial bolus injection of 1 mgtkg followed by continuous intravenous infusion of 50 mg/kglhr. The animals received surgical carotid 25 endarterectomy, bilateral brachial artery or bilateral femoral artery Fogarty balloon catheter angiopla~lies.
The DEGR-FVlla was administered for 7 or 30 days by continuous infusions via venous catheter using the tether system. Thirty days after surgery the animals were ane~ll,eti~ed with halothane and underwent in situ pressure-perfusion fixation with 4%
30 par~rur,,,aldehyde containing 0.1% glularaldeyde for 30 min. At that time, vascular segments (containing the sites previously injured) were harvested using procedures of Harker et al., Circulation 83:41-44 (1991) and Hanson et al., Hype~lension 18:1170-1176 ....

CA 022~6761 1998-12-01 W O 97/47651 PCTnDK97/00251 (1991). The specimens were post-fixed in vitro (4% paraformaldehyde containing 0.1%
glutaraldehyde), cryopreserved and processed for mor~,ho,~ llic analysis of lesion extent.
Eleven normal mature baboons (Paio anubis) were studied. Six animals received DEGR-FVlla infusions (50 mglkg/hr) and the remaining five were control animals that did not 5 receive DEGR-FVlla. The animals were dewo~",ed and observed to be dise~se free for three months prior to use. All procedures were approved by the Institutional Animal Care and Use Committee and were in co",~' -nce with procedures and methods outlined by NIH
Guide for the Care and ~se of Laboralory Animals, as well as the Animal Welfare Act and related institutional policies. Invasive procedures were carried out under h~'alhane 10 anesthesia after induction by ketamine (10 mg/kg intramuscularly) and valium (0.5 mg/kg intravenously). For subsequent short-term immobilization in performing ex~ eri"lenlal procedures poslo,l~erati~/ely, ketamine hydrocl~lDride (5-20 mg/kg intramuscularly) was used.
Carotid enda,lereclu",y was performed through a midline neck incision using the technique of Hanson et al., Hype,lansion 18:1170-1-176 (1991) and Krupski et al., Circulation 84:1749-1757 (1991), incorporated herein by reference. Endallarectomy was used as a vascular injury model because of its clinical relevance, and because VLF induced by endarterectomy of normal arteries has been shown to be reproducible. In brief, the common carotid artery was dicsected free of surrounding tissues from the clavicle proximally to the carotid bifurcation distally. The common carotid artery was cross-clamped using atraumatic 20 vascular clamps placed at each end of the exposed vessel three minutes after a bolus injection of heparin sulfate (100 Ulkg intravenously; Elkins-Simm Inc., Cherry Hill, NJ) and divided 1 cm proximal to the distal crossclamp. The proximal arterial segment was then everted over curved forceps. After maximal eversion was obtained, a pair of polypropylene stay sutures (7-0) was placed on either side ploxi"~ally and a second pair placed distally in 25 the lumen-exposed segment. The enda,lart:ctomy was then pel~,r",ed beginning 1 cm from the divided end of the everted vessel segment and continued for a measured distance of 1 cm. This procedure involves mechanical removal of the normal intima and a partial thickness of media using forceps and a surgical mic~oscope (32X ",agni~icaliGn). Following endarterectomy, the vessel was returned to its normal configuration, and an end-to-end 30 anastomosis pel ror"led with 7-0 polypropylene suture and continuous tect n j~ ~e under 2.5-fold magnification, and the wound closed in layers.

. ~

CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/00251 For morphometric analysis of VLF, sections embedded in paraffin and stained for connective tissue components (~DI~gen, elastin) and with hematoxylin-eosin, wereevaluated using a Zeiss Photoscope coupled with image analysis system (Thomas Optical Measurement Systems, Columbus, GA) consisting of high resolution (580 lines) CCDmicroscope camera coupled to a high resolLtion (700 lines) monitor, an IBM 386 chip, 80 MB
computer with high resolution graphics digitablet for image ~cquisition and storage.
Quar,tildlive image analysis was pe, r~,r",ed using a morphometric software driver (Optimas, ~ioscan, Inc., Edmonds, WA). Arterial cross-sections were analyzed with respect to the total area of neointimal proliferative lesion and corresponding area of arterial media. For statistical analysis, comparisons between groups were made using the Student's t test (two tailed) for paired and u"pa;,ed data.
The results showed that the intimal area was siy,lifican~ly decreased in the animals treated with DEGR-Factor Vlla for seven days and studied at 30 days as compared to control animals who had undergone the same vascular injury but who did not obtain any DEGR-Factor Vlla (Fig. 4). A similar result was found in the animal treated with DEGR-Factor Vlla for 30 days and examined at 30 days.
Preliminary studies with a balloon angiographic brachial artery model suggested no measurable benefit of DEGR-Factor Vlla therapy. This model, however, has not been shown in baboons to be a ploll"ori,botic model in which tissue factor plays a key role.
Studies with the femoral artery balloon injury in the baboon did show a statistically signiricant benefit from DEGR-Factor Vlla as compared to controls, as shown in Fig. 5.

