CA2253836A1 - Antithrombotic materials and methods - Google Patents

Abstract

Antithrombotic materials and methods are provided for the treatment of thrombotic disorders, in which therapeutically effective amounts of BPI protein products are administered.

Description

CA 022~3836 1998-11-09 A~l l l l'll~Ol\/IBOTIC MATERIALS AND l\~IETHODS

BACKGROUND OF THE INVENTION
The present invention relates generally to therapeutic compositions and treatment methods utili7ing bactericidal/permeability-increasing protein (BPI) 5 protein products for the treatment of thrombotic disorders.
The coagulation, or blood clotting process is involved both in normal hemostasis, in which the clot stops blood loss from a damaged blood vessel, and in abnormal thrombosis, in which the clot bloc~s circulation through a blood vessel. During normal hemostasis, the platelets adhere to the injured blood 10 vessel and aggregate to form the primary hemostatic plug. The platelets then stimulate local activation of plasma coagulation factors, leading to ge"el~tion of a fibrin clot that rein~,-;es the platelet aggregate. Later, as wound healing occurs, the platelet agg~ ;ate and f~brin clot are degraded by specifically activated proteinases. During the pathological process of thrombosis, the same mechq-ni~m.c 15 create a platelet/fibrin clot that occludes a blood vessel. Arterial thrombosis may produce i.cchP.mic necrosis of the tissue supplied by the artery, e.g., myocardial infarction due to thrombosis of a coronary artery, or stroke due to thrombosis of a cerebral artery. Venous thrombosis may cause the tissues drained by the vein to become edem~tous and inflqme~, and thrombosis of a deep vein may result in a 20 pulmonary embolism.
An increased ter--lency toward thrombosis acco~ nip~s surgery, trauma, many il~ ory disorders, mqlignqn~y, pregllancy, obesity, vascular disorders and prolonged immobilization. Inherited thrombotic ten-lenries, which are much rarer, are being increasingly recognized and include deficiencies of the 25 protein C-protein S system, deficiencies of alllill,ru"lbin m (Al~), dysfibrinogenP.miq~, and other disorders of the fibrinolytic system. The evaluatio of hypercoagulable risk involves chPcking for a family history of thromboembolism, and for other systemic predisposing diseases or conditions thatfavor localized vascular stasis (such as prolonged immobilization, p,Ggnancy, or30 m~lign~n~y~ and ev~ tin~ possible labG,~to,y abnormalities, such as thrombocytosis, elevated blood or plasma viscosity, and elevated plasma levels of CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 coagulation factors or flbrin degradation products. Levels of ATm, protein C, orprotein S levels, may also be measured, although hypercoagulability due to such abnormalities is uncommon compared to factors such as stasis or localized injury.
Severe derangements of the coagulation process are seen in 5 dissemin~ted intravascular coagulation (DIC), a syndrome characterized by the slow formation of fibrin microthrombi in the microcirculation and the development of concomitant fibrinolysis. The net result of these processes is the consumption of platelets and clotting factors in the thrombotic process, and the proteolyticdigestion of several clotting factors by the fibrinolytic process, leading to 10 decreased coagulability of the patient's blood. DIC never occurs as a primarydisorder; it is always secondary to another disorder. These primary disorders fall into three general categories: (1) release of procoagulant substances into the blood, as may occur in amniotic fluid embolism, abruptio pl~çnt~, cerlain snake bites, and various m~lign~ncies, (2) contact of blood with an injured or abnorrnal 15 surface, as may occur in extensive burns, infections, heat stroke, organ grafts, and during extracorporal circulation, and (3) generation of procoagulant-active substances within the blood, as may occur if red or white blood cell or plateletmembranes become damaged and release thromboplastic substances, e.g., during leukemia treatment, hemolytic transfusion reactions and microangiopathic 20 hemolytic anemia. Bacterial endotoxins on, associated with or released from gram-negative bacteria also have thromboplastin-like pl.,pe,lies that initiate clotting.
Intravascular clotting occurs most frequently with shock, sepsis, cancer, obstetric complications, burns, and liver disease. There are no speci~lc25 symptoms or signs unique to DIC. Bleeding, however, is much more evident thanthrombosis. The rate and extent of clotting factor activation and con~u,llpLion, the concentration of naturally occurring inhibitors, and the level of fibrinolytic activity determine the severity of the bleeding tendency. In some patients there is no clinical evidence of bleeding or thrombosis, and the syndrome becomes app~t;lll 30 only as a con~equence of abnorrnal blood coagulation tests. Many patients develop only a few petechiae and ecchymotic areas and bleed a little more than usual from venipuncture sites. More pronounced forrns of diffuse intravascular clotting may CA 022~3836 1998-11-09 wo 97/42967 PCTIUS97/08017 become evident as a result of severe gastrointestinal hemorrhage or genitourinary bleeding. In sorne inst~nce~ bleeding may cause death. Hemorrhage caused by the DIC syndrome can be especially life thre~rening in association with obstetric complications or in conjunction with surgery.
The endpoint of the coagulation process is the generation of a powerful serine protease, thrombin, which cleaves the soluble plasma protein fibrinogen so that an insoluble meshwork of fibrin strands develops, enmeshing red cells and platelets to form a stable clot. This coagulation process can be triggered by injury to the blood vessels and involves the rapid, highly controlled 10 interaction of more than 20 different coagulation factors and other proteins to amplify the initial activation of a few molecules to an a~ )riately sized, fullydeveloped clot. Most of the coagulation proteins are serine proteases that show a high degree of homology (Factors II, VII, IX, and X); others are cofactors without enzyme activity (Factors V and Vm). These proteins circulate as inactive 15 zymogens in amounts far greater than are required for blood clotting. Both the injured vessel wall and platelet aggregates provide specialized surfaces that localize and catalyze the coagulation reactions.
The coagulation cascade can be initi~ted via two different activation pathways: the intrinsic pathway, involving contact with injured tissue or other 20 surfaces, and the extrinsic pathway, involving tissue factor t;,.~ulessed on injured or inflamed tissue. Both pathways converge into a common pathway when Factor X
is activated at the platelet surface. [See, e.g., Cecil's Essentials of Medicine, 3rd ed., WB Saunders Co., Pennsylvania (1983); Goodman & Gilman, The Pharmacological Basis of Therapeutics, 9th ed., McGraw-Hill, NY (1996).] The 25 intrinsic pathway begins when Factor XII is activated to XIIa by contact with the altered or injured blood vessel surface or with another negatively charged surface, such as a glass tube. Cofactors or promoters of Factor XII activation include prekallikrein, high molecular weight kininogen, and Factor XI. These proteins form a surface-localized complex which optimally activates Factor XII. The 30 activated Factor XIIa then converts the complex-bound Factor XI to its activeforrn, XIa, and also converts prekallikrein to its active form, kallikrein, which then cleaves high molecular weight kininogen to forrn bradykinin. In turn, Factor CA 022~3836 1998-ll-09 W 097/42967 PCTrUS97/08017 XIa requires calcium ions (Ca2+) to activate Factor IX to IXa. Factor XIa may also activate Factor VII (in the extrinsic pathway) as well. Activated Factor XIa also cleaves plasminogen to form plasmin, which is the main protease involved inthe fibrinolytic mech~ni~m~ that restrain blood clotting. In the presence of Ca2+
S and phospholipid, Factor IXa activates Factor X to Xa, which is the first step in the common pathway. Factor X activation usually takes place at the plasma membrane of stimulated platelets but also may occur on the vascular endothelium.In the extrinsic pathway, the release of tissue factor from injured tissues directly activates Factor VII to VIIa. Tissue factor is present in activated 10 endothelium and monocytes as well as in brain, vascular adventitia, skin, andmucosa. Factor VIIa then activates Factor X to Xa in the presence of Ca2+ . In addition, the tissue factor, Factor VII, and Ca2+ form a complex that can activate Factor IX (in the intrinsic pathway).
The activated Factor Xa (the first step in the common pathway) then 15 activates ~ ",bin (Factor II) to generate the protease thrombin. Assembly of the plasma plo~h~lllbinase complex on the surface of activated platelets in the presence of Factor V, another cofactor, enhances the efficiency of pr~,ll"~,l,lbin activation to thrombin on the platelet surface. Thrombin cleaves fibrinogen, which is a large, asymmetric, soluble protein with a molecular weight of about 340 20 kilodaltons con~i~ting of three pairs of polypeptide chains: Acl!, B~B, and y.
Thrombin first removes small peptides from the A~x chain of fibrinogen to form Fibrin I, which polymerizes end to end; further thrombin cleavage of small peptides from the B~B chain leads to forlnation of Fibrin II molecules, which polymerize side to side and are then cross-linked via the y subunits by the plasma 25 glut~mir~q~e (Factor XIII) to form an insoluble fibrin clot.
Thrombin has multiple critical actions during coagulation in addition to the cleavage of fibrinogen to fibrin. It activates platelets, exposing their procoagulant activity (e.g., binding sites for the prulhrolllbinase complex) andinduces the release of platelet-aggl~g~tillg substances such as thromboxane, Ca2+, 30 ADP, von Willebrand factor, fibronectin, and thrombospondin. Thrombin cleavesFactors VIII and Va, thus augmenting the coagulation cascade, and also cleaves plasma gh~ e, the enzyme which cross-links fibrin and stabilizes the fibrin CA 022~3836 1998-11-09 WO 97/42967 PCTrUS97tO8017 clot. Thrombin acts on the endothelium by binding to the surface protein thrombomodulin to activate protein C, which is a potent inactivator of Factors Va and VIIIa and also stimulates fibrinolysis. Thrombin also causes endothelial cell contraction. Conversely, endothelium can bind and inactivate thrombin, and in some cases can generate the vasodilatory substance prostacyclin in response to thrombin. Thus, thrombin activation contributes to the limitation as well as theinitiation of clotting.
There are two commonly used tests for measuring the coagulability of blood: the activated partial thromboplastin time (API~ or Pl-r) and the 10 prothrombin time (PI). Blood generally clots in vitro in four to eight minutes when placed in a glass tube. Clotting is prevented if a chel~tin~ agent such as ethylenedi~minetetraacetic acid (EDTA) or citrate is added to bind Ca2+.
Recalcified plasma, i.e., plasma in which Ca2+ has been replenichP~, clots in two to four minutes. The clotting time after recalcification is shortened to 26 to 33 15 seconds by the addition of negatively charged phospholipids and a particulatesubstance such as kaolin (~h1minllm silicate); this post-recalcification clotting time is the APTT. Alternatively~ recalcified plasma will clot in 12 to 14 seconds after addition of "thromboplastin," a saline extract of brain that contains tissue factor and phospholipids; this post-recalcification clotting time is the PT.
An individual with a prolonged APl~ and a normal PT is considered to have a defect in the intrinsic coagulation pathway, because all of the components of the APTT test (except kaolin) are in~rincic to the plasma. A patient with a prolonged PT and a normal APTT has a defect in the extrinsic coagulation pathway, since thromboplastin is extrinsic to the plasma. Prolongation of both the APTT and the PT suggests a defect in a common pathway.
Whereas the blood coagulation pathways involve a series of enzymatic activations of serine protease zymogens, downregulation of blood clotting is influenced by a variety of natural anticoagulant mech~ni.sms, including antithrombin m (ATm), the protein C-protein S system, and fibrinolysis. Normal vascular endothelium promotes the activation of these anticoagulant mech~ni.sm.s by acting as a source of heparin-like substances that enhance ATm activation, a CA 022~3836 1998-ll-09 W097/42967 PCT~US97/08017 source of thrombomodulin, a cofactor in protein C activation, and a source of the tissue plasminogen activators that initiate fibrinolysis.
The anticoagulant ATm is a plasma protease inhibitor that is specific for plasmin, the enzyme that dissolves clots. All-l also binds all the 5 serine protease procoagulant proteins (Factor Xa as well as thrombin). Complexes of ATm and protease are rapidly cleared by the liver and the reticuloendothelialsystem. The activity of ATm is enhanced by heparin or heparin-like substances.
Other enzymes that play a role in limiting the coagulation process include the nonspecific plasma protease inhibitors ~I-antitrypsin, ~2-plasmin inhibitor, and10 ~2-macroglobulin, which rapidly inactivate any circul~ting serine proteases including thrombin and plasmin.
The final stage of the coagulation process is fibrinolysis, or clot dissolution. The endpoint of the fibrinolytic system is the generation of the enzyme plasmin, which dissolves intravascular clots by digesting fibrin.
15 Fibrinolysis is initi~ted during clotting by the action of thrombin. When complexed to thrombomodulin in the endothelium, thrombin activates protein C, which ini~iat~s the release of tissue plasminogen activator (tPA) from the bloodvessel wall. Protein C, together with its cofactor protein S, also inactivates Factors Va and VIIIa, thus dampening the coagulation cascade. The tPA then 20 cleaves a circul~ting proenzyme, plasminogen, to form the active protease, plasmin, which digests fibrin. Plasmin is a relatively nonspecific protease; it not only digests fibrin clots but also digests other plasma proteins, including several coagulation factors.
The fibrinolytic system is regulated in a manner so that unwanted 25 fibrin thrombi are removed, while fibrin in wounds persists to m~in~in hemostasis. The tPA is released from endothelial cells in response to various signals, including stasis produced by occlusion of the blood vessel. This released tPA exerts little effect on circulating plasminogen because tPA is rapidly cleared from blood or inhibited by circulating inhibitors, pl~minogen activator inhibitor-1 30 and pl~min~gen activator inhibitor-2. Both pl~minogen and its activator tPA
bind to fibrin. The activity of tPA is actually enhanced by this binding to fibrin, CA 022~3836 1998-11-09 W O 97/42967 PCT~US97/OB017 so that the generation of plasmin is localized to the vicinity of the blood clot. In addition, fibrin-bound plasmin is protected from inhibition.
~ Four main types of theMpies are used to prevent or treat thrombosis: antiplatelet agents, anticoagulant agents (heparin), vitamin K
5 antagonists (coumarin derivatives) and thrombolytic agents. Each type of agentinterferes with clotting at a different site in the coagulation pathway [See, generally, Goodman & Gilman, The Pharmacological Basis of T~ dl)t;U~iCS, 9th ed., McGraw-Hill, NY (1996).] Dipyridamole is another agent sometimes used to prevent or treat thrombosis; it is a vasodilator that, in combination with warfarin 10 (a coumarin derivative), inhibits embolization from prosthetic heart valves and, in combination with aspirin, reduces thrombosis in patients with thrombotic disorders.
The antiplatelet agents include aspirin and other non-steroidal anti-infl~mm~tory agents such as ibuprofen, which are all ~dmini.ct~red orally. Aspirin 15 acts by irreversibly inhibiting platelet cyclooxygenase and thus blocking production of thromboxane A2, an inducer of platelet ag~,c;galion and potent vasoconstrictor.
In general, antiplatelet agents are used as prophylaxis against arterial thrombosis, because platelets are more important in initi~ting arterial than venous thrombi.Antiplatelet therapy also reduces the risk of occlusion of saphenous vein bypass20 grafts.
The ~ntico~gulant agents include heparin and its derivatives, which act by accelerating the activities of ATIII in inhibiting thrombin geneMtion and in antagonizing thrombin's action. Low molecular weight preparations of heparin such as dalteparin and enoxaparin may also be effective for anticoagulation.
25 Heparin increases the rate of the thrombin-antithrombin reaction at least a thousandfold by serving as a catalytic template. Heparin can only be ~mini~teredparenteMlly and has an immediate anticoagulant effect. It is used to prevent andtreat arterial and venous thrombosis, as well as to keep blood fluid during extracorporeal circulation, such as with renal hemodialysis or during 30 cardiopulmonary bypass, and to keep vascular access catheters patent. Heparintherapy is also standard in p~tient~ undergoing percutaneous tr~n.shlmin~l coronary angioplasty.

CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 Bleeding is the primary adverse effect of heparin. Major bleeding occurs in 1 % to 33 % of patients who receive various forms of heparin therapy.
Purpura, ecchymoses, hematomas, gastrointestinal hemorrhage, hem~turia, and retroperitoneal bleeding are regularly encountered complications of heparin 5 therapy. Frequently bleeding is most pronounced at sites of invasive procedures.
If bleeding is severe, the effects of heparin can be counteracted by giving 1 mg of protamine sulfate for each 100 units of heparin. Another side effect, thrombocytopenia, also occurs in 1% to 5 % of patients receiving heparin, but subsides when heparin is discontinued.
The vitamin K antagonists (coumarin derivatives) are sometimes referred to as oral anticoagulants although they do not actually directly inhibit the coagulation c~cad~. These agents include 4-hydroxycoum~rin, warfarin sodium, dicumarol, phenprocoumon, indan-1, 3-dione, acenocoumarol, and ~ni~inrlione.
They interfere with the hepatic synthesis of Factors II, VII, IX, and X and proteins C and S, which are all involved in the coagulation process, and therefore have a slow onset of anticoagulant effect that spans several days. They are given orally; once the dose is established for an individual patient, they can provide a steady level of anticoagulation. Vitamin K antagonists are used for both the prevention and tre~tm.ont of arterial and venous thrombosis.
Bleeding is the major adverse effect of vitamin K antagonists.
Especially serious episodes involve sites where irreversible damage may result from compression of vital structures (e.g., intracranial, pericardial, nerve sheath, or spinal cord) or from massive internal blood loss that may not be diagnosed rapidly (e.g., ga~lloi~ l, inl-dpeliloneal, r~lr~e-iloneal). The risk of intracerebral or subdural hematoma in patients over 50 years of age taking an oral anticoagulant over a long term may be increased ten-fold. For continued or serious bleeding, vitamin Kl (phytonadione) is an effective antidote. Since reversal of anticoagulation by vitamin K1 requires the synthesis of fully carboxylated coagulation proteins, significant improvement in hemostasis does not occur for several hours, regardless of the route of ~lmini~tration, and 24 hours or longer may be needed for maximal effect. Warfarin is conll,.;n~liç~ted in women who are or may become pregnant because the drug passes through pl~rfnt~l CA 022~3836 1998-11-09 W O 97/42967 rCTrUS97/08017 _ 9 _ barrier and may cause fatal hemorrhage in the fetus. Warfarin treatment during pregnancy may also cause spontaneous abortion, still birth and birth defects.
The thrombolytic agents include tPA, streptokinase, urokinase prourokinase, anisolylated plasminogen streptokinase activation complex (APSAC),S and animal salivary gland plA~minogen activators, all of which act by accelerating fibrinolysis. The thrombolytic drugs are used to Iyse freshly formed arterial and venous thrombi; they are not emcacious in dissolving thrombi that have been present for more than a few hours. The intravenous a~lmini~tration of these agents is now accepted as useful therapy in the management of deep vein thrombosis, 10 pulmonary embolism, acute myocardial infarction, and peripheral arterial thromboembolism .
The major toxicity of all thrombolytic agents is hemorrhage, which results from two factors. Therapy with thrombolytic drugs tends to dissolve bothpathological thrombi and fibrin deposits at sites of vascular injury. In addition, a 15 systemic Iytic state results from systemic formation of plasmin, which produces fibrinogenolysis and destruction of other coagulation factors. Massive fibrinolysis is initiated, and the inhibitory controls of the process are overwhelmed. The systemic loss of fibrinogen and platelet dysfunction caused by the thrombolytic agents also produces a hemorrhagic tendency. Thus, the use of thrombolytic 20 agents is contr~in-lic~te~l in situations where there is active bleeding or a risk of major hemorrhage.
If heparin is used concurrently with either streptokinase or t-PA, serious hemorrhage will occur in 2 % to 4% of patients. Intracranial hemorrhage is by far the most serious problem; it occurs in a~lo~ llately 1% of cases, and 25 the frequency is the same with all three thrombolytic agents. R~ll~e~iloneal hemorrhage is also a serious complication. The frequency of hemorrhage is less when thrombolytic agents are utilized to treat myocardial infarction compared with pulmonary embolism or venous thrombosis; this dirrelc;nce may be due to the duration of therapy (1 to 3 hours for myocardial infarction, compared to 12 to 72 30 hours for pulmonary embolism and venous thrombosis).
In general, venous thrombosis and its potential for life~ .nil-g pulmonary embolism are prevented and treated with heparin or warfarin. Low-.

CA 022~3836 1998-11-09 W O 97/42967 rCT~US97/08017 dose subcutaneous heparin is frequently used as prophylaxis against venous thrombosis in surgical patients but is ineffective in those at highest risk, forexample, after hip fracture. Warfarin reduces mortality from pulmonary embolism and can be given more safely to immobilized or post-surgical patients in low-dose or stepwise regimens. Once a venous thrombosis has developed, however, full-dose heparin treatment for S to 10 days ovellappu~g with full-dose warfarin treatment for 4 to 5 days is neces~ry to prevent clot progression and/or pulmonary embolism. Thrombolytic agents have been used to treat pulmonary embolism and deep venous thrombosis, but their efficacy in reducing mortality 10 remains to be established. Aspirin offers little value in treating venous thromboembolism .
For acute arterial thrombosis, thrombolytic therapy is generally the treatment of choice. The goals of thrombolytic therapy are to achieve rapid reperfusion of the thrombosed vessel and m~int~in patency of the vessel; these 15 objectives are based on the premise that rapid and sllst~ined restoration of blood flow reduces associated complications. However, multiple episodes of vessel reocclusion typically follow thrombolytic therapy. Although widely used as an adjunct to thrombolytic therapy, heparin does not accelerate thrombolysis or prevent reocclusion of the vessel. [Klement et al., Thrombosis Haemostasis, 20 68:64-68 (1992).] In p~ti~n~.~ with a fresh cor~ aly thrombosis, intravenous thrombolytic therapy can permit rapid reperfusion of the thrombosed coronary artery, thus preserving cardiac function and reducing mortality, if ~11ministered within a few hours of the onset of symptoms. Thrombolytic agents can also re-establish the patency of thrombosed peripheral arteries if ~dmini~tered within a25 few hours after acute thrombosis. In some in~t~n-~es, e.g., for coronary artery thrombosis, the thrombolytic agent is ~lmini~tered locally by selective cath~;lel,,~lion of the involved vessel. When given systemic~lly rather than locally, a thel~;uLic effect is evident if the thrombin time is greater than twice normal. Such treatment should generally be followed by heparin and then oral anticoagulants to prevent further clot promulgation or recurrence. Following thrombolytic therapy and before the thrombin time has returned to its normal range, heparin is generally given to fully anticoagulate the patient for five to ten CA 022~3836 l998-ll-09 W O 97/42967 PCTrUS97/08017 days. Warfarin may be started before the heparin is stopped, depending on whether prolonged anticoagulation will be required in the management of the patient's disorder. Aspirin is ineffective in the immediate setting, but is useful for long-term prophylaxis against arterial thrombosis. Recent studies suggest that the S concurrent ~dminictration of low doses of aspirin improves the efficacy of thrombolytic therapy of myocardial infarction. Patients with symptomatic strokesare acutely anticoagulated with heparin and followed indefinitely with warfarin.Aspirin is recommended for prophylaxis of stroke in patients with cervical bmits, asymptomatic carotid stenosis, or a history of transient ischemic attacks and minor 10 stroke.
Considerable controversy continues to surround the use of heparin in DIC. Heparin is usually reserved for fillmin~nt, explosive forms of diffuse intravascular clotting, in which massive defibrination is accompanied by fibrinogen levels of less than 100 mg/dL and replacement therapy is not controlling the 15 hemorrhage. In these cases, heparin is given as a continuous intravenous infusion at a rate of 10 to 15 units/kg/hour. If the patient is in immç(li~te danger of dying from hemorrhage, 5000 to 10,000 units of heparin are given intravenously as a bolus and heparin is then continued at an infusion rate of 1000 units per hour.
Heparin can also be useful in treating unstable angina and patients 20 undergoing elective cardioversion for atrial fibrillation of greater than 2 days duration. Warfarin and aspirin are useful for prophylaxis of cerebral embolism, particularly in p~ient~C at risk because of atrial fibrillation. More than 50% of patients with cerebral embolism have atrial fibrillation. Warfarin is also recommPnded for treating patients with mechanical heart valves, for whom the 25 associated risk of embolism is 2 % to 6 % per patient per year despite anticoagulation, patiçnts with rh~um~tic mitral valve disease, in whom the rate of associated thromboembolic complications is 1.5% to 4.7% per year, and patients with a history of thromboembolism. Aspirin is recommended for p~tientc with mitral valve prolapse.
Anti-thrombotic agents are also used routinely to prevent the occlusion of extracorporeal devices: intravascular c~nnul~.c (heparin), vascularaccess shunts in hemodialysis patients (aspirin), hemodialysis m~çhines (heparin), CA 022~3836 1998-11-09 Wo 97/42967 PcT/uss7lo8ol7 and cardiopulmonary bypass machines (heparin). In addition, they have been utilized in the treatment of certain renal diseases (heparin/warfarin) and small-cell lung cancer (warfarin).
BPI is a protein isolated from the granules of m~mm~ n S polymorphonuclear leukocytes (PMNs or neutrophils), which are blood cells essential in the defense against invading microor~ni.cm.s. Human BPI protein hasbeen isolated from PMNs by acid extraction combined with either ion exchange chromatography [Elsbach, J. Biol. Chem., 254: 11000 (1979)] or E. coli affinity chromatography [Weiss, et al., Blood, 69:652 (1987)]. BPI obtained in such a 10 manner is referred to herein as natural BPI and has been shown to have potentbactericidal activity against a broad spectrum of gram-negative bacteria. The molecular weight of human BPI is approximately 55,000 daltons (55 kD). The amino acid sequence of the entire human BPI protein and the nucleic acid sequence of DNA encoding the protein have been reported in Figure 1 of Gray et al., J. Biol. Chem., 264:9505 (1989), incorporated herein by reference. The Grayet al. amino acid sequçnce is set out in SEQ ID NO: 1 hereto. U.S. Patent No.
5,198,541 discloses recombinant genes encoding and methods for expression of BPI proteins, including BPI holoprotein and fragments of BPI.
BPI is a strongly cationic protein. The N-terminal half of BPI
20 accounts for the high net positive charge; the C-terminal half of the molecule has a net charge of -3. [Elsbach and Weiss (1981), supra.] A proteolytic N-terminal fragment of BPI having a molecular weight of about 25 kD possesses essentially all the anti-bacterial efflcacy of the naturally-derived 55 kD human BPI
holoprotein. [Ooi et al., J. Bio. Chem., 262: 14891-14894 (1987)]. In contrast to 25 the N-terminal portion, the C-terminal region of the isolated human BPI protein displays only slightly (letect~ble anti-bacterial activity against gram-negativeorg~nicm.c. [Ooi et al., J. E:~cp. Med., 174:649 (1991).] An N-terminal BPI
fragment of approximately 23 kD, referred to as "rBPI23," has been produced by recombinant means and also retains anti-bacterial activity against gram-negative30 organisms as well as endotoxin-neutralizing activity. [G~zano-Santoro et al., Infect. Imm~m. 60:4754-4761 (1992).]

