WO1989012680A1

WO1989012680A1 - USE OF MODIFIED tPA TO DETECT BLOOD CLOTS

Info

Publication number: WO1989012680A1
Application number: PCT/US1989/002656
Authority: WO
Inventors: Burton E. Sobel
Original assignee: Washington University
Priority date: 1988-06-20
Filing date: 1989-06-16
Publication date: 1989-12-28
Also published as: AU3876789A

Abstract

Modified forms of tPA which lack clot-dissolving activity but retain their ability to bind fibrin are useful as carriers of scintographic labels to permit the detection and location of blood clots in the circulation system of animals. These tracers can be injected intravenously, and then detected by scintographic imaging.

Description

USE OF MODIFIED tPA TO DETECT BLOOD CLOTS

Technical Field

The invention relates to diagnosis of subjects with circulation problems, specifically detection of blood clots. More particularly, it concerns the use of an enzymatically inactive form of tissue plasminogen activator bearing a scintographic label to locate the clot and permit its detection.

Background Art

The present standard procedure for detection of blood clots is angiography, an invasive procedure requiring catheterization which is not only traumatic, but can lead to bleeding complications and can interfere with subsequent therapy (Rao, A.K., et al, J Am Coll Cardiol (1988) 1_1:1-11). It has been attempted to find alternatives using radiopharmaceuticals as clot-imaging agents. These agents include iodine-125 labeled fibrinogen (Kakkar, B.V., et al, Lancet (1970) 1_:540-542) ; indium-Ill labeled platelets (Riba, A.L., et al, Circulation (1979) 6j0:767-775; Yamada, M. , et al, Brit Heart J (1984) !51.:298-305; Thakur, M.L., Thromb Res (1976) 345-357); technetium 99 and indium-11 labeled plasmin (Persson, B. , et al, Radiopharmacol (1981) ^:1<*7-156); monoclonal antibodies or fragments, specific for platelets labeled with technetium 99 (Som, P., et al, J Nucl Med (1986) 1315-1320; Oster, Z.H., et al, Proc Natl Acad Sci (USA) (1985) 82_:3465-3468; Peters, A.M., et al, Brit Med J (1986) 293;1525-1527) , monoclonal antibodies raised against fibrin and labeled with iodine-125 (Liau, C.S., et al, (1987) :49-54); and streptokinase labeled with iodine-131 (Goodman, L.R., et al, Invest Radiol (1973) 8:377-383). Indium-Ill labeled platelets and technetium 99 labeled red blood cells have also been studied (Fox, K.A.A. , et al, J Am Coll Cardiol (1984) 4_:975-986).

Attempts have been made to use tissue plas inogen activator as a carrier for label. Indium-Ill labeled recombinant tPA has been considered as a potential imaging agent (Hnatovich, D.J., et al, Eur J Nucl Med (1987) L3:467-473). The use of this enzymatically active form has the disadvantage of simultaneously dissolving the clot, thus blurring the image, and this form is furthermore more likely to become complexed with plasminogen activator inhibitors circulating in the blood, further complicating the results. The use of modified tissue plasminogen activators which have enhanced fibrin- binding characteristics has been suggested by Pannekoek, H., et al, EPO publication no. 234051, published 2 September 1987; however, enzymatic activity in the modified forms of tPA suggested by these workers is retained.

The ability to detect the presence and location of clots in the context of apparent heart attack is, of course, important. If the damage is due to causes other than clots, the expensive and potentially dangerous therapy of clot dissolution can be avoided. On the other hand, the detection of the presence of thrombi permits the administration of the appropriate therapy with confidence. Accordingly, the methods and materials of the present invention, which offer an improved and specific method to detect the presence of thrombi in animal subjects, offer important diagnostic tools for patients with cardiovascular difficulties.

Disclosure of the Invention

The invention provides methods and materials which permit the identification and localization of thrombi occurring in the circulatory system of individuals without simultaneously blurring the image by disintegration of the clot. The clot is labeled using a radioactive isotope which is detectable by ex vivo detectors and is homed to the clot by virtue of a fibrin- binding agent which lacks the enzymatic activity to dissolve the clot. This fibrin-binding agent is a modified form of tissue plasminogen activator which lacks the enzymatic activity normally associated with this molecule.