EXAMPLE XV
Effect of DEGR-Factor Vlla on tPA-lnduced Thrombolysis Ongoing coronary tllro"~bus formation during acute myocardial i~rar~;tiGn is primarily mediated by tissue factor (TF) in complex with Factor Vlla through the extrinsic co~gu~ation pathway. The effect of adjunctive coagu~ation cascade inhibition at different points in the extrinsic pathway on the efficiency of tissue plasminogen activator (TPA) thrombolysis was deter",i, led.
Thirty-six dogs with electrically-induced coronaly thrombus undergoing thrombolysis with tPA (1 mg/kg over 20 min) were given 1 of 4 adjunctive treatments: 9 received tick anticoagulant peptide (TAP), a selective factor Xa inhibitor, at 30 mg/kg/min for 90 min. TF-.. .. . ... . .. .

CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/002~1 FactorVlla co",Flex was inhibited by reco",~-.,anl tissue factor pdlhwdy inhibitor (TFPI) (100-150 mg/kg/min for 90 min) in 9 dogs and by DEGR Vlla (1-2 mg/kg bolus) as acol"pelilive antagonist of activated Factor Vlla in 9 dogs. Nine dogs received a saline control. Dogs were observed for 120 minutes after thrombolysis for reocclusion. The effects of these agents on the efficiency of thrombolysis are shown below in Table 18 (data as mean + SD).

T~hle 18.
Saline DEGR-FVlla TFPI TAP
Time to reflow 32 + 13 20 ~ 7* 21 + 6* 18 + 10 (min) Reflow duration 62 + 4~ 70 ~ 48 91 i 35* 120 (min) Cycle flow 70% 89% 56% 0%
variations Reocclusion 70% 78% 67% 0%
~Value dirrere"l from saline control at a level 0.05 of significance.
These data indicate that extrinsic pathway inhibition by either Factor Xa or TF-Factor Vlla blockade by DEGF Vlla or TFPI accelerated tPA-induced thrombolysis.
Selective inhibition of Factor Xa more efficiently ma;ntai"ed arterial patency following successful reperfusion.

EXAMPLE XVI
Modified Factor Vlla Inhibits Intravascular Thrombus Formation Without Affecting Systemic Coagulation To determine whether inhibition of Factor Vll binding to TF would result in antithrombotic effects cycle flow variations (CFVs) due to recurrent thrombus formation were initiated by placing an external constrictor around endothelially-injured rabbit carotid arteries (Fotts' model). Carotid biood flow was measured continuously by a Doppler flow ....

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 56 probe placed proximally to the consl~ i~.lor. After positioning the consl. i- lor around the artery CFVs dcveloped with a mean frequency of 11+2 cycles/hr in 6 of 6 rabbits whereas carotid blood flow velocity averaged 5+2% of baseline values at the nadir of CFVs. After CFVs were observed for 30 min the animals received an infusion of human ,eco",~..,ant active site-5 blocked (Phe-Phe-Arg chloror"ethylketone) Factor Vlla (FVllai) (0.1 mg/kglmin for 10 min).
The Factor Vllai completely abolished CFVs in 6 of 6 animals (CFV frequency = 0 cycles/hr;
pcO.05; carotid blood flow velocity = 106.9% of the baseline values; p=NS vs. baseline).
Thirty minutes following inhibition of CFVs human recombinant FVlla was infused at the doses of 0.1 mg/kg/min for 10 min. Infusion of the Factor Vlla restored CFVs in all animals 10 thus indicating that Factor Vlla binding to TF was co",pelili~/e. Prothrombin times activated partial ~I,romboplastin times and Q vivo platelet aggregation in response to ADP and thrombin were not different after FVllai infusion as compared to baseline values. Thus FVII-Vlla plays an important role in initiating ll,-ur"bus formation in vivo. Adminisl,dlion of Factor Vllai exerts potent antill,rombotic effects in this model without affecting systemic 1 5 co~gul~tion.

EXAMPLE XVII
Inhibition of Microarterial Thrombosis by Topical Administration of Modified Factor Vlla In vascular surgery microvascular reconstructive surgery or replantation surgerythe most common cause of failure is thrombosis at the anastomotic sites. The risk of occlusive thrombus formation is highly inc,~ased when vessels have been subjected to trauma exhibit pathological changes or when i"lar~,ositional vein grafts are used.
Therefore anlill,rombotic intervention in context with surgery is frequently used. The substances which are currently available and used on this indication are adl"i"i~,ler~d parenlerally (heparin dextran) or orally (ASA) and are all associated with hemorrhagic side-effects. Moreover heparin and especially ASA are only partially effective in preventing (arterial) thrombus formation. Based on these drawbacks a need exists to preventthrombosis in vascular surgery using an agent that binds to and is effective in anastol"otic regions and sites of vascular trauma by means of a substance that can be delivered locally thereby avoiding undesirable systemic side-effects. In this Example topical admi"i;.l,dlion of active-site inactivated Factor Vlla at arterial trauma sites was used to produce an CA 022~6761 1998-12-01 a"lill,r~.",bolic effect without inducing a tendency for increased bleeding or other hemostatic defect.