CA 022~3836 1998-11-09 W O 97/42967 PCTrUS97/08017 The bactericidal effect of BPI has been reported to be highly specific to gram-negative species, e.g., in Elsbach and Weiss, Inflammation.
Basic Principles and Clinical Correlates, eds. Gallin et al., Chapter 30, Raven Press, Ltd. (1992). The precise mechanism by which BPI kills gram-negative bacteria is not yet completely elucidated, but it is believed that BPI must first bind to the surface of the bacteria through electrostatic and hydrophobic interactions between the cationic BPI protein and negatively charged sites on LPS. In susceptible gram-negative bacteria, BPI binding is thought to disrupt LPS
structure, leading to activation of bacterial enzymes that degrade phospholipids and 10 peptidoglycans, altering the permeability of the cell's outer membrane, and initiating events that ultimately lead to cell death. [Elsbach and Weiss (1992),supra]. LPS has been referred to as "endotoxin" because of the potent infl~mm~ ry response that it stimulates, i.e., the release of me li~tQrs by hostinfl~mm~tory cells which may Illtim~tP.ly result in irreversible endotoxic shock.
BPI binds to and neutralizes lipid A, reported to be the most toxic and most biologically active component of LPS.
In addition to BPI's bactericidal and endotoxin binding/neutralizing activities, BPI has been shown to bind and neutralize heparin. Co-owned U.S.
Patent No. 5,348,942 was issued September 20, 1994 with claims directed to methods of neutralizing the anticoagulant effects of heparin with BPI protein products (i.e., their procoagulant activity). There has been no suggestion or use of BPI as an anticoagulant or thrombolytic agent, nor any suggestion of its use for the prophylaxis or treatment of thrombotic disorders.
There exists a need in the art for methods and compositions capable of exerting anticoagulant or thrombolytic effects without severe adverse side effects, and methods and compositions capable of improving the therapeutic effectiveness of existing anticoagulant or thrombolytic agents, which ideally could reduce the required dosages of such existing agents.

SIJMMARY OF THE INVENTION
The present invention provides novel methods for slowing clot formation and for enhancing clot dissolution using BPI protein products, and .

CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 further provides methods for treatment of thrombotic disorders by ~dmini~trationof BPI protein products in therapeutically effective amounts.
According to the invention, a BPI protein product such as rBPI21 is ~(lmini.~tered to a subject suffering from thrombotic disorder in an amount 5 effective to treat such disorder, including prophylactic and therapeutic treatment.
BPI protein products reduce the adverse effects of thrombotic disorder by activities that include slowing or delaying clot formation (i.e., anticoagulant activity) or by enhancing, accelerating or increasing clot dissolution (i.e., thrombolytic activity).
In another aspect of the invention, methods are provided for the 10 treatment of thrombotic disorder by concurrent ~dmini~tration of a BPI protein product with a thrombolytic agent, including tPA, streptokinase, urokinase, prourokinase, APSAC, animal salivary gland plasminogen activators, other plasminogen activators, and derivatives of such plasminogen activators. According to this aspect of the invention, the thrombolytic agent dissolves the clot, while the 15 BPI protein product enhances the dissolution activity of the thrombolytic agent and/or delays clot formation, thus delaying, decreasing or preventing r~ olllbosis. The BPI protein products are effective with both endogenous levelsand therapeutic levels of thrombolytic agents such as plasminogen activators.
This aspect of the invention also provides methods for decreasing 20 the dose of a thrombolytic agent required for a desired thel~eulic or prophylactic effect in a patient, such as for dissolving a blood clot, by concurrent allmini~tration of BPI protein product and the thrombolytic agent.
The invention further provides methods for accelerating reperfusion and/or delaying or preventing reocclusions in a subject treated with a thrombolytic 25 agent, e.g., tPA, by concurrent admini~tration of BPI protein product with the thrombolytic agent.
Additionally contemplated is use of a BPI protein product in the preparation of a medicament for the treatment of a thrombotic disorder, or in the preparation of medicaments for slowing clot formation or enh~ncing clot 30 dissolution in blood. Further contemplated is use of a BPI protein product in the preparation of a medicament for co-~(lmini.~tration with a thrombolytic agent in the treatment of a thrombotic disorder, as well as in the preparation of a medicament CA 022~3836 1998-11-09 for enhancing reperfusion or reducing reocclusion in a subject treated with a thrombolytic agent.
As will be appreciated from the following detailed description, methods of the present invention provide safer and more effective treatment of 5 thrombotic disorders than conventional therapies. By reducing the dosage of a~ h,olllbotic agent required to achieve a desired therapeutic effect, BPI protein products can reduce or elimin~te the potential side effects often associated with conventional antithrombotic agent therapies, while not il,le,r~lh~g with the antithrombotic activity of those agents. Importantly, use of such BPI protein 10 products can enhance the antithrombotic activity of such agents by slowing clot formation or enh~ncing clot dissolution.
Numerous additional aspects and advantages of the invention will become al~pal~nl to those skilled in the art upon consideration of the followingdetailed description of the invention which describes presently prepared 15 embodiments thereof.

DETAILED DESCRIPTION OF THE INVENTION
Thrombotic disorders, including acute vascular diseases, such as myocardial infarction, stroke, pelipbeldl arterial occlusion, deep vein thrombosis, pulmonary embolism, and other blood system thromboses, constitute major health 20 risks. Such disorders are caused by either partial or total occlusion of a blood vessel by a blood clot, which consists of fibrin and platelet aggregates.
Therapeutic intervention with agents that prevent or delay clot formation (i.e.,anticoagulants) or with agents that dissolve blood clots (i.e., thrombolytics) is associated with numerous limitations, complications, risks and side effects. Most 25 significant are the bleeding side effects associated with therapeutic doses of such agents and the complications associated with l~lhlulllbOSiS and reocclusion following reperfusion. It has now been unexpectedly discovered that admini~tration of BPI protein products ef~ectively slows clot formation and enhances clot dissolution in blood. The ~lminictered BPI protein product present30 in the blood during clot formation delays clotting time and/or may change the character of the clot that is formed to a looser, less stable clot.

CA 022~3836 1998-11-09 W 097/42967 PCT~US97/08017 It is contemplated that BPI protein products may be ~lmini~tered alone or concurrently with other antithrombotic (anticoagulant or thrombolytic) agents. Anticoagulant agents are agents with the pharmacological effect of slowing clot formation, such as (l~ltep~rin and enoxaparin, the coumarin derivative oral anticoagulants such as warfarin, and aspirin. Thrombolytic agents are agents with the pharmacological effect of enhancing clot dissolution, and include plasminogen activators such as t-PA, streptokinase, urokinase, proutokinase, APSAC, animal salivary gland plasminogen activators and derivatives thereof.
BPI protein products used according to methods of the invention unexpectedly have the property of making blood clots more susceptible to dissolution or Iysis either at endogenous levels or added levels of plasminogen activators, such as tPA. Whether the tPA is present prior to clot formation or after clot formation, the BPI protein product enhances clot dissolution, e.g., accelerates clot dissolution or Iysis, or provides more complete clot dissolution or Iysis. Thus, BPI protein products are useful in methods for the treatment of thrombotic disorders, for dissolving or Iysing clots in thrombotic patients, fordelaying or inhibiting hard clot formation or supplementing thrombolytic therapyin the patients.
The previously described biological activities of BPI protein products, including bactericidal, endotoxin binding and neutralizing, heparin binding and neutralizing activities, do not suggest or even hint at the anticoagulant or thrombolytic activities of BPI protein products and the therapeutic uses thatarise from these unexpected and previously undiscovered activities. In particular, the activity of BPI protein products as agents for treatment of thrombotic disorders is particularly surprising in view of the previously discovered activity of BPI
protein products to bind and neutralize heparin (see, co-~sign~d U.S. Patent No.5,348,942).
The term "treatment" as used herein encompasses both prophylactic and therapeutic treatment of thrombotic disorders.
The term "thrombotic disorder" as used herein encompasses conditions associated with or resulting from thrombosis or a tendency towards thrombosis. These conditions include conditions associated with arterial CA 022~3836 1998-11-09 Wo 97/42967 PCT/USg7/08017 thrombosis, such as coronary artery thrombosis and resulting myocardial infarction, cerebral artery thrombosis or intracardiac thrombosis (due to, e.g.Jatrial fibrillation) and resulting stroke, and other peripheral arterial thrombosis and occlusion; conditions associated with venous thrombosis, such as deep venous 5 thrombosis and pulmonary embolism; conditions associated with exposure of the patient's blood to a foreign or injured tissue surface, including rii~e~cecl heart valves, mechanical heart valves, vascular grafts, and other extracorporeal devices such as intravascular c~nn~ , vascular access shunts in hemodialysis patients, hemodialysis m~chin~.s and cardiopulmonary bypass machines; and conditions 10 associated with coagulopathies, such as hypercoagulability and dic.~emin~ted intravascular coagulopathy that are not the result of an endotoxin-initi~t~d coagulation c~cca~le.
"Concurrent ~lmini.stration," or "co-~lmini~tration" or "co-tr~tment," as used herein includes a~1minictration of the agents, in conjunction or lS combination, together, or before or after each other. The BPI protein product and other alllill-,olllbotic (including anticoagulant or thrombolytic) agents may be~dministe.red by dirl~lGnl routes. Por example, the BPI protein product may be ~lministered intravenously while the antithrombotic agent is ~dmini~tered intramuscularly, intravenously, subcutaneously or orally. Alternatively, the BPI20 protein product may be ~lminictered~ e.g., in an aerosolized or nebulized form while the anlilllrumbotic agent is ~dmini~t~red, e.g., intravenously. The BPI
protein product and alllilllloll~botic agent are preferably both ~mini~tered intravenously, in which case they may be given sequentially in the same intravenous line, or after an intermediate flush, or in dirrel~ l intravenous lines.
25 The BPI protein product and alllilhl~lllbotic agent may be ~lminictered simult~neously or sequenti~lly, as long as they are given in a manner sufflcient to allow both agents to achieve effective concentrations at the site of thrombosis.During sequential arlministration of BPI protein product and alllilhr()lllbotic agent, it is also co,llelll~lated that a time period varying from minutes to hours may 30 intervene between the a~lmini~tration of the agents.
Conventional antithrombotic agents are expected to be a~lministered in dosages and by routes consistent with the usual clinical practice. The typical CA 022~3836 1998-11-09 dosages and ~lmini.ctration regimens for some of these anticoagulant and thrombolytic agents, when ~dmini.ctered as monotherapy, are discussed below.
Naturally, these dosages vary as determined by good medical practice and the clinical condition of the individual patient.
S The dosing of warfarin must be indivi-iu~li7ed according to the patient's sensitivity to the drug as in(lic~t~cl by its effect on the prothrombin time (PT) ratio. The loading dose is typically 2 to 5 mg/day and most patients are satisfactorily m~int:~in~d at a dose of 2 to 10 mg/day. Warfarin is generally given orally but may be a~lmini.stered intravenously if the patient cannot take the drug orally .
Urokinase is indicated for Iysis of acute pulmonary emboli and coronary artery emboli, and is also used to restore patency to intravenous c~nnul~
and c~theters. The drug is typically ~-~mini~tered in an initial dose of 2,000 units/lb over a period of 10 minutes followed by a continuous infusion of 2,000 units/lb/hr for 12 hours. The total dose of urokinase given will range from 2.25million to 6.25 million units, depending on the weight of the patient. When it is used to clear intravenous c~nmll~ or catheters, urokinase is given as a single injection of 5,000 units in a volume of 1 mL.
Streptokinase is indicated for use in the management of acute myocardial infection, lysis of intracoronary thrombi, arterial thrombosis or embolism, deep vein thrombosis, pulmonary embolism, and for clearing blocked ~nnul~e or c~theters. For treatment of acute myocardial infarction, 1.5 million units may be given by intravenous infusion over 60 minutes. Alternatively, it may be given by intracoronary infusion of a 20,000 unit bolus followed by 2,000 units/min. over 60 minutes. For other non-myocardial infarction indications, a dosage of 250,000 units by intravenous infusion over 30 minutes is a~ iate for the great majority of patients.
Anistreplase (also known as ASPAC) is generally a-1mini~tered in a single dose of 30 units by intravenous injection over 2 to 5 minutes. Its use isindicated in the management of acute myocardial infection and for the lysis of coronary artery thrombi.