In one aspect, the invention is directed to a method to detect blood clots in animal subjects which comprises administering to the animal a composition comprising a tissue plasminogen activator (tPA) molecule modified so as to lack proteolytic activity and conjugated to a label detectable by ex vivo scintographic imaging. The label is bound, preferably covalently, to the tPA, which binds specifically to the clot, but can be rapidly cleared from the system. In other aspects, the invention is directed to pharmaceutical compositions containing the labeled modified tPA and to the modified labeled tPA analogs, themselves.

Brief Description of the Drawings

Figure 1 shows a diagram of tissue plasminogen activator and the location of the various domains.

Figure 2 shows the amino acid sequence of tPA and the cDNA encoding it. Modes of Carrying Out the Invention

A. Definitions

As used herein, "tissue plasminogen activator" or "tPA" refers to the peptide sequence, described by Goeddel, D.B, et al, European application 0093619, published in 1983. The basic peptide structure disclosed in this reference is shown for convenience in Figures 1 and 2. It is well understood that this sequence is glycosylated in its native form, and when produced recombinantly in non-bacterial systems. As used herein, tPA includes substances related to the illustrated peptide sequence regardless of the level and nature of glycosylation so long as ability to bind fibrin or otherwise to bind to blood clots in maintained.

As indicated in Figure 1, the protein sequence comprises, starting at the N-terminus, a finger (F) region, two kringle regions ("K-l" and "K-2"), and a serine protease region at the C-terminus. The numbering used herein refers to the numbering of the peptide sequence as shown in Figure 1. It is also known that tPA is converted by plasmin or trypsin into a two-chain form wherein the two chains are linked through a disulfide bond. The N-terminal portion of the chain, MW 39 kd, contains the F and K-l and K-2 regions; the C-terminal portion, MW 33 kd, contains the serine protease activity.

The secondary structure shown in Figure 1 has been proposed by Pennica, D., et al. Nature (1983) 301:214-221. In addition to the native sequence referenced above, the tPA protein from which the invention modifications are made may be itself altered in amino acid sequence in nonsubstantial ways which do not materially diminish the ability of the tPA to bind to fibrin.or to blood clots otherwise. Numerous alterations of this native structure have been proposed by others for various reasons. For example, EP application 231,624, published 12 August 1987 to Upjohn, and incorporated herein by reference, discloses and claims a number of tPA analogs, all of which, however, contain an active enzyme site.

This series of analogs is included within the scope of the definition of tPA herein. Other publications incorporated herein by reference are as follows: EP publication 231,883, published 12 August 1987 to Saga i Chemical Research Center and several other applicants, describes hybrid plasminogen activators containing enzymically active domains from prourokinase polypeptides. The modified tPA portions of these hybrids are also included within the definition. European application publication no. 225,286, published 10 June 1987 and assigned to Ciba- Geigy, discloses a number of mutants which lack one or more glycosylation sites. Other enzymatically active mutants are disclosed in EPO publication no. 227,462, published 1 July 1987 assigned to Chiron. Addition of a finger domain while retaining enzymatic activity has been described in Kokai 289181/87, assigned to the Beacham Group.

The literature describing modifications of the basic molecule while retaining enzymatic activity is vast, and it is known that the fibrin-binding capacity of this molecule can be enhanced or modified in a variety of ways. All of these modified forms are included in the basic tissue^"plasminogen activator molecule to be modified according to the invention to destroy enzymatic activity so long as they retain the desirable characteristics of capability of specific fibrin or blood clot binding and acceptable clearance rates from the circulation, lack of immunogenicity, and enzymic inactivity. B. Radionuc1ides