Methods:
Swedish loop rabbits of either sex weighing -3.5 kg were fed a standard pellet diet and given water ad libitum. They were observed to be disease-free at the laboratory for at 10 least one week before use.
A marginal ear vein was cannulated and anaesll,esia induced with sodium pentobarbital, 18 mg/kg, and maintained by repeated injections.
Skin flaps were raised on both ears, and 3 cm long segments of the central arteries (outer .)iameter ~1mm) prepared. All branches were ligated with 10-0 sutures and cut. The 15 operative field was superfused with isotonic saline and covered with thin plastic films. To keep blood-flow high and constant and counteract vasospasm, the animals were placed on heat pads and kept slightly hyperthermic at a body temperature of -39.5~ C (normal body temperature -38.5~ C) and three drops of lidocaine 10 mg/ml were applied t~p c-"y to the vessels after manipulating them (after reperfusion and after testing patency at 30 minutes 20 after reperfusion).
Vessels on both sides were simultaneously placed in double microvascular clamps (S&T 2V, S&T Marketing Ltd., Neuhausen, Switzerland), thereby isolating 7 mm artery segments between the clamps. Longitudinal arteriotomies (7 mm) were performed, whereafter the clamps were approximated, the vessels repositioned and the vascular lumina 25 everted and flattened, exposing deep layers of the tunica media. The a,lerioto",ies were closed with continuous 10-0 monofilament nylon sutures (Ethilon 10-0 BV-75-3, Ethicon Ltd., Edinburgh, U.K.). All surgical procedures were carried out by one surgeon using a high-quality operating microscope (Wild M-650, Leica-Heerbrugg, Heerbrugg, Sw;kerland).
Vessels were simultaneously reperfused by opening the vascular clamps. They 30 were quickly covered by saline-soaked gauze pads, and inspected once a minute. The time until complete cessation of arteriotomy bleeding was recorded.

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 58 At 30 and 120 min. after reperfusion, vessel patency was ~ssessed using a standard microsurgicai empty/refill test: Vessels were gently occluded distal to the trauma area with a pair of mi.;,urur.;eps, and emptied dow"sl,t:al" with another pair of forceps. After release of the first pair of forceps, vessel refilling was assessed and vessels classified as patent or occluded. Occluded vessels showed no refilling, while patent vessels showed rapid or slow refilling, the latter being referred to as "reduced patency". After the final patency test, vessels were excised and opened longitudinally, whereafter the thrombotic material was removed and wei~l,ed.

10 Compounds:
Recombinant chemically-inactivated human factor Vlla (Vllai) at a concenl,dtion of 3.1 mg/ml or vehicle were stored as 200 ml aliquots in coded vials.

Experimental protocol:
Twenty rabbits were treated as follows in a blind random fashion, each rabbit serving as its own control: After performing the deep arterial trauma as described in the method section above, the exposed trauma site on one ear was superfused during 5 minutes with Vllai solution (a total of 0.5 mg) and on the other ear with vehicle. The superfused trauma fields were allowed to incubate with the solutions during an additional 5 minute period, whereafter all surplus of solution was flushed away with isotonic saline. The arteriutor"ies were then closed and the vessels reperfused.

Statistical methods:
Patency results were compared using the sign test, and thrombus weights and arteriotomy bleeding data with the Wilcoxon test. Two-sided p values were presented.

Results:
The administration of Vllai gave a distinct antill,ror,lbotic effect as measured by patency rates. In the Vllai group the vessel patency was 85% at 30 minutes and 75% at 120 30 after reperfusion. Corresponding values in the vehicle group were 40% and 30%respectively. The difference is statistically significant (p=0.008 and p=0.004 respectively).
Median thrombus weights were 0.3 mg in the Vllai group and 0.5 mg in the vehicle group, ... ....

CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/00251 although this difference was not siy"iricanl (p=0. 19). Median arteriotomy bleeding times were 1.5 minutes in the Vllai group and 2 minutes in the vehicle group. The groups are statistically indistingu;.,h3~'~ (p=1).
This Example demonst,dtes that topical admi"isllation of Vllai at arterial trauma 5 sites produces an anlill,-or"bolic effect without inducing an increased bleeding tendency.
This presents a highly attractive mode of treatment for preventing thrombotic comp' cdlions due to surgery microsurgery on blood vessels angioplasty or other trauma.

10 E)CAMPLE XVIII
I ncal ~I 6'ic~tion of FVllai Reduces Thrombus-Wei~ht and Improves Patency This Example demonsL,dles that local ~Fpl. ~ fion of chemically inactivated FVlla (FVllai) reduces thrombus weighl and improves vascular patency.
Twenty anestl,eti,ed rabbits were used in this Example. The jugular veins were 15 mobilized and a 10 mm segment was isol~ted between clamps. The thrombus was introduced by a comb:.,alion of chemical (aetoxysclerol) destruction of the endothelium of the isol ted segment and a semi-lesl,iclil-g ligature placed caudally of the segment. In a blinded rando",i~ed fashion one side was treated with 0.5 mg chemically inactivated FVlla (FVllai) and the other with the buffer. The test sul,slance was in,ected to the isolated 20 segment and incub~ted for 10 min. after the chemical destruction. 30 and 120 min. Iater patency was conL,,l'sd with an empty/refill test. Possible thrombus was weighed after sacrifice.

medianrange of 30 min. 120 thromb.thromb. p-value patencyp-value min. p-value weightwei!Jhls patency FVllai 0.85 mg0-22.3 mg 90 % 85 %
- 0.035 0.0070 0.070 buffer 9.3 mg0-26.8 mg 55 % 50 %

.

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00251 The results showed that local ar,~ ~tion of inactivated FVlla siyllirica.,lly reduced thrombus-v ei~l,t and improved patency in the venous li~ -lbosis model.