CA 022~3836 1998-11-09 W O 97/42967 PCTrUS97/08017 The drug tPA is dosed based upon patient weight, with the total dose not e7~cee-1ing 100 milligrams. For patients weighing more than 67 kg, the recommended dose is a 15 mg initial intravenous bolus followed by a continuous infusion of 15 mg over 30 minutes, and further followed by 35 mg infused over 5 the next 60 minutes. For patients weighing less than or equal to 67 kg, the recommended dose is a 15 mg initial intravenous bolus followed by 0.75 mg/kg (not to exceed 50 mg) over 30 minutes, and further followed by 0.5 mg/kg (not toexceed 35 mg) over the following 60 minutes.
Therapeutic compositions comprising BPI protein product may be ~lminictered systemically or locally into the involved vessel. Systemic routes of ~dministration include oral, intravenous, intramuscular or subcutaneous injection (including into a depot for long-terrn release), intraocular and retrobulbar, intrathecal, intraperitoneal (e.g., by intraperitoneal lavage), intrapulmonary using aerosolized or nebulized drug, or transdermal. The p~,rell~d systemic route is intravenous ~imini~tration. In some inct~ncec, e.g., for coronary artery or peripheral artery thrombosis, it is advantageous to a~1minicter the BPI protein product regionally by selective catheterization of the involved vessel. When given parenterally, BPI protein product compositions are generally injected in doses ranging from 1 llg/kg to 100 mg/kg per day, preferably at doses ranging from 0.1mg/kg to 20 mg/kg per day, and more preferably at doses ranging from 1 to 20 mg/kglday. The treatment may continue by continuous infusion or intermittent injection or infusion, at the same, reduced or increased dose per day for as long as determined by the treating physician. Those skilled in the art can readily optimize effective dosages and monotherapeutic or concurrent a~lminictr~tion regimens forBPI protein product and/or other anlilillu,llbotic agents, as deterrnined by good me~lic~l practice and the clinical condition of the individual patient.
When BPI protein product is concurrently ~lmini.ctered with antithrombotic agents, the BPI protein product and the all~ lulllbotic agents may each be a~lminictered in amounts that would be sufficient for monotherapeutic effectiveness, or they may be ~(lmini.ctered in less than monothel~eulic amounts.
It is expected that BPI protein products are capable of improving the therapeutic effectiveness of existing anticoagulant or thrombolytic agents, which would reduce . .

CA 022~3836 1998-11-09 wo 97/42967 PCT/US97/08017 the dosages needed to exert their desired anticoagulant or thrombolytic effects.This, in turn, decreases the risk of adverse side effects associated with the use of thrombolytic agents, including, for example, undesirable internal or external bleeding.
BPI protein products may improve the therapeutic effectiveness of other alllilhrol"botic agents in a variety of ways. For example, lowering the dosage of the antilllr~ lbotic agent required for therapeutic effectiveness reduces toxicity and/or cost of treatment, and thus allows wider use of the agent.
Alternatively, concurrent ;~lministration may produce an increased, more rapid or more complete anticoagulant or thrombolytic effect than could be achieved with either agent alone.
It is further contemplated that BPI protein product compositions are useful, in vitro or in vivo in restoring or m~int~inin~ patency of c~nn~
catheters and tubing obstructed by clotted blood or fibrin, in m~int~inin~ the anticoagulation of blood, e.g., in blood bags, and in m~int~ining blood fluidity in, e.g., hemodialysis and extracorporeal circulation, and around foreign implants, e.g., heart valves or prosthetics.
As used herein, "BPI protein product" includes naturally and recombinantly produced BPI protein; natural, synthetic, and recombinant biologically active polypeptide fragments of BPI protein; biologically active polypeptide variants of BPI protein or fragments thereof, including hybrid fusion proteins and dimers; biologically active polypeptide analogs of BPI protein or fr~ments or variants thereof, including cysteine-substituted analogs; and BPI-derived peptides. The BPI protein products ~lmini.stPred according to this invention may be generated and/or isolated by any means known in the art. U.S.
Patent No. 5,198,541, the disclosure of which is incorporated herein by reference, discloses recombinant genes encoding and methods for ~ ,ression of BPI proteins including recombinant BPI holoprotein, referred to as rBPI50 and recombinant fragments of BPI. Co-owned, copending U.S. Patent Application Ser. No.
07/885,501 and a continn~tion-in-part thereof, U.S. Patent Application Ser. No.
08/072?063 filed May 19, 1993 and coll~;sl)onding PCT Application No. 93/04752 filed May 19, 1993, which are all incorporated herein by reference, disclose novel CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 methods for the purification of recombinant BPI protein products expressed in and secreted from genetically transformed m~mm~ n host cells in culture and discloses how one may produce large quantities of recombinant BPI products suitable for incorporation into stable, homogeneous pharm~ceutical preparations.S Biologically active fragments of BPI (BPI fragments) include biologically active molecules that have the same or similar amino acid sequence as a natural human BPI holoproLein, except that the fragment molecule lacks amino-terminal amino acids, internal amino acids, and/or carboxy-terminal amino acids of the holoprotein. Nonlimhing examples of such ~gments include a N-terminal 10 fragment of natural human BPI of approximately 25 kD, described in Ooi et al., J.
E~p. Med., 174:649 (1991), and the recombinant expression product of DNA
encoding N-terminal amino acids from I to about 193 or 199 of natural human BPI, described in Gazzano-Santoro et al., Infect. Immun. 60:4754-4761 (1992), and referred to as rBPI23. In that publication, an ~ cs~ion vector was used as a15 source of DNA encoding a recombinant expression product (rBPI23) having the 31-residue signal sequence and the first 199 amino acids of the N-te~ll,inus of the mature human BPI, as set out in Figure 1 of Gray et al., supra, except that valine at position 151 is specified by GTG rather than GTC and residue 185 is glutamic acid (srecifi~d by GAG) rather than Iysine (specified by AAG). Recombinant holoprotein (rBPI) has also been produced having the sequence (SEQ ID NOS: 1 and 2) set out in Figure 1 of Gray et al., supra, with the exceptions noted for rBPI23 and with the exception that residue 417 is alanine (specified by GCT) rather than valine (specffled by Gl~I). Other examples include dimeric forms of BPI fr~pmçnt~, as described in co-owned and co-pending U.S. Patent Application Serial No. 08/212,132, filed March 11, 1994, and corresponding PCT Application No. PCT/US95/03125, the disclosures of which are incorporated herein by reference. Preferred dimeric products include dimeric BPI protein products wherein the monomers are amino-terminal BPI fragments having the N-terminal residues from about 1 to 175 to about 1 to 199 of BPI holoprotein. A particularly preferred dimeric product is the dimeric form of the BPI fragment having N-terminal residues 1 through 193, design~ted rBPI42 dimer.

, . . .. .

CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 Biologically active variants of BPI (BPI variants) include but are not limited to recombinant hybrid fusion proteins, comprising BPI holopr~leill or biologically active fragment thereof and at least a portion of at least one other polypeptide, and dimeric fonns of BPI variants. Examples of such hybrid fusion 5 proteins and dimeric forms are described by Theofan et al. in co-owned, copending U.S. Patent Application Serial No. 07/885,911, and a continuation-in-part application thereof, U.S. Patent Application Serial No. 08/064,693 filed May 19, 1993 and corresponding PCT Application No. US93/04754 filed May 19, 1993, which are all incorporated herein by reference and include hybrid fusion 10 proteins comprising, at the amino-terminal end, a BPI protein or a biologically active fragment thereof and, at the carboxy-terminal end, at least one constant domain of an immunoglobulin heavy chain or allelic variant thereof. Similarly configured hybrid fusion proteins involving part or all Lipopolysaccharide Binding Protein (LBP) are also contemplated for use in the present invention.
Biologically active analogs of BPI (BPI analogs) include but are not limited to BPI protein products wherein one or more amino acid residues have been replaced by a different amino acid. For example, co-owned, copending U.S.
Patent Application Ser. No. 08/013,801 filed February 2, 1993 and corresponding PCT Application No. US94/01235 filed February 2, 1994, the disclosures of which are incorporated herein by reference, discloses polypeptide analogs of BPIand BPI fragments wherein a cysteine residue is replaced by a dirre.~"l amino acid. A pr~;~e~ d BPI protein product described by this application is the expression product of DNA encoding from amino acid 1 to approximately 193 or 199 of the N-terminal amino acids of BPI holopr()tein, but wherein the cysteine at residue number 132 is substituted with alanine and is design~tçd rBPI211\cys or BPI21. Other examples include dimeric forms of BPI analogs; e.g. co-owned and co-pending U.S. Patent Application Serial No. 08/212,132 filed March 11, 1994, and corresponding PCT Application No. PCT/US95/03125, the disclosures of which are incorporated herein by reference.
Other BPI protein products useful according to the methods of the invention are peptides derived from or based on BPI produced by recombinant or synthetic means (BPI-derived peptides), such as those described in co-owned and CA 022~3836 1998-11-09 wo 97/42967 PCT/USg7/08017 co-pending U.S. Patent Application Serial No. 08/504,841 filed July 20, 1995 and in co-owned and copending PCT Application No. PCT/US94/10427 filed September 15, 1994, which corresponds to U.S. Patent Application Serial No.
08/306,473 filed September 15, 1994, and PCT Application No. US94/02465 filed March 11, 1994, which corresponds to U.S. Patent Application Serial No.
08/209,762, filed March 11, 1994, which is a continuation-in-part of U.S. PatentApplication Serial No. 08/183,222, filed January 14, 1994, which is a continl-~tion-in-part of U.S. Patent Application Ser. No. 08/093,202 filed July 15, 1993 (for which the corresponding international application is PCT Application No. US94/02401 filed March 11, 1994), which is a contim~tion-in-part of U.S.
Patent Application Ser. No. 08/030,644 filed March 12, 1993, the disclosures of all of which are incorporated herein by reference.
Presently preferred BPI protein products include recombinantly-produced N-terminal fr~ments of BPI, especially those having a molecular weight of approximately between 21 to 25 kD such as rBPI21 or rBPI23, or dimeric forms of these N-terminal fragments (e.g., rBPI42 dimer). Additionally, preferred BPI protein products include rBPI50 and BPI-derived peptides.
The ~lmini.ctration of BPI protein products is preferably accomplished with a pharm~celltical composition comprising a BPI protein productand a pharm~ceutic~lly acceptable diluent, adjuvant, or carrier. The BPI proteinproduct may be ~(lminictered without or in conjunction with known surfactants, other chemotherapeutic agents or additional known anti-microbial agents. One pharm;tceuti-~l composition cont~ining BPI protein products (e.g., rBPI50, rBPI23) comprises the BPI protein product at a concentration of 1 mg/ml in citrate buffered saline (5 or 20 mM citrate, 150 mM NaCI, pH 5.0) comprising 0.1 % by weight of poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% by weight of polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, DE). Another pharm~eutic~l composition cont~ining BPI protein products (e.g., rBPI21) comprises the BPI protein product at a concentration of 2 mg/mL in S
mM citrate, 150 mM NaCl, 0.2% poloxamer 188 and 0.002% polysorbate 80.
Such combinations are described in co-owned, co-pending PCT Application No.
US94/01239 filed February 2, 1994, which corresponds to U.S. Patent Application CA 022~3836 1998-11-09 W O 97/42967 PCT~US97/08017 Ser. No. 08/190,869 filed February 2, 1994 and U.S. Patent Application Ser. No.
08/012,360 ~lled February 2, 1993, the disclosures of all of which are incorporated herein by reference.
Other aspects and advantages of the present invention will be 5 understood upon consideration of the following illustrative examples. Example 1 addresses the effects of BPI protein product on clot formation and clot lysis/dissolution under varying conditions in tube assays. Example 2 addresses the effects of BPI protein products on clot formation and clot lysis/dissolution, asmonitored by turbidity measurements, under varying conditions in microtiter plate 10 assays. Example 3 addresses the effects of BPI protein product in vivo in a rat thrombus model with concurrent i--lmini~tration of tPA.