Radionuclides which are detectable by ex vivo scintigraphy are those whose presence can be detected at their location inside the body by means of counting equipment, typically scintillation counting equipment placed outside the body. In general, the results of the ionizing radiation are recorded photographically; hence, the term "scintigraphy", and typically the manner of imaging the position of the radionuclide would be handled in this manner. However, this term is used for convenience and is not meant to exclude, necessarily, other methods of obtaining a scan of the body which may be recorded in other forms by utilizing appropriate algorithms in connection with an array of detectors. In order to detect the location of label ex vivo, the radiation must be capable of penetrating the skin. Hence, the radionuclide must emit gamma or positron radiation, or extremely high energy beta particles. Weak beta emitters, such as carbon-14 and tritium are not included. However, useful radionuclides include indium- Ill, cobalt-60, technetium 99 and iodine-125. These isotopes have been used in medical imaging for some years, and techniques for attaching them to the appropriate specific homing molecule are well known. See, for example, Hnatowich, D.J., et al, Eur J Nucl Med (1987) 1^3:467-473 (supra); Halpern, S.E., et al. Cancer Res (1983) 4_3_:5347; Hnatowich, D.J., et al, Int J Appl Radiat Isot (1982) 33_: 321 Krejcarek, C.E., et al, Biochem Biophvs Res Comm (1977) IJ SiM - Meares, C.F., et al. Anal Biochem (1984) 142:68. In the context of the present invention, these radionuclides will be conjugated, using techniques known per se, to the modified tPA molecules of the invention to provide the specific conjugate used for imaging. C. Appropriate tPA Molecules

The modified tPA of the present invention is inactive enzymatically in activating plasminogen to plas in, but retains its ability to bind fibrin or other material characteristic of blood clots. In addition, it has desirable qualities which should be present to an extent which enhance the practicality of the method. These include a short half-life in the circulation, in order to avoid large amounts of radioactivity in the blood pool; lack of antigenicity; inability to bind to circulating inhibitors or to endothelial cells; sufficient clearance to prevent interference with subsequent therapeutic fibrinolysis with tPA or other activators and ability to be conjugated conveniently to a level of high specific activity with the appropriate radionuclides, such as technetium 99 or other isotopes suitable for gamma camera scintillation detection or positron emission tomography. The molecules of tPA should not be adversely affected by the radiation. While the tPA should be, in general, specific for fibrin, cross-reactivity with other cells and substances associated with blood clots such as activated platelets is beneficial rather than harmful.

The modified tPA may be produced conveniently by manipulation of the gene and construction of expression vectors for recombinant production of the protein. While production in bacteria is theoretically possible, clearly production in eukaryotes is preferred as the molecule requires a certain amount of host translational processing, and glycosylation may be helpful. While it is known that the deglycosylated form of tPA is enzymatically active, and that it is capable of binding fibrin, the glycosylation pattern has favorable effects on clearance rates and thus may be helpful in providing the appropriate characteristics. The enzymatically inactive forms of tissue plasminogen activator can be conveniently produced by manipulation of the gene using site-directed mutagenesis or by truncation of the gene using restriction enzyme cleavage. One convenient form is the truncated protein constructed by deletion of codons encoding Leu₄₁₁-Pro_t.«₇ by cleavage of the cDNA at an Sstl/SacI site. Other preferred muteins comprise deletion of or replacement of the serine residue at position 478, preferably with a conservative amino acid substitution such as by alanine, glycine, or threonine, most preferably threonine. Other enzymic activity-destroying mutations in the serine protease region are also included in the scope of the invention.

D. Administration and Use

The modified tPAs generally produced by standard recombinant techniques are conjugated to the appropriate radionuclide and formulated for injection into the subject. Animals, particularly humans, are appropriate subjects. The tPA variants are first purified using a combination of chromatographic procedures, including an immunoaffinity purification after an initial ion exchange chromatography step, and characterized for purity by SDS- PAGE and tested for residual enzymic activity and for freedom from endotoxins.

The purified tPA is labeled with a suitable radiono lide, for example with carrier-free sodium 1-125 by the chloramine-T method of Hunter, W.M. , et al. Nature (1962) 194:495, or with technetium 99 using the method of Baker, R.J., et al, Eur J Nucl Med (1985) 1O:155-159. In the Baker method, stannous chloride is mixed with a saline solution containing TcO. and the pH of the solution brought to approximately 11.3. The tPA is then added in the solution, incubated at room temperature for one hour, the pH is then adjusted to 7.1 and unbound technetium 99 removed by gel filtration. In the alternative, the tPA is labeled with indium-Ill using 1-2 millicuries of InCl, according to the method of Hnatowich (supra). The labeled enzymatically inactive tPA is then infused, preferably intravenously, over a suitable time period, approximately 15 min - 1 hr, preferably around 20- 30 min, in an amount sufficient to effect detection of any clots, in an amount depending of the specific activity of the labeled compound. Approximately 1 millicurie of the label should be sufficient, and dosages of less than several milligrams should be required. Immunogenic effects are not expected from what is fundamentally a native protein, and therefore relatively high dosage levels are expected to be tolerated. Subjects are monitored over the next several hours for accumulation of the label at the location of clots, as detected by the standard and appropriate ex vivo imaging techniques. The presence of clots in the circulatory system is apparent by the accumulation of the labeled conjugate.