EXAMPLE XIX
~lication of FVllai Reduces the Area of Risk and improves reperfusion Methods 10 ExperimentalPreparation Twenty-eight New Zealand white rabbits of both sexes (3.2-3.8 kg) were studierl Briefly, the animals were anesll,esked with a mixture of ketamine (35 mg/kg) and xylazine (5mg/kg) administered intramuscularly, inh~h~ted and venlilaled with aconstant volume r~sFiiator (harvard Apparatus Co., Car"bridge, MA). Polyethylene catheters were placed in the aorta 15 through the left carotid artery and into a jugular vein for monitoring arterial pressure and acll"i"i~L,d~ion of drugs, respectively. A thordcoto",y was pelrc",)ed through the fifth left i"ler.;oslal space and the pericardium opened. A polyethylene catheter was placed into the left atrial appendage for later injections of coloured microsperes. The large ,narg;nal branch of the circumflex coronary artery was tel"porarily occluded approximately 0.3 cm from its 20 origin with a surgical suture snare. Coronary artery occlusion was maintained for 30 min, at which time the ligature was releascd and reperfusion allowed for additional 5.5 hours.
Systemic arterial pressure (Statham P23 DB pressure transducer) was recorded contineously during the experimant (Gould Instruments).
Experimental Protocol 25 At the moment of reperfusion, the animals were ,dndomly assigned to one of the fcllo~r.;.,g treatment groups: A control group received a 5-ml bolus of saline into the left atrium; a group treated with human recombinant, active site-blocked factor Vlla (FVllai, Novo Nordisk AJS, Gentofte, Denmark, 1 mg/kg bolus into the left atrium); a group treated with human recombinant activated factor Vlla (FVllai, Novo Nordisk A/S, Gentofte, Denmark, 1 mg/kg 30 bolus into the left atrium).
Assessment of Area of Risk, Infarct Size, and No-Reflow phenomenon CA 022~6761 1998-12-01 To esli",ale the distribution of tissue perfusion at the end of the experi",ent ("no-reflow"
phenomenon, NR) the animals received an injection of a 6% solution of thioflavin S (1 mgtkg) via the left atrial catheter. To permit the assessment of the area of risk of i"rar~;ti"g, the coronary artery was reoccluded immediately after injecting thioflavin, and a solution of monastral blue (E.l.DuPont; 1 mg/kg) was in,ect~ ' through the left atrial catheter. The heart was thereafter immediately excised and the left ventricle was ~issected free from all other structures and weighed. The left ventricle was frozen at -70 ~C for 30 min and cut into 8 to 10 slices parallel to the atrioventricular groove. Contours of the normally perfused myocardium as well as of the area of risk, according to the distribution of monasteral blue, 10 were traced onto a l~ansparent plastic sheet. Myocardial slices were then observed under ultraviolet light and the normally perfused myocardium (fluorescent) was then easily differentiated and separaled from the ischemic myocardium (nonfluorescent) according to the thioflavin distribution. These areas were also traced onto l,ansparent plastic sheets. The slices were next incuh~ted in a 2% solution of triphenyllt:l,a~a' Im chloride (TTC, Sigma 15 Chemicals) for 10 min at 37 ~C, to visualize the area of necrosis. Again, a transparent plastic sheet was used to trace the contours of the normal myocardium (rrC-positive), and of the infarcted portion (TTC-negative).
The following variables were c~lculated: 1 ) the area of risk of i"rarution, as a percent of the left ventricle, assessed at the end of the reperfusion period (monastral blue distribution) 20 (AR); 2) infarct size (IS), as a percent of the area of risk that actually evolved to necrosis by the TTC alai"il-g crilerion; 3) the peruenlage of the area of risk that did not receive blood flow at the end of the reperfusion period (no-reflow phenomenon, NR).
Regional Myocardial Blood Flow measurements.
Regional myocardial blood flow (RMBF) was measured in all rabbits in each treatment 25 group. Differentially coloured plastic microsperes (Blue, Red, and Yellow, triton Technology, san Diego, CA) were used to measure RMBF 20 min after occlusion and 10 min and 5 hrs after reperfusion.
Microsperes were 15 + 1 ,u in size and suspended in 10% dextran solution with 0.01%
Tween 80. To ensure adequate dispersion, the microsperes were sonicated in an ultrasonic bath for 5 min immediately before use. Approximately 500,000 microsperes were injected (0.5 -1.0 ml total volume) into the left atrial catheter. One minute before mic,uspere injection, reference arterial blood flow w;;l,d~d~val was begun and continued for 1 min after CA 022~6761 1998-12-01 WO 97/47651 PCTnDX~7/00251 62 the injection. Tissue s~",r'es (100 to 300 mg) from the center of the ischemic areas and from nonischemic regions were taken accordi"g to TTC staining. The n,iclusper~s were then recovered from tissue by digestion in a 4 M KOH solution at 72 ~C for 3 hrs and from reference blood sar, ;-les by digestion in 16 M KOH at room temperature for 3 hrs and 5 subsequent mic,uri;tldtion accordi"g to the instructions provided by the manufacturer. The dyes were then recovered from the spheres within a known volume of a solvent (dimethylformamide) and their concenl,alions deter",i"ed by spe~;t,ophotometry at optimal wave lenghts for each dye according to the manufacturer's instructions. The composite spectrum of each dye solution was resolved into the spectra of the single constituents by a 10 matrix inversion technique. Blood flow to each myocardial sample was calculated by the formula: RMBF = Fr x AmlAr where RMBF = myocardial blood flow in ml/min AM =
absorbance in myocardial sample and AR = absorbance in reference blood sample.
Myocardial blood flow was divided by the sample wet weight and expressed as ml/min/g.
Coagulation Studies 15 To determine the effects of FVllai and FVlla admin;~ lion on systemic coagulation prothrombin time (PT) and activated partial thromboplastin time (aPTT) were measured at baseline and 30 minutes after drug adl"i,)isl,~lion. Blood san,ples (4.5 ml) were collected in 0.5 ml sodium citrate (3.8%) and centrifuged at 2000 9 for 10 min at 4 ~C to separate the plasma. PT and aPTT were measured in duplicate within 2 hrs from blood ccllection.
20 Statistical Analyses Results are expressed as mean ~ SD of the mean. Analysis of variance was used for multiple comparisons among groups. Differences for individual groups were tested with Student's t-test for unpaired observations with Bonferroni's correction. For comparisons of hemodynamic variables as well as regional myocardial blood flow among groups a two-way 25 analysis of variance with a design for lepe~led measures was used.