E~'FECTS OF BPI PROTEIN PRODUCT ON
CLOT FORMATION AND CLOT LYSIS/DISSOLUIION
A tube assay was used to determine the effects of a BPI protein product on clot formation and on clot Iysis or dissolution under a variety of conditions using human plasma samples. Unless otherwise noted the human plasma used in these assays was prepared from human blood drawn from a variety of donors into ACD Vacutainer~ tubes (Becton Dickinson, Moul,lainview, CA) 20 cont~ining citrate as an anticoagulant, and was stored frozen at -70 C. For the preparation of platelet rich plasma (PRP), the anticoagulated blood was centrifuged at approximately 180 x g for 5 minutes and the plasma removed following this low-speed centrifugation. For the plepa~ ion of platelet poor plasma (PPP), the anticoagulated blood was centrifuged at approximately 460 x g for 10 minutes and25 the plasma removed following this higher speed centrifugation.
In an initial experiment to determine whether BPI protein products interacted with plasma proteins, ACD plasma pooled from two human donors was passed over a column cont~ining BPI23 that was conjugated to Sepharose via cyanogen bromide. Approximately 40 mL of plasma was passed through the 30 rBPI23-Sepharose column to allow binding of plasma components. The column was washed with phosphate buffered saline (lOmM phosphate, 0.15 M NaCl, pH

CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 7.2) until the OD280 of the wash was <0.02. The bound plasma components were eluted with high salt (1.5 M NaCl) and the protein eluate was analyzed by SDS-PAGE. Amino acid sequence analysis of several protein bands showed that pro~}llv.llbin and fibrinogen were bound by rBPI23.
The ability of exemplary BPI protein products to delay clot formation (i.e., anticoagulant activity) and/or to enhance the dissolution or lysis of a clot once formed (i.e., thrombolytic activity) was evaluated. Such anticoagulant and/or thrombolytic activity demonstrates the utility of BPI protein products for the treatment of thrombotic disorder in a subject suffering from such disorder.
10 The effects of BPI protein products were evaluated in PPP and PRP under a variety of conditions as follows.

A. Effect of Different Surface Environments Clot formation and lysis were evaluated in the presence and absence of rBPI21 in polypropylene or glass test tubes. For all experiments in this and subsequent examples, the rBPI21 used was formulated at 2 mg/mL in 5 mM
citrate, 150 mM NaCl, 0.2% poloxamer 188 (Pluronic F-68, BASF Wyandotte, Parsip~any, NJ), and 0.002% polysorbate 80 (TWEEN 80, ICI Americas Inc., Wilmington, DE). Other BPI protein products used herein were similarly formulated at 1 mg/mL. The 0.1% HSA-TBS used was 0.1% human serum albumin (HSA) [Alpha Therapeutics, Los Angeles, CA] in Tris-buffered saline (TBS) [0.02M Tris, 0.15 M NaCl, pH 7.4]. For the clot formation part of this e,~c.i---ent, the following reagents were mixed together in the following order:(1) 160 ~4L of PPP (Donor RL); (2) 40 ,uL of BPI21 (either 10 or 250 ,ug/mL in 0. 1 % HSA-TBS); and (3) 200 ~4L of 40 mM CaCl2 in TBS, pH 7. 8.
The tubes were allowed to stand at room temperature, and every 1 minute the tubes were checked by gently tipping the tube and visually inspecting it for clot formation. The time in minutes to clot formation after CaC12 addition was measured.

CA 022~3836 1998-ll-09 W 097/42967 PCTrUS97/08017 Minutes to Clot Formation (After CaCl2 Addition) Tube Type Control rBPI21 rBPI21 (0.1% HSA-TBS) 1 ,ug/mL 25 ~g/mL
Polypropylene 12 14 24 Glass 6 6 9 Clot formation was faster in glass tubes than in polypropylene tubes. However, rBPI21 prolonged clotting times in both glass and polypropylene tubes. The higher rBPI21 concentration tested (25 ~g/mL) produced the longest delay in clotting time.
For the clot dissolution part of this experiment, 33 minutes after CaC12 addition, 44 ~4L of tPA [Calbiochem, San Diego, CA] (600 units/mL, or 1 ,ug/mL) were added to each tube to provide a final tPA concentration of 60 units/mL (or 100 ng/mL). This is in the range of elevated endogenous concentrations observed for tPA in certain physiologic states/conditions ~See, e.g., von der Mohlen e~ al., Blood, 85:3437-3443 (1995); Suffradini, et al., New Engl.J. Med., 320:1165-1172 (1989)]. The tubes were incub~ted for 10 minutes at room temperature, then placed in a 37~C water bath. Each tube was checked for clot dissolution/lysis by visual inspection.
After 5 minutes, the rBPI21-treated clots in glass tubes had detached from the sides of the tube while all other clots remained adhered to the side of the tube. At 3.5 hours the clot in the 25 ,ug/mL BPI21 polypropylene tube was smaller, approximately 1/3 the size of the clots in the 1 ,~g/mL rBPI21 and control polypropylene tubes. Clot dissolution/lysis times were faster in glass than in polypropylene tubes. The presence of 25 ~g/mL rBPI21 accelerated the dissolution of the clot. In all subse~uent experiments, polypropylene tubes wereutilized to minimi7e the protein adsorption effects of the glass.

B. Effect of Calcium Ion Concentration ~ Clot formation and lysis were evaluated in the presence or absence of rBPI21 with varying concentrations of calcium (10, 15, 20 mM) to determine CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 optimum calcium concentration. For the clot formation part of this experiment, the following reagents were mixed together in a polypropylene test tube in the following order: (1) 60 ~L of 0.1% HSA-TBS; (2) 100 ~bL of PPP (Donor PC);
(3) 40 ~uL of rBPI21 (either 10 or 250 ,ug/mL in 0.1% HSA-TBS); and (4) 200 ,uL
of 20, 30, or 40 mM CaC12 in TBS, pH 7.8.
The tubes were allowed to stand at room temperature and were checked every 1 minute for gel clot formation by visual inspection. At all calcium concentrations tested, increased rBPI21 concentrations correlated with increasedclotting time. The higher rBPI21 concentration produced the longest delay in clotting time.
For the clot dissolution part of this e~e-inlent, 35 minlltes after CaC12 addition, 44 ~4L of tPA (600 units/mL, or 1 ,~4g/mL) were added to each tube to provide a final tPA concentration of 60 units/mL or 100 ng/mL. The tubes were incubated in a 37~C water bath and checked approximately every twenty minutes for clot dissolution/lysis by visual in~pectil-n.
rBPI21 accelerated the dissolution of the clot. Again, this was most apparent at the 25 ,,lg/mL BPI2~ concentration. Optimal clot formation and clot Iysis was observed using 10 mM calcium. In all subsequent e,y~.i,l,ents, 10 mM
CaC12 was used for initiation of clotting and Iysis.

C. Effect of Pre-Clot and Post-Clot Addition of tPA
Clot formation and lysis were evaluated in the presence or absence of rBPI21 with pre-clot or post-clot addition of tPA. For the clot formation part of this t;~ue~ ent, the following reagents were mixed together in a polypropylene test tube in the following order: (1) 60 ,uL of 0.1 % HSA-TBS; (2) 100 ~L4L of PPP (Donor PC); (3) 40 ,uL of rBPI21 (either 10 or 250 ~g/mL in 0.1% HSA-TBS); (4) for pre-clot only, 44 ~4L of tPA (600 units/mL, or 100 ng/mL); and (5)200 ,~L of 20 mM CaC12 in TBS, pH 7. 8.
The tubes were allowed to stand at room temperature and were checked every 1 minute for gel clot formation by visual inspection. The time in minutes to clot formation after CaC12 addition were measured. A delay in clot formation was observed only for 25 ~g/mL rBPI21 with pre-clot tPA addition.

.. . . . ..

CA 022~3836 1998-11-09 wo 97/42967 PCT/US97/08017 For the clot dissolution part of this experiment, 35 minutes after CaC12 addition, 44 ,uL of tPA (600 units/mL, or 1 ~bg/mL) were added to the post-clot tubes. The final tPA concentration in all tubes (pre- and post-clot) was 60units/mL or 100 ng/mL. All tubes were incubated in a 37~C water bath and checked approximately every twenty minutes for clot dissolution/lysis by visual inspection. Time to clot dissolution in hours (for post-clot addition of tPA) or in minutes (for pre-clot addition of tPA) was evaluated. In this experiment, clot Iysis when the tPA was added pre-clot was at least 6 times faster than post-clot. The rBPI21 at 25 ,ug/mL appeared to accelerate clot Iysis time. Because of the 10 unexpectedly rapid dissolution of the clot under conditions of pre-clot tPA
addition, precise times for clot dissolution were not assessed in this experiment.
These results demonstrated that clot Iysis was dramatically accelerated by the addition of tPA prior to clot formation rather than after clot formation.

D. Effect of Pre-Clot Addition of Varying Concentrations of tPA
Clot formation and Iysis were evaluated in the presence or absence of rBPI21 with pre-clot addition of tPA to a final concentration of 0, 6, or 60 units/mL. For the clot formation fimal part of this experiment, the following reagents were mixed together in a polypropylene test tube in the following order:
(1) 60 ,uL of 0.1 % HSA-TBS; (2) 100 ~L of PPP (Donor RL); (3) 40 ~L of BPI21 (either 10 or 250 llg/mL in 0. 1 % HSA-TBS; (4) 44 ,uL of tPA (600 units/mL, or 100 ng/mL) or buffer control (0.1% HSA-TBS); and (5) 200 ,uL of 20 mM CaC12 in TBS, pH 7.8.
The tubes were allowed to stand at room telllp~ lul~, and were checked every 1 minute for gel clot formation by visual inspection. The minutes to clot formation after CaC12 addition were measured. Adding increasing amounts of tPA (0, 6, 60 units/mL) did not significantly alter the time to clot formation for the conditions tested.

CA 022~3836 l998-ll-09 W O 97/42967 PCT~US97/08017 tPA Minutes to Clot Formation Concentration (After CaC12 Addition) (units/mL) Control rBPI2l rBPI21 (0.1 % HSA-TBS) ~ 1 ~bg/mL ~ 25 ~4g/mL

6 18 l9 45 For the clot dissolution part of this experiment, 53 minutes after CaC12 addition, all tubes were placed in a 37~C water bath and checked for clot dissolution/lysis by visual inspection. Time to clot dissolution in hours or minutes lO was evaluated. With no tPA, no clot Iysis was observed. A tPA dose response effect was observed, with the higher tPA concentration (60 units/mL) producing the most rapid clot dissolution. Under these conditions with this donor's plasma, rBPI21 had a much greater effect on the delay of clot formation while having minim~l effect on clot dissolution. It is a~a~ t that individual plasma donors can 15 have significantly dirr~,lcl~l clotting and dissolution times (compare results in parts A-C above). These dirre~ ces could be due to different concentrations of crucialclotting factors in individual donor plasma.

E. Effect of rBPI21 and tPA When Added Pre-Activation and Post-Activation Clot formation and Iysis were evaluated when both rBPI21 and tPA
were added prior to calcium addition (pre-activation or pre-calcium) and 4 minutes after calcium addition (post-activation or post-calcium). The following reagentswere mixed together in a polypropylene test tube in the following order: (1) 60 L4L of 0.1 % HSA-TBS; (2) 100 ,uL of PPP (Donor PC); (3) 84 ,~4L of rBPI21 (either 5, 25 or 125 ~4g/mL) with tPA (300 units/mL, 50 ng/mL), or buffer control (0.1% IISA-TBS); and (4) 200 ~L of 20 mM CaCl2 in TBS, pH 7.8.
The tubes were allowed to stand at room temperature, and were checked every 1 minute for gel clot formation by visual inspection. The minutes to clot formation after CaCl2 addition were measured.

CA 022~3836 1998-ll-09 W O 97/42967 PCTrUS97/08017 Minutes to Clot Formation Timing of~After CaC12 Addition) rBPI21 and tPA additionControl rBPI21 rBPI21 rBPI
(0.1% ~ I ,ug/mL ~ S ~g/mL ~ 25 ~bg/m~
HSA-TBS) Pre-Calcium 7 7 8 4 min Post- 7 7 8 8 Calcium In this experiment, 25 ,ug/mL rBPI21 slowed clot formation only when present prior to calcium addition. No significant effect was observed on rate of clot formation when the rBPI21 was added 4 minutes after calcium addition.
For the clot dissolution part of this experiment, 15 minutes after CaC12 addition, all tubes were placed in a 37~C water bath and checked approximately every minute for clot dissolution/lysis by visual inspection. The minutes to clot dissolution were measured.