Examples

The following examples are intended to illustrate but not to limit the invention.

Example 1 Preparation of Recombinant Modified tPA

Site-specific utagenesis to replace the TGC codon encoding ser₄_„ with AGC encoding threonine was conducted using a 2.2 kb BamHI insert containing the entire native tPA cDNA subcloned into M13 mplδ using a 17- mer. The mutated insert was then subcloned into the BamHI site of the commercially available vector PSVL (Pharmacia} and the resulting vector transfected into COS 7 cells using DEAE/Dextran. The media were analyzed for tPA by an ELISA assay after 72 hr, and the presence of 20-90 ng/ml tPA was detected by ELISA assay. The tPA was inactive as judged by fibrin autography. Control cultures transfected with corresponding vectors containing DNA encoding native tPA showed similar amounts of production by ELISA but the tPA produced in these cultures was active in the fibrin autography assay, which shows the presence of active tPA even in the presence of physiological inhibitors which are normally present in the serum media supplement. In addition, a truncated form of tPA was constructed by deletion of bases encoding amino acids Leu.., to Pro--- by specific cleavage of the cDNA at the SstI restriction site which occurs at position 1421 of the cDNA sequence, and subsequent religation. This variant was inserted into PSVL, and the vector transfected into COS cells with similar results.

Immunoprecipitation of tPA from all three media, subsequent SDS-PAGE and Western blot confirmed that only cells transformed with vectors containing native tPA produced tPA in the medium as a 100 kd inhibitor complex, while the media from cells containing either mutant showed tPA present mostly in free form of molecular weight 64 kd and 33 kd for the thr.-g and truncated forms respectively.

Example 2

Ability to Bind Fibrin The lack of effect of inactivation of tPA on binding ability is shown in this example. Native tPA was inactivated by isolation of the active site using the peptide inhibitor PPACK (D-phenylalanyl-L-prolyl-L- arginine chloromethylketone) as described by Fry, E.T.A. et al Blood (1988) (in press). Both native untreated and inactivated tPA were labeled with 1-125. Blood clots were formed from human blood as described by Chandler, A.B., Lab Invest (1958) 2 ^{110-1 4 and} thrombosis was complete within an hour. The thrombi were washed and the labeled tPA preparations were added to a final concentration of 1/ug/ml. The mixtures were incubated for 30 min and the unbound tPA removed by washing. The thrombi were counted in a gamma counter; the counting procedure was that described by Cano-Angles, E. Annal Biochem (1986) 153:201- 210.

The amount of tPA bound to the clot was 63% greater for inactivated tPA than was obtained with the active molecule. It was inferred that the difference in affinity was due to constant turnover of the thrombus surface during fibrinolysis induced by active tPA. Also the inactive variant would not be expected to form complexes with plasma inhibitors, thus increasing the availability of the variant.

Example 3 Stable Production of Variants The variants are substituted for the native tPA sequences in the expression vector pSTH-MDH. The construction of pSTH-MDH containing the native sequence is as follows:

This vector contains expression systems for both tPA and DHFR under control of the SV40 promoter. The DHFR expression system is derived from pSV-DHFR; that for tPA from pSV-tPA17. pSV-tPA17 is constructed from three components: pSVoriHBV3'_f ρtPA-BAL17, and pMON-1068. PSV-DHFR is obtained from ligation of segments from pSVoriHBV3' and two segments of pUC-DHFR. The construction of these intermediates, the vectors containing the separate expression systems, and the integration of the tPA and DHFR expression systems onto a single vector is as follows: pSVoriHBV3' contains the origin of replication and early and late promoters of SV40 upstream of the 3' termination sequences from the hepatitis B surface antigen gene with insertion sites for a foreign gene between them. pSVoriHBV