Results Twenty-eight rabbits underwent the surgical procedure; two animals died during coronary occlusion for ventricular fibrillation before treatment group allocation and two additional rab-30 bits died during reperfusion (one in the control and one in the FVlla-treated group). These animals were excluded from subsequent statistical analysis. Therefore eight animals in each treatment group were included in the study.
.

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 Hemodynamic Measurements:
In all treatment groups, coronary occlusion induced a slight decrease in heart rate and mean arterial pressure. No difr~r~nces were found among the three groups in heart rates and me-an arterial pressures during the course of the ex~Jeri",enlal periods (Table 1).Assessment of area of Risk, Infarct Size, and the N~Reflow Phenomenon:
Coronary occlusion produced an area of risk of infarc~ion assessed by injection of monastral blue at the end of the e~,ueri",ent which was similar in the three treatment groups (31.6 6.3, 28.2 i 4.1, and 29.2 + 5.3% of the left ver,l~i~'e in control, FVllai, and FVlla-treated animals, respectively, p=NS).
After 30 min of coronary occlusion and 5.5 hrs of reperfusion, the amount of thearea at risk that evolved toward necrosis averaged 59.8 _ 12.8% in the control group (Fig.
7). Adl"in;~l,dtion of FVllai significantly reduced infarct size to 28.1 + 11.3% of the area at risk p~0.01 by ANOVA, Fig. 1), while FVlla adl"ini~l,dlion was associated with a significant increase in infarct size to 80.1 + 13.1 % of the area of risk (p~0.01 vs. controls and FVllai-15 treated rabbits, Fig. 7).
In control rabbits, 24.4 + 2.7% of the area of risk showed a perfusion defect, as de-termined by thioflavine S distribution at the end of the experiment (no-reflow phenomenon).
The extent of this area of no-reflow was significantly reduced by FVllai and significantly in-creased by FVlla to 11.1 ~ 6.1 and 61.9 + 13.8% of the area of risk, respectively (p~0.01, Fig. 8).
Previous studies have est~hl;shed that the amount of the myocardial tissue that shows a perfusion defect during post-ischemic reperfusion is related to various parameters;
the most important are the extent of the area of risk, the magnitude of infarct size, and the amount of residual collateral flow during occlusion. Controlling for these var ~!es allows for more precise assessment of the effects of interventions on the no-reflow phenomenon. In the present study, when the no-reflow area in control rabbits was correlated to these para-meters, a close relationship was observed, which fits a multiple linear regl~ssion equation:
NR (% of the left ventricle [LV]) = -14.62 + 0.75(AR) + .07(1S) + 3.69(RMBF); r2 = 0.98; F
test = 109.3, (0.37)(0.3)(3.69), where NR is no-reflow area, AR is the area at risk, and RMBF
is collateral blood flow (in ml/min/g). The number in pare"ll,eses indicate the standard errors of the coefficients. With an r2 value of 0.98, this model accounted for ~95% of the variation in the no-reflow area that was observed in control animals in this study.