Minutes to Clot Lysis Timing of(After Placement in Water Bath) 15rBPI21 and tPA Control rBPI21 rBPI21 rBPI21 addition (0.1 % ~ 1 ~g/mL ~ 5 ,ug/mL ~ 25 ,ug/mL
HSA-TBS) Pre- 58 54 53 51 Calcium 204 min Post- 50 45 44 40 Calcium Clot dissolution appeared to be faster when rBPI21 and tPA were added post-calcium compared to pre-calcium. A small rBPI21 dose-response effect on clot dissolution was deteGte(l for both pre- and post-calcium groups. These results 25 indicated that the timing of rBPI21 and tPA addition relative to calcium addition (clot activation) influenced clot forrnation and dissolution.
In an additional experiment using higher plasma concentrations, clot formation and Iysis were evaluated when rBPI21 and tPA were added pre-c~lcil-m and 3 minutes post-calcium. The following reagents were mixed together in a CA 022~3836 1998-11-09 wo 97/42967 PCT/US97/08017 polypropylene test tube in the following order: (1) 160 ~bL of PPP (Donor PC);

(2) 84 ,uL of BPI21 (either 5, 25 or 125~ug/mL) with tPA (300 units/mL, or 50 ng/mL) or buffer control (0.1% HSA-TBS); and (3) 200 ,uL of 20 mM CaC12 in TE~S, pH 7.8.
The tubes were allowed to stand at room temperature and were checked every 1 minute for gel clot formation by visual inspection. The minutes to clot formation after CaC12 addition were measured. In this experiment, 25 ~ug/mL rBPI21 slowed clot formation (~50% prolongation) when present prior to calcium addition. A slight effect ( ~ 22 % prolongation) was observed on clot 10 formation when 25 ~g/mL rBPI21 was added 3 minutes after calcium addition.
For the clot dissolution part of this experiment, 15 minutes after CaC12 addition, all tubes were placed in a 37~C water bath and checked for clot dissolution/lysis by visual inspection. The minutes to clot dissolution were measured. A small rBPI21 dose-response effect on clot dissolution was again detected for both pre- and post-calcium addition groups. Only minim~l differences in clot dissolution were observed between pre- and post-calcium addition of rBPI21 and tPA. Although a larger dirre.~,nce in clot dissolution between pre- and post-calcium addition groups was observed in a previous experiment, that result may have been due to the different plasma concentration utili7ed These results confirmed those of the previous e,.~el;l-lents that the timing of rBPI21 and tPAaddition relative to calcium addition (clot activation), as well as plasma concentration, influenced clot formation and dissolution.

F. Effect of Clottin,e Telllpelalule Clot formation and lysis were evaluated at several clotting temperatures. For the clot formation part of this experiment, the following reagents were mixed together in a polypropylene test tube in the following order:
(1) 60 ,uL of 0.1% HSA-TBS; (2) 100 ~L of PPP (Donor PC); (3) 40 ,~4L of rBPI21 (either 10, 50 or 250~bg/mL) or buffer control; (4) 44 ~L of tPA (600 units/mL, or 100 ng/mL) or buffer control (0.1% HSA-TBS); and (5) 200 ~bL of 20 mM CaC12 in TE~S, pH 7.8.

CA 022~3836 1998-11-09 W 097/42967 PCT~US97/08017 The tubes were incubated at room temperature (R.T.) or 37~C and were checked every 1 minute for gel clot formation by visual inspection. The minutes to clot formation after CaC12 addition were measured.

Minutes to Clot Formation Temperature(After CaC12 Addition) 5for clotting (Timing of tPA Control rBPI21 rBPI21 rBPI21 Addition) (0.1 %~ I ,ug/mL ~ S ~bg/mL ~ 25 ~g/mL
HSA-TBS) R.T. 7 7 8 10 (Pre-clot tPA) 10 37~C 5 5 6 8 (Pre-clot tPA) 37~C 5 5 6 7 (Post-clot tPA) Clot formation proceeded more rapidly at 37~C than at R.T. However, 25 ,ug/mL
rBPI21 delayed clot formation at both temperatures. Under the conditions tested in this ~e,i,.lent, the lower concentrations of rBPI21 did not appear to have aneffect on rate of clot formation.
For the clot dissolution part of this experiment, 15 minutes after CaC12 addition, 44 ~L of tPA (600 units/mL, or 1 ~g/mL) were added to post-clot tubes to provide a final concentration of 60 units/mL (or 100 ng/mL). The tubes were placed in a 37~C water bath and checked for clot dissolution/lysis by visual inspection. Time to clot dissolution in hours (for post-clot addition of tPA) or in minutes (for pre-clot addition of tPA) was measured.

CA 022~3836 l998-ll-09 W O 97/42967 PCTrUS97/08017 Minutes/Hours to Clot Lysis Temperature(After Placement in Water Bath) for clotting (Timing of tPA Control rBPI21 rBPI21 rBPI
Addition)(0.1% HSA- ~ 1 ~g/mL ~ 5 ,ug/mL --25 TBS) ,ug/mL
R.T. 38 min. 33 min. 37 min 38 min.
(Pre-clot tPA) 37~C 45 min. 30 min. 31 min. 29 min.
(Pre-clot tPA) 37~C 10-12 7-8 hours# 7-8 hours~ 6 hours#
(Post-clot tPA) hours#
- Time estim~ted based on cJot size at the 7 hour time point.
Clot Iysis was greatly accelerated when tPA was added pre-clot formation compared to post-clot formation. For pre-clot tPA addition, rBPI21 provided a greater acceleration of clot lysis time when the clot was formed at 37~C compared to at R.T.(~ 35 % versus ~ 3 %). All subsequent experiments were performed at 37~C.

G. Effect of BPI21 rBPI50. and rLBP
Clot formation and lysis were evaluated with rBPI21, rBPI50, and rLBP. The following reagents were mixed together in a polypropylene test tube inthe following order: (1) 60 ,uL of 0.1% HSA-TBS; (2) 100 ~L of PPP (Donor PC);(3) 40 ~L of rBPI21, rBPI50, or rLBP (either 10, 50 or 250 ,ug/mL) or buffer control; and (4) 200 ~L of 20 mM CaC12 in TBS, pH 7.8.
The tubes were incubated in a 37~C water bath and were checked every 1 minute for gel clot formation by visual inspection. The minutes to clot formation after CaC12 addition were measured.

CA 022~3836 1998-11-09 W O 97/42967 PCT~US97/08017 Minutes to Clot Formation (After CaCl2 Addition) Control 1 ~bg/mL 5 ~bg/mL of 25 ,ug/mL
Tested Protein(0.1% HSA- of tested tested of tested TBS) protein protein protein rBPI21 6 5 7 8 rBPI50 6 4 6 6 rLBP 6 5 5 5 25 ~g/mL rBPI21 slowed the rate of clot formation, while rBPI50 and rLBP
clotting times were compaMble to the controls.
For the clot dissolution part of this experiment, 15 minutes after CaC12 addition, 44,uL of tPA (600 units/mL, 1 ~g/mL) were added to all tubes to provide a final concentration of 60 units/mL (100 ng/mL). Then tubes were placed in a 37~C water bath and checked for clot dissolution/lysis by visual inspection. The minutes to clot dissolution were measured.

Hours to Clot Lysis (After Placement in Water Bath) Tested Protein Added Control 1 ~bg/mL of 5 ,ug/mL of 25 ~L~g/mL
(0.1% HSA-tested proteintested protein of tested TBS) protein rBPI21 13-14 7 7 7 15rBPI50 13-14 10 11 7 rLBP 13-14 13-14 8 11 * - Time estim~t~-d based on clot size at 11 hours.
At all concentrations tested, rBPI21 decreased clot Iysis time by a~lvxilllately50%. rBPI50 decreased clot Iysis by approximately 29% at 1 ,~4g/mL, 19% at 5 ~4g/mL and 505~ at 25 ,ug/mL. rLBP decreased clot Iysis time by approximately 57% at 5 ~g/mL, approximately 29 % at 25 ~ug/mL, and had no discernable effect at 1 ,ug/mL. These results show that, on a mass basis, rBPI21 was more potent than rBPI50 and rLBP in enhancing the effects of tPA on clot dissolution.

CA 022~3836 1998-ll-09 W 097/42967 PCTrUS97/08017 H. Effect of Presh Platelet Rich Plasma (PRP) and Platelet Poor Plasma (PPP) Clot formation and Iysis were evaluated in the presence and absence of rBPI21 with freshly collected platelet rich plasma (PRP) and platelet poor 5 plasma (PPP) from the same donor. The following reagents were mixed together in a polypropylene test tube in the following order: (1) 120 ~4L of fresh PRP
or PPP (Donor EL); (2) 80 ~L of BPI21 (either 5, 25 or 125 ~g/mL) or control (0.1 % HSA-TBS) and tPA (300 units/mL); and (3) 200 ~L of 20 mM CaC12 in TBS, pH 7.8.
The tubes were incubated in a 37~C water bath, and were checked every 1 minute for gel clot formation by visual inspection. The minutes to clot formation after CaC12 addition were measured.

Minutes to Clot Formation (After CaC12 Addition) Type of Plasma Control rBPI21 rBPI21 rBPI21 (0.1% HSA- 1 ,ug/mL S ~g/mL25 ~g/mL
TBS) Platelet Poor 21 24 36 55 15Platelet Rich 15 16 21 37 A direct rBPI21 dose-response effect on rate of clot formation was observed for both PPP and PRP; higher concentrations of rBPI21 produced greater prolongation of clot formation. For all conditions tested in this experiment, PRP
exhibited faster clotting times than PPP.
For the clot dissolution part of this eh~ ent, incubation was continued at 37~C without further addition of reagents. The minutes to clot dissolution were measured.

CA 022~3836 1998-ll-09 W O 97/42967 PCTrUS97/08017 Minutes to Clot Lysis (After CaCl2 Addition) Type of Plasma Control rBPI21 rBPI21 rBPI21 (0.1% HSA- 1 ,ug/mL 5 ,ug/mL 25 Ibg/mL
TBS) Platelet Poor 71 59 66 86 Platelet Rich 81 65 66 81 For all conditions tested, the fresh PRP had longer clot dissolution times than S fresh PPP. For each plasma type (PRP or PPP), clot dissolution was fastest at the 1 ~g/mL concentration of rBPI21. The time from clot formation to clot dissolution is calculated and shown below.

Duration of Clot (Time of Clot Pormation Subtracted From Time of Clot Lysis) Type of PlasmaControl rBPI21 rBPI21 rBPI21 (0.1% HSA- 1 ~g/mL 5 ,ug/mL 25 ~g/mL
TBS) Platelet Poor 50 35 30 31 10Platelet Rich 66 49 45 44 In this experiment, a dose response effect was observed for clot formation; increasing rBPI21 concentration resulted in increasing clotting times in both PRP and PPP. However, a rBPI21 dose response effect was not clearly observed for clot dissolution. Overall, faster clot dissolution times were observed l5 for rBPI21 at 1 ~g/mL. However, if the time between clot formation to clot dissolution is measured, then there are only modest dirÇelc;nces in clot dissolution times. From these results, it appeared that at lower rBPI21 concentrations, greater effects were observed on clot dissolution rather than clot formation. As rBPI
concentration was increased, greater effects were observed on clot formation ~ 20 rather than clot dissolution.

CA 022~3836 1998-11-09 wo 97/42967 PCT/USg7/08017 I. Effect of rBPI2l on Freshly Collected Blood Clot formation was evaluated with rBPI2~ and freshly collected blood. For this experiment, blood was collected into four siliconized 3 mL
Vacutainer~ tubes contAining either 50, 100, 200 ~g/ml rBPI21 or control 5 formulation buffer. Each tube was inverted several times after blood collection and placed on ice until blood had been collected for all tubes. (Collection time of each tube was less than thirty seconds.) All tubes were placed in a 37~C water bath and checked every one minute by gently tipping the tube and visually inspecting it for clot formation.
Minutes to Clot Formation (After Placement in 37~C Water Bath) Control rBPI21 rBPI21 rBPI
Type of Plasma(0-1% HSA-50 ~g/mL 100 200 TBS) ~4g/mL ~g/mL
Whole Blood 6 1 l 16 30 (Donor PM) Increasing rBPI21 concentration slowed the rate of clot formation in a dose dependent manner, but did not completely prevent clot formation.

EXAl\~PLE: 2 EFFECTS OF BPI PROl~N PRODUCTS ON CLOT FORMATION
AND CLOT LYSIS IN MICROTITER PLATE ASSAYS
A 96-well microtiter plate assay was used to evaluate the effects of a BPI protein product on clot formation under a variety of conditions using human plasma samples. These assays confirmed the results of tube assays described in Example l. For these assays, human plasma, either PRP or PPP, was prepared as described in Example 1.
For the plate-based assay, all experiments were conducted at 37~C
and total volume per well was ~ 200 ~L in the presence or absence of tPA as a pre-clot addition or ~ 250 ,uL where tPA (50 ,uL) was added post-clot formation to the 200 ~L cont~inin~ well.

CA 022~3836 1998-11-09 Wo 97/42967 PCT/USg7/08017 A. Effect of BPI Protein Product and Pre-Clot Addition of tPA: Fresh PRP and PPP
Clot formation was evaluated when rBPI21 and tPA were added to fresh PRP or PPP prior to calcium addition. The following reagents were added 5 to each well of a 96 well microtiter plate (e.g., Dynatec, Chantilly, VA, CoStar, Cambridge, MA) in the following order: (1) 60 ,uL of 0.1% HSA -TBS; (2) 50 ~uL of fresh PRP or PPP (Donor PM); (3) 20 ,llL of rBPI21 (1000, 250, 50, 10 or 2 ,ug/mL); (4) 20 ~uL of tPA (600 units/mL, 100 ng/mL); and (5) 50 ~L of 20 mM
CaC12 in TBS, pH 7.4.
Immediately after CaC12 addition, the turbidity of the wells was measured as the optical density at 405 nm at various times (in this experiment at 2 minute intervals for 2 hours) by using an automatic plate reader (Vmax Plate Reader, Molecular Devices, Menlo Park, CA). The entire plate was sc~nne~
within 5 seconds. The OD405 versus time data at 2 minute intervals over a 2 hour period was plotted. The rate of clot forrnation was measured as a change inOD405 over time, i.e., as the clot forrned the OD405 increased. After the peak OD405 was achieved, the pre-clot addition of tPA allowed dissolution of the clotas measured by a decrease in OD405 over time.
In this experiment, rBPI21 completely prevented clot formation of both PRP and PPP at concentrations of 5, 25 and 100 ~g/ml. At 1 ,~4g/mL, rBPI21 effectively delayed time to clot as measured by a delay in tirne to peak OD405 levels [clot time] and a decrease in peak OD405 intensity [clot density].
At 0.2 ,L~g/mL, rBPI21 clot formation time was comparable to the control.

B. Effect of Various BPI Protein Products and Control Protein with Pre-Clot Addition of tPA:Frozen PPP
Clot formation was evaluated when rBPI21, rBPI50, rBPI42, LBP
and th;lumatin (a control cationic protein having a similar size and charge as rBPI21) and tPA were added to PPP prior to calcium addition. The following reagents were added to each well of a 96 well microtiter plate in the following order: (1) 60 ,uL of 0.1 % HSA - TBS; (2) 50 ,uL of PPP that had been frozen at -70~C (Donor PM); (3) 20 ~L of protein product (rBPI21, rBPI50, rBPI42, LBP

CA 022~3836 1998-11-09 Wo 97/42967 PCT/US97/08017 and th~llm~tin each at 1000, 250, 50, 10 or 2 ~g/mL); (4) 20 ~L of tPA (600 units/mL, 100 ng/mL); and (5) 50 ,uL of 20 mM CaC12 in TBS, pH 7.4.
Tmmerli~tely after CaC12 addition, the turbidity of the wells was measured as the OD405 at various times as described in part A above. In this S experiment, the time to clot formation was determined as the minutes to clot formation as measured by the time at which the OD405 had reached its peak value.

Minutes to Clot Formation Protein 0.2 1.0 5.0 25.0 100.0 Product Control ,ug/mL ~bg/mL ~g/mL,ug/mL llg/mL

BPI21 34 32 34 50 Sot No clot rBPI50 34 34 34 40 46t No clot rBPI42 40 28 24 20 30t No clot Th~llm~tin40* 32 34 32 34 34 t peak height subst~nti~lly decreased (e.g., 3-4 fold) from control * higher effective concentration of citrate in formulation buffer control At 100 ~g/mL, rBPI21, rBPI50, rBPI42 completely prevented clot formation (as measured by no detect~ble increase in OD405 over the 2 hour kinetic plate reader20 analysis), and at 25 ,ug/mL, these BPI protein products subst~nti~lly prevented clotting (as measured by a slight OD405 increase over the 2 hours). At 5 ~g/mL, rBPI21 and rBPI50 effectively delayed time to clot as measured by a delay in time to peak OD405 levels. LBP and th~llm~tin did not affect time to clot fonnation in this experiment. The lack of effect by the cationic control protein th~um~tin 25 indicated that the delay to clot formation by BPI protein products was not simply a charge effect due to their cationic pl~e,lies.

CA 022~3836 1998-11-09 W O 97142967 PCT~US97/08017 C. Effect of BPI Protein Product and Pre-Clot Addition of Various Plasminogen Activators: Frozen PPP
Clot formation was evaluated when rBPI21 and a plasminogen activator (tPA, urokinase or streptokinase) were added to PPP prior to calcium 5 addition. The following reagents were added to each well of a 96 well microtiter plate in the following order: (1) 60 ~L of 0.1% HSA -TBS; (2) 50 ~L of PPP
that had been frozen at -70~C (Donor PM); (3) 20 ~L of rBPI21 (1000, 250, 50, 10 or 2 ~g/mL); (4) 20 ,uL of PA (tPA, 1000 ng/mL; for urokinase, 100 and 1000 ng/mL; for streptokinase, 100 and 1000 ng/mL); and (5) 50 ,uL of 20 mM CaC12 10 in TBS, pH 7.4.
Immediately after CaC12 addition, the turbidity of the wells was measured as the OD405 as described in part A above. In this experiment, rBPI
at 100 ~ug/mL completely prevented clot formation (as measured by no detect~hle increase in OD405 over the 2 hour kinetic plate reader analysis), and at 25 ~bg/mL, it subst~nti~lly prevented clotting under conditions where tPA, urokinase or streptokinase was present in the pre-clot mixture. Effects on decreasing clotformation were observed with S ,ug/mL rBPI21. At 1 and 0.2 ~g/mL, rBPI21 clot formation time was comparable to the control. At the concentrations of urokinasetested (10 and 100 ng/mL), clot dissolution did not occur following clot formation, as it did with 100 ng/mL tPA and 100 ng/mL (but not 10 ng/mL) streptokin~e.

D. Effect of BPI Protein Product on Plasma from Various Donors: Fresh PPP
Clot forrnation was evaluated when rBPI21 (but not tPA) was added to fresh PPP prior to calcium addition. The following reagents were added to each well of a 96 well microtiter plate in the following order: (1) 80 ,~4L of 0.1%
HSA -TBS; (2) 50 ~L of fresh PPP (Donors MW, RD and RL); (3) 20 ,llL of rBPI21 (1000, 250, 50, 10 or 2 ,ug/mL); and (4) 50 ~L of 20 mM CaC12 in TBS, pH 7.4.
Immediately after CaC12 addition, the turbidity of the wells was ~ 30 measured as the OD405 as described in part A above. In this experiment, rBPI
at various concentrations slowed or prevented clot formation. Some individual CA 022~3836 1998-ll-09 variation was observed in the response among the three donor plasma samples simultaneously tested. For all three donors, rBPI21 at 25 and 100 ,llg/mL
completely prevented clot formation of fresh PPP. At 5 ,ug/mL, rBPI21 also completely prevented clot formation of the PPP from one donor (RL) and S subst~nti~lly prevented clotting of the PPP from the other two donors (MW and RD). At I ,ug/mL, rBPI21 substantially prevented clotting of the PPP from one donor (RL), and reduced clotting was observed in the PPP of the other two donors(MW and RD). A slight effect was observed even at 0.2 ~g/mL rBPI21 with one donor; generally at 0.2 ~g/mL rBPI21, clot formation was comparable to control.

10 E. ~;ffect of Multiple BPI Protein Products After Thrombin-Driven Clot Formation and Post-Clot tPA Addition: Fresh PPP
Clot Iysis was evaluated when rBPI21 and tPA were added to fresh PPP with 0.125 units/mL thrombin and calcium. The following reagents were added to each well of a 96 well microtiter plate in the following order: (1) 80 ,~L
of 0.1% HSA -TBS; (2) 50 ~uL of PPP that had been frozen at -70~C (Donor PM);

(3) 20 ,uL of rBPI21 or rBPI42 (50, 10 or 2 ~g/mL); and (4) 50 ~4L of 20 mM
CaC12 in TBS, pH 7.4 with 0.5 units/mL thrombin.
After thrombin and CaC12 addition, clot formation was allowed to occur and 50 ~4L of tPA (300 units/mL, 500 ng/mL) with 0.5 units/mL of heparin 20 in TBS, pH 7.4 were added. After clot formation and tPA addition (~ 15 minutes), the turbidity of the wells was measured as the OD405. In this initial experiment with thrombin-driven clot formation, rBPI21 or rBPI42 with post-clot addition of tPA allowed dissolution of the clot as measured by a decrease in OD405 over time. Increasing the concentration of rBPI21 or rBPI42 (0.2, 1 and 5 25 ~g/mL) with a constant concentration of tPA (100 ng/mL) resulted in enhanced clot dissolution (i.e., the rate at which the OD405 decreased was ~2-4 fold faster).

CA 022~3836 1998-11-09 W O 97/42967 rCTrUS97/08017 EFFECTS OF BPI Pl~Ol~N PRODUCT ON tPA-INDUCED CLOT LYSIS
IN A RAT THROMBOSIS MODEL
A rat thrombosis model was used to determine the effects of a BPI
5 protein product (rBPI21) with a thrombolytic agent ~tPA) on clot lysis and reocclusion after thrombolytic therapy. The methods of Klement et al., l~rombosls Haemostasis, 68:64-68 (1992), were modified as described herein to determine the effects of rBPI21 in 5 mM citrate, 150 mM NaCI, 0.2% poloxamer 188, 0.002% polysorbate 80 on tPA-induced clot Iysis in vivo. In this experiment, 10 two groups of rats (5 for the vehicle-treated group and 5 for the BPI21-treated group) were anestheti7ed with k~t~min~/Rompum, and catheters were placed in both jugular veins for adminictration of therapeutic agents. The right common carotid artery was e~nnul~tP,d to measure blood pressure and heart rate. A midline incision was made in the lower abdomen to expose the terminal aorta and iliac 15 vessels, which were tli~sectPd free of connective tissue and separated from the vena cava, where possible, and the great veins. All of the numerous small branches from the aorta that were observed were ligated in order to isolate a section of the vessel approximately 1 cm in length. The left iliac artery was c~nnul~t~d with a 20 gauge blunt needle sealed with a rubber septum. An 20 ultrasonic flow transducer cuff was placed around the right iliac artery in order to measure blood flow with a flow meter [Crystal Biotech, Holliston, MA]. The flow signal was recorded on a chart recorder.
The following procedures were carried out in order to injure the aorta and thus provide a surface for clot formation. The 20 gauge needle was 25 advanced into the aorta and slowly moved against the vessel wall along a 1 to 1.5 cm length. This was repeated 8 times. The needle was removed and the same area of the aorta was pinched 4 times with smooth forceps. Subsequently, an area of stenosis was produced in the terminal aorta by gently tightening siL~ thread at two locations. Right iliac blood flow was measured at this time and de.cign~tPd 30 as the "pre-occlusion" level.
At this point, the rBPI21-treated rats were ~lmini~ctered 20 mg/kg rBPI21 as a 2 mg/m~ solution in 5 mM citrate 0.2% poloxamer 188, 150 mM

CA 022~3836 1998-11-09 W 097/42967 PCT~US97/08017 NaCI (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% polysorbate 80 (Tween 80, ICI Americas Inc., Wilmington, DE), pH 5 as a bolus over 30 seconds (4 mL/kg) intravenously into one of the jugular veins, followed by a constant infusion of rBPI21 at 20 mg/kg/hr that was continued until the end of the 5 experiment. The vehicle-treated rats received the same volumes; the vehicle solution was 5 mM citrate 150 mM NaC1, 0.2% poloxamer 188, (Pluronic F-68, BASF Wyandotte, Parsippany, NJ) and 0.002% polysorbate 80 (Tween 80, ICI
Americas Inc., Wilmington, DE), pH 5. Next, the right iliac artery was occluded with a small metal clip placed just above the blood flow cuff. Since the left iliac 10 artery was already sealed by the needle catheter, blood was then trapped above the occlusion. Finally, the aorta was occluded above the injured site, producing a stenotic area with stasis. The occlusions were m~int:-ined for 30 minutes and then removed. Confirmation that a clot had formed was provided by the failure to record flow through the right iliac artery.
Five minutes after removal of the occlusions, t-PA was ~dminictered into the other jugular vein (in which rBPI21 had not been atlmini~tered) as a bolus of 1 mg/kg followed by an infusion of 1 mg/kg over 1 hour. After 1 hour, both the tPA and the rBPI21 (or vehicle) infusions were discontinued and the experiment was te~ ed. Clot Iysis was defined as a return of blood flow 20 through the right iliac artery to 50% of the pre-occlusion level. The length of time until clot Iysis was recorded, as well as the time until the first reocclusion after the initial clot lysis. The number of times that the vessel became reoccluded over the 1 hour period of t-PA treatment was also recorded. Reocclusion was defined as a reduction in blood flow to 10% of the pre-occlusion level. St7~ti~ti('~
25 comparisons between the buffer- and rBPI21-treated groups were done with Student's T test. Neither t-PA nor rBPI21 produced any adverse systemic effects,other than a rise in blood pressure due solely to the aortic occlusion.
The results revealed no significant difference in the mean time to clot lysis, which was 14.4 + 5.1 minutes for the buffer-treated group and 16.1 +30 4.2 minutes for the rBPI21-treated group. Multiple reocclusion episodes occurred in all rats. The time to the first reocclusion was not significantly dirre~nl between the two groups. However, the number of reocclusions was st~ti~ti~ ~lly CA 022~3836 1998-11-09 Wo 97/42967 PCT/USg7/08017 significantly reduced in rBPI21-treated rats (p<0.05); the rBPI21-treated rats averaged 4 + 1.6 reocclusion episodes, while the buffer-treated rats averaged 9 +
1.7 reocclusion episodes.