CA 022~6761 1998-12-01 W 0 97t47651 PCT~DK97/00251 64 The equation coefficients that were obtained from the data of control rabbits in the multiple rey,ession equation were then used to c~c~ e the expected no-reflow areas for individual animals within each intervention group (Fig. 9). The actually observed no-reflow areas in rabbits receiving FVllai were Siyl ,ir,carilly smaller than the expected ones c~ atcd 5 applying the e~U~tion obtained from the multiple reg,~ssion analysis. Indeed, in FVllai-treated rabbits, for any given eYpeçted area of no-reflow, the actual observed value was smaller, such that all animals in this group distributed below the regression line obtained for control animals (Fig. 9). On the contrary, FVlla-treated rabbits showed just the opposite, i.e., for any given expected area of no-reflow, the actual observed value was significantly bigger, 10 as all animals distributed above the regression line of control animals (Fig. 9). Taken toget-her, these data indicate that the reduction in the no-reflow phenomenon observed in FVllai treated animals is nor entirely accounted for by the reduction in infarct size, and suggest that activation of the extrinsic co~gu'~tion casc~de during post-ischemic reperfusion contribute to the occurrence of the no-reflow phenomenon.
Regional Myocardial Blood Flow Measurements:
In control animals RMBF to the non-ischemic myocardium averaged 1.20 ml/minlg of tissue throughout the study (data not shown). In the same group of animals, RMBF to the ischemic myocardium was 0.08 + 0.02, 1.43 + 0.28, and 0.98 + 0.19 ml/min/g of tissue 20 min after coronary occlusion and 10 min and 5 hrs after perfusion, respectively. The various drug tre-20 atment used in the presenl study did not significantly change RMBF during the experimental period both in the normal and in the ischemic myocardium as cor"pared to control animals (Fig. 10).
Coagu/ation Studies:
To study possible systemic effects of FVllai, which may pred;spose to an increased risk of 25 bleeding, PTs and aPTTs were measured in blood samples collected at 30 minutes of CFVs, and after FVllai and FVlla administration. At the end of the 30 min CFVs period, PTs and aPTTs averaged 8.2 + 0.6 seconds and 25 + 3 seconds, respectively. A slight increase in PTs to 10.1 + 0.6 seconds was observed after FVllai admini Lldlion (Fig. 11). This increase, however, did not reach statistic~l siyl ,iricance (p=0.09 by ANOVA and student's t-test with 30 the Bonferroni's correction). APTT did not change siy"ificar~ly after FVllai administration (Fig. 10). FVlla administration resulted in a significant shortening in both PTs and aPTTs only with respect to the values obtained after FVllai ad",.r,i~l,alion (Fig. 11).

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 Table 1:
Hemodynamic variables during coronary artery occlusion-reperfusion Heart Rate (b/min) Time after Control SQ29548 Dazoxiben R68070 ASA
occlusion 0 175i5 170i4 178i6 169i5 30min 169i4 165+5 173i5 164i6 1 hr 169i6 167+4 171 ~5 166i5 2hr 173i5 172i5 175i6 170~5 3hr 170+4 168i5 177i5 168i6 4hr 175~5 168i6 174i5 16gi5 5hr 175i5 172+2 173i5 172i6 6hr 168i5 170+4 174i5 168+5 Mean arterial pressure (mm Hg) Time after Control SQ29548 Dazoxiben R68070 ASA+R680 occlusion 70 0 75i4 78i3 78+4 73+3 7 30min 69i4 65i4 71 i3 67+4 6 1hr 69i3 67i4 71+4 69~4 6 2hr 73+4 75+4 75i5 72i4 7 3hr 74i4 75i3 77~5 75~3 7 4hr 75i5 78+4 76i4 73i4 7 5hr 73i4 74i5 78~5 72i4 7 6hr 73+5 76i4 74i5 73i5 7 CA 022~6761 1998-12-01 W O 97/47651 PCTADK~7/00251 66 Table ll Regional myocardial blood flow (ml/min/g of tissue) during coronary occlusion and reperfusi-on Control FVllai FVlla Normal myocardi-um 20 min CAO 1.19 + 0.22 1.03 + 0.19 1.27 ~ 0.24 10 min REP 1.22 + 0.15 1.10 ~ 0.17 1.19 + 0.20 2 hrs REP 1.17 i 0.18 1.07 + 0.16 1.22 + 0.19 Ischemic myocar-dium 20 min CAO 0.09 + 0.05 0.08 + 0.05 0.08 + 0.04 10minREP 1.53iO.12 1.65+0.18 1.24+0.14 2hrsREP 0.89+0.14 1.23+0.15 0.72+0.13 CAO = Coronary Artery Occlusion; REP = Reperfusion EXAMPLE XIX
A~plication of FVllai Reduces Infarct ci~ and Area at Risk of Infarction Tissue factor exros~re occurs during reperfusion of post-ischemic hearts within the coronary v~scl~latllre, leading to a decrease in coronary blood flow.
To assess whether tissue factor exposure might contribute to myocardial injury via activation of the coagulation and reduction in coronary blood flow during post-ischemic re-perfusion, NZW rabbits underwent 30 min coronary occlusion followed by 5.5 hrs of reperfu-sion. At reperfusion, the animals randomly received: saline (n=8); human recombinant, acti-ve site-blocked factor Vlla (FVllai, 100 ~lg/kg/min for 10 minutes into the left atrium, n=8) or human recombinant activated factor Vlla (FVlla, 100 !lg/kg/min into the left atrium, n=8).
Regional myocardial blood flow (RMBF) was measured using coloured microspheres at 20 min of ischemia, and 10 min and 2 hrs following reperfusion. The area at risk of i"rar~;tion (AR), infarct size (IS), and the no-reflow area (NR) were determined at the end of the expe-.. . .

CA 022~676l l998-l2-Ol riment by monastral blue and thioflavin distribution and by TTC staining. FVllai resulted in a sig"ificant reduction in both IS and NR with respect to conl~ols (28.1 i 11.3% and 11.1 i 6.1% of AR vs. 59.8 i 12.8% and 24.4 i 8.2% of AR, respectively, p~0.01), while FVlla re-sulted in a significant increase in both IS and NR to 80.1 i 13.1 % and 61.9 i 13.8% of AR, 5 respectively, p~0.01 vs. Controls. No differences in blood pressure, heart rate, AR, and RMBF at 20 min of ischemia were observed among groups. RMBF was significantly higher at 2 hrs of reperfusion in FVllai-treated animals, while it was lower in FVlla-treated rabbits.
Thus, TF-mediated activation af the co~g~ tion importantly contributes to the occurrence of myocardial injury during post-ischemic reperfusion.

FVllai (AR) Control (AR) FVlla (AR) IS 28.1 i 11.3 59.8 i 12.8 80.1 i 13.1 NR 11.1 i 6.1 24.4 + 8.2 61.9 + 13.8 AR = The area at risk of infarction; IS = infarct size (IS); NR =the no-reflow area Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be obvious that certain changes and modifications may be practiced within the scope of the appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: ZymoGenetics, and Novo Nordisk A~S

(ii) TITLE OF lNvhNllON: Modified Factor VII
(iii) N~MBER OF SEQUENCES: 4 (iv) CORRESPON~N~ ADDRESS:
(A) ADDRESSEE: Novo Nordisk A/S, Corporate Patents (B) STREET: Novo Allé
(C) CITY: DK-2880 Bagsvaerd (D) CO~N 1 KY: Denmark (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.24 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 08/475,845 (B) FILING DATE: 07-JUN-1995 (C) CLASSIFICATION:

CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 (2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2422 base pairs (B) TYPE: nucleic acid (C) sTR~n~n~ s single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: N

(iv) ANTI-SENSE: N

15 (ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 28... 1420 (D) OTHER INFORMATION: /codon_start= 28 /product= "Factor VII"
(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:l:

Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln Gly Cys Leu Ala Ala GTC TTC GTA ACC CAG GAG GAA GCC CAC GGC GTC CTG CAC CGG CGC CGG l5l Val Phe Val Thr Gln Glu Glu Ala His Gly Val Leu His Arg Arg Arg -15 -l0 -5 CA 022~676l l998-l2-Ol W O 97/476Sl PCT~DK97/00251 Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu Glu Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pro Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp Asp Gln Leu Ile Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Ser 30 Cys Arg Cys His Glu Gly Tyr Ser Leu Leu Ala Asp Gly Val Ser Cys Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile Pro Ile Leu Glu Lys .. . . .. ..... . . . ... .

CA 022~676l l998-l2-Ol W O 97/47651 PCTADK~7/00251 Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Leu Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile Trp Val Val Ser Ala 15 Ala His Cys Phe Asp Lys Ile Lys Asn Trp Arg Asn Leu Ile Ala Val Leu Gly Glu ~is Asp Leu Ser Glu His Asp Gly Asp Glu Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser Thr Tyr Val Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu His Gln Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu Arg CA 022~676l l998-l2-Ol W 0 97/47651 PCTnDK97/00251 Thr Leu Ala Phe Val Arg Phe Ser Leu Val Ser Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Met Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Ser Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe Cys Ala Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro CA 022~676l l998-l2-Ol WO 97/47651 PCT~DK97/00251 AGAGAAAGCC AAGGCTGCGT CGAACTGTCC TGGCACCAAA TCCCATATAT ~ GCAG 1456 ATGAGAGGCA GAGGCAGACA GGCGCTGGAC AGAGGGGCAG GGGAGTGCCA AG~ll~lC~l 1696 AAGGCGGTTG TTTAGCTCTC A~llll~lGG TTCTTATCCA TTATCATCTT CACTTCAGAC 2236 l~lCC~llCG CTGGGTGCCG GGCTGCACAG ACTATTCCCC ACCTGCTTCC CAGCTTCACA 2356 ATAAACGGCT GCGTCTCCTC CGCACACCTG TGGTGCCTGC CACCCAAAAA P~a~U~AAA 2416 .. . .. . . ....

CA 022~6761 1998-12-01 W O 97147651 PCTADK97/002Sl (2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 444 amino acids (B) TYPE: amino acid ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: protein 10 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Val Ser Gln Ala Leu Arg Leu Leu Trp Leu Leu Leu Gly Leu Gln ~5 Gly Cys Leu Ala Ala Val Phe Val Thr Gln Glu Glu Ala His Gly Val -20 -15 -l0 Leu His Arg Arg Arg Arg Ala Asn Ala Phe Leu Glu Glu Leu Arg Pro -5 l 5 lo Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu Gln Cys Ser Phe Glu Glu Ala Arg Glu Ile Phe Lys Asp Ala Glu Arg Thr Lys Leu Phe Trp Ile Ser Tyr Ser Asp Gly Asp Gln Cys Ala Ser Ser Pro Cys Gln Asn Gly 30 Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr Ile Cys Phe Cys Leu Pro Ala Phe Glu Gly Arg Asn Cys Glu Thr His Lys Asp Asp Gln Leu Ile CA 022~6761 1998-12-01 W O 97/47651 PCT~DK~7/00~51 Cys Val Asn Glu Asn Gly Gly Cys Glu Gln Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Ser Cys Arg Cys His Glu Gly Tyr Ser Leu Leu Ala 110 llS 120 Asp Gly Val Ser Cys Thr Pro Thr Val Glu Tyr Pro Cys Gly Lys Ile 10 Pro Ile Leu Glu Lys Arg Asn Ala Ser Lys Pro Gln Gly Arg Ile Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys Pro Trp Gln Val Leu Leu Leu Val Asn Gly Ala Gln Leu Cys Gly Gly Thr Leu Ile Asn Thr Ile Trp Val Val Ser Ala Ala His Cys Phe Asp Lys Ile Lys Asn Trp Arg Asn Leu Ile Ala Val Leu Gly Glu His Asp Leu Ser Glu His Asp Gly 25 Asp Glu Gln Ser Arg Arg Val Ala Gln Val Ile Ile Pro Ser Thr Tyr Val Pro Gly Thr Thr Asn His Asp Ile Ala Leu Leu Arg Leu Hls Gln Pro Val Val Leu Thr Asp His Val Val Pro Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu Arg Thr Leu Ala Phe Val Arg Phe Ser Leu Val Ser CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/00251 76 Gly Trp Gly Gln Leu Leu Asp Arg Gly Ala Thr Ala Leu Glu Leu Met 5 Val Leu Asn Val Pro Arg Leu Met Thr Gln Asp Cys Leu Gln Gln Ser Arg Lys Val Gly Asp Ser Pro Asn Ile Thr Glu Tyr Met Phe Cys Ala Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys Lys Gly Asp Ser Gly Gly Pro His Ala Thr His Tyr Arg Gly Thr Trp Tyr Leu Thr Gly Ile Val Ser Trp Gly Gln Gly Cys Ala Thr Val Gly His Phe Gly Val Tyr Thr 20 Arg Val Ser Gln Tyr Ile Glu Trp Leu Gln Lys Leu Met Arg Ser Glu Pro Arg Pro Gly Val Leu Leu Arg Ala Pro Phe Pro CA 022~6761 1998-12-01 W O 97/47651 PCT~DK97/002S1 (2) INFORMATION FOR SEQ ID NO:3:

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(A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ili) HYPOTHETICAL: N

(iv) ANTI-SENSE: N

1~

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TGGGCCTCCG GCGTCCCCCT T 2l (2) INFORMATION FOR SEQ ID NO:4:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

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.. ..

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Claims (28)

1. A method for inhibiting thrombus formation in a patient comprising administering topically to a vascular site susceptible to thrombus formation in the patient a therapeutically effective dose of a composition comprising Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX.

2. A method according to claim 1, wherein the modification comprises reaction of the Factor VII with a serine protease inhibitor.

3. A method according to claim 2, wherein the protease inhibitor is an organophosphor compound, a sulfanyl fluoride a peptide halomethyl ketone, or an azapeptide.

4. A method according to claim 3, wherein the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloro-methyl ketone Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloromethyl-ketone.

5. The method according to any of claims 1 to 4, wherein the site of thrombus formation is associated with surgery, microsurgery, angioplasty or trauma.

6. A method for maintaining or improving vascular patency in a patient comprising administering locally to a vascular site susceptible to decreased patency a therapeutically effective dose of a composition comprising Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX.

7. A method according to claim 6 wherein the modification comprises reaction of the Factor VII with a serine protease inhibitor.

8. A method according to claim 7, wherein the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide.

9. A method according to claim 8, wherein the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloro-methyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloromethyl-ketone.

10. The method according to any of claims 6 to 9, wherein the site of decreased patency is associated with surgery, microsurgery, angioplasty or trauma.

11. Use of Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X
or IX, for the manufacture of a composition for prevention or minimizing of myocardial injury associated with post-ischemic reperfusion.

12. Use according to claim 16, wherein the modification comprises reaction of the Factor VII
with a serine protease inhibitor.

13. Use according to claim 17, wherein the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide.

14. Use according to claim 18, wherein the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloromethylketone.

15. Use according to any of claims 16 to 19, wherein the myocardial injury is myocardial necrosis.

16. A method for preventing or minimizing myocardial injury associated with post-ischemic reperfusion in an individual, comprising administering to the individual a composition which comprises a pharmacologically acceptable Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX.

17. The method according to claim 21, wherein the modification comprises reaction of the Factor VII with a serine protease inhibitor.

18. The method according to claim 22, wherein the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide.

19. The method according to claim 23, wherein the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloro-methylketone.

20. The method according to any of claims 21 to 24, wherein the myocardial injury is myocardial necrosis.

21. Use of Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X
or IX, for the manufacture of a composition for improving regional myocardial blood flow during post-ischemic reperfusion.

22. Use according to claim 26, wherein the modification comprises reaction of the Factor VII
with a serine protease inhibitor.

23. Use according to claim 27, wherein the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide.

24. Use according to claim 28, wherein the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloromethylketone.

25. A method for improving regional myocardial blood flow during post-ischemic reperfusion in an individual, comprising administering to the individual a composition which comprises a pharmacologically acceptable Factor VII having at least one modification in its catalytic center, which modification substantially inhibits the ability of the modified Factor VII to activate plasma Factor X or IX.

26. A method according to claim 30, wherein the modification comprises reaction of the Factor VII with a serine protease inhibitor.

27. A method according to claim 31, wherein the protease inhibitor is an organophosphor compound, a sulfanyl fluoride, a peptide halomethyl ketone, or an azapeptide.

28. A method according to claim 32, wherein the protease inhibitor is a peptide halomethyl ketone selected from Dansyl-Phe-Pro-Arg chloromethyl ketone, Dansyl-Glu-Gly-Arg chloromethyl ketone, Dansyl-Phe-Phe-Arg chloromethyl ketone and Phe-Phe-Arg chloro-methylketone.