Numerous modifications and variations of the above-described 5 invention are expected to occur to those of skill in the art. Accordingly, only such limitations as appear in the appended claims should be placed thereon.

CA 022~3836 l998-ll-09 W 097/42967 PCTrUS97/08017 SEQUENCE LISTING

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~ Ammons, William Steve (ii) TITLE OF lNv~NllON: Antithrombotic Materials and Methods (iii) NUMBER OF SEQUENCES: 2 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Marshall, O'Toole, Gerstein, Murray & Borun (B) STREET: 6300 Sears Tower, 233 South Wacker Drive (C) CITY: Chicago (D) STATE: Illinois (E) COUNTRY: United States of America (F) ZIP: 60606-6402 (v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) CO~I~ul~: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Relea6e #1.0, Version #1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) All~k~Y/AGENT INFORMATION:
(A) NAME: Jeffrey S. Sharp (B) REGISTRATION NUMBER: 31,879 (C) R~:~R~NG~:/DOCKET NUMBER: 27129/33249 PCT
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 312/474-6300 (B) TELEFAX: 312/474-0448 (C) TELEX: 25-3856 (2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1813 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA

(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 31..1491 (ix) FEATURE:
(A) NAME/KEY: mat peptide (B) LOCATION: 124..1491 CA 022~3836 l998-ll-09 W 097/42967 PCTrUS97/08017 (ix) FEATURE:
(A) NAME/KEY: misc_feature (D) OTHER INFORMATION: "rBPI"

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
CA&GCCTTGA GGTTTTGGCA GCTCTGGAGG ATG AGA GAG AAC ATG GCC AGG GGC 54 Met Arg Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val Asn Pro Gly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Glu Phe Gln Leu Pro Ser Ser Gln Ile Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser Ile Ser Asn Ala Asn Ile Lys Ile Ser Gly AAA TGG AAG GCA CAA AAG AGA TTC TTA A~A ATG AGC GGC AAT TTT GAC 438 Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser CAC ATC AAC AGT GTC CAC GTG CAC ATC TCA AAG AGC A~A GTC GGG TGG 582 His Ile Asn Ser Val His Val His Ile Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys Ile Glu Ser Ala Leu Arg Asn Lys ATG AAC AGC CAG GTC TGC GAG A~A GTG ACC AAT TCT GTA TCC TCC AAG 678 Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys CTG CAA CCT TAT TTC CAG ACT CTG CCA GTA ATG ACC A~A ATA GAT TCT 726 Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met Thr Lys Ile Asp Ser CA 022~3836 l998-ll-09 W 097/42967 rCTAUS97/08017 Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro Pro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Arg Leu Thr Thr ~ys Phe Phe Gly Thr Phe Leu Pro Glu Val Ala Lys Lys Phe Pro Asn Met Lys Ile Gln Ile His Val Ser Ala Ser Thr Pro Pro His Leu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu Leu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu Leu Leu Gln Asp Ile Met Asn Tyr Ile Val CCC ATT CTT GTG CTG CCC AGG GTT AAC GAG AAA CTA CAG A~A GGC TTC 1398 Pro Ile Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val Val Tyr Lys TGAAGGCACC AGGGGTGCCG GGGGCTGTCA GCCGCACCTG TTCCTGATGG G~l~lGGGGC 1551 ACCGGCTGCC TTTCCCCAGG GAAlC~l~lC CAGATCTTAA CCAAGAGCCC CTTGCAAACT 1611 CA 022~3836 1998-11-09 W O 97/42967 PCT~US97/08017 AACTTCTGGT ~ CATG TG 1813 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 487 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Arg Glu Asn Met Ala Arg Gly Pro Cys Asn Ala Pro Arg Trp Val Ser Leu Met Val Leu Val Ala Ile Gly Thr Ala Val Thr Ala Ala Val ~sn Pro Gly Val Val Val Arg Ile Ser Gln Lys Gly Leu Asp Tyr Ala Ser Gln Gln Gly Thr Ala Ala Leu Gln Lys Glu Leu Lys Arg Ile Lys Ile Pro Asp Tyr Ser Asp Ser Phe Lys Ile Lys His Leu Gly Lys Gly His Tyr Ser Phe Tyr Ser Met Asp Ile Arg Glu Phe Gln Leu Pro Ser ~er Gln Ile Ser Met Val Pro Asn Val Gly Leu Lys Phe Ser Ile Ser ~sn Ala Asn Ile Lys Ile Ser Gly Lys Trp Lys Ala Gln Lys Arg Phe Leu Lys Met Ser Gly Asn Phe Asp Leu Ser Ile Glu Gly Met Ser Ile Ser Ala Asp Leu Lys Leu Gly Ser Asn Pro Thr Ser Gly Lys Pro Thr Ile Thr Cys Ser Ser Cys Ser Ser His Ile Asn Ser Val His Val His ~le Ser Lys Ser Lys Val Gly Trp Leu Ile Gln Leu Phe His Lys Lys ~le Glu Ser Ala Leu Arg Asn Lys Met Asn Ser Gln Val Cys Glu Lys Val Thr Asn Ser Val Ser Ser Lys Leu Gln Pro Tyr Phe Gln Thr Leu Pro Val Met Thr Lys Ile Asp Ser Val Ala Gly Ile Asn Tyr Gly Leu Val Ala Pro Pro Ala Thr Thr Ala Glu Thr Leu Asp Val Gln Met Lys CA 022~3836 l998-ll-09 W 0 97/42967 PCTtUS97tO8017 Gly Glu Phe Tyr Ser Glu Asn His His Asn Pro Pro Pro Phe Ala Pro ~ro Val Met Glu Phe Pro Ala Ala His Asp Arg Met Val Tyr Leu Gly Leu Ser Asp Tyr Phe Phe Asn Thr Ala Gly Leu Val Tyr Gln Glu Ala Gly Val Leu Lys Met Thr Leu Arg Asp Asp Met Ile Pro Lys Glu Ser Lys Phe Arg Leu Thr Thr Lys Phe Phe Gly Thr Phe Leu Pro Glu Val ~la Lys Lys Phe Pro Asn Met Lys Ile Gln Ile His Val Ser Ala Ser ~hr Pro Pro His Leu Ser Val Gln Pro Thr Gly Leu Thr Phe Tyr Pro Ala Val Asp Val Gln Ala Phe Ala Val Leu Pro Asn Ser Ser Leu Ala Ser Leu Phe Leu Ile Gly Met His Thr Thr Gly Ser Met Glu Val Ser Ala Glu Ser Asn Arg Leu Val Gly Glu Leu Lys Leu Asp Arg Leu Leu ~eu Glu Leu Lys His Ser Asn Ile Gly Pro Phe Pro Val Glu Leu Leu ~ln Asp Ile Met Asn Tyr Ile Val Pro Ile Leu Val Leu Pro Arg Val Asn Glu Lys Leu Gln Lys Gly Phe Pro Leu Pro Thr Pro Ala Arg Val Gln Leu Tyr Asn Val Val Leu Gln Pro His Gln Asn Phe Leu Leu Phe Gly Ala Asp Val Val Tyr Lys , . .... .

Claims (18)

WHAT IS CLAIMED IS:

1. A method for slowing clot formation in blood comprising administering to a subject a BPI protein product in an amount effective to delay or prevent clot formation in the blood.

2. A method for enhancing clot dissolution in blood comprising administering to a subject a BPI protein product in an amount effective to enhance clot dissolution in the blood.

3. A method for treating a thrombotic disorder selected from the group consisting of arterial thrombosis coronary artery thrombosis, myocardial infarction, cerebral artery thrombosis, stroke, intracardiac thrombosis, peripheral arterial thrombosis or occlusion, venous thrombosis, pulmonary embolism, thrombosis associated with exposure of blood to a foreign or injured tissue surface, hypercoagulability, non-endotoxin-associated coagulopathies, andnon-endotoxin-associated disseminated intravascular coagulopathy, comprising administration of a pharmaceutically effective amount of a BPI protein product to a subject suffering from the thrombotic disorder.

4. A method for treating a thrombotic disorder selected from the group consisting of arterial thrombosis, coronary artery thrombosis, myocardial infarction, cerebral artery thrombosis, stroke, intracardiac thrombosis, peripheral arterial thrombosis or occlusion, venous thrombosis, pulmonary embolism, thrombosis associated with exposure of blood to a foreign or injured tissue surface, hypercoagulability, non-endotoxin-associated coagulopathies, andnon-endotoxin-associated disseminated intravascular coagulopathy, comprising co-administration of a pharmaceutically effective amount of a BPI protein product and a thrombolytic agent to a subject suffering from the thrombotic disorder.

5. The method of claim 4 wherein the amount of the thrombolytic agent is less than that required for a desired pharmaceutical effect when the thrombolytic agent is administered as a monotherapy.

6. A method for enhancing reperfusion or reducing reocclusion in a subject treated with a thrombolytic agent comprising co-administration of apharmaceutically effective amount of a BPI protein product and the thrombolytic agent.

7. A method for decreasing the dose of a thrombolytic agent required to establish reperfusion or to reduce reocclusion in a subject comprising co-administration of a BPI protein product and a thrombolytic agent, the dosage of the thrombolytic agent being less than that required for a desired pharmaceutical effect when the thrombolytic agent is administered as a monotherapy.

8. A method of slowing clot formation in blood comprising contacting the blood with an amount of BPI protein product effective to delay orprevent clot formation in the blood.

9. A method for enhancing clot dissolution in blood comprising contacting the blood with an amount of BPI protein product effective to dissolve or lyse the clot.

10. The method of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9 wherein the BPI protein product is an amino-terminal fragment of BPI protein having a molecular weight of about 21 kD to 25 kD.

11. The method of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9 wherein the BPI protein product is rBPI23 or a dimeric form thereof.

12. The method of claims 1, 2, 3, 4, 5, 6, 7, 8 or 9 wherein the BPI protein product is rBPI21.

13. Use of a BPI protein product in the preparation of a medicament for the treatment of a thrombotic disorder selected from the group consisting of arterial thrombosis, coronary artery thrombosis, myocardial infarction, cerebral artery thrombosis, stroke, intracardiac thrombosis, peripheral arterial thrombosis or occlusion, venous thrombosis, pulmonary embolism, thrombosis associated with exposure of blood to a foreign or injured tissue surface, hypercoagulability, non-endotoxin-associated coagulopathies, and non-endotoxin-associated disseminated intravascular coagulopathy.

14. Use of a BPI protein product in the preparation of a medicament for co-administration with a thrombolytic agent in the treatment of athrombotic disorder selected from the group consisting of arterial thrombosis, coronary artery thrombosis, myocardial infarction, cerebral artery thrombosis, stroke, intracardiac thrombosis, peripheral arterial thrombosis or occlusion, venous thrombosis, pulmonary embolism, thrombosis associated with exposure of blood to a foreign or injured tissue surface, hypercoagulability, non-endotoxin-associated coagulopathies, and non-endotoxin-associated disseminated intravascular coagulopathy.

15. Use of a BPI protein product in the preparation of a medicament for enhancing reperfusion or reducing reocclusion in a subject treated with a thrombolytic agent.

16. Use of a BPI protein product in the preparation of a medicament for slowing clot formation in blood.

17. Use of a BPI protein product in the preparation of a medicament for enhancing clot dissolution in blood.

18. The use according to any one of claims 13 through 17 wherein the BPI protein product is selected from the group consisting of amino-terminal fragments of BPI protein having a molecular weight of about 21 kD to 25 kD, rBPI23 or dimeric forms thereof, and rBPI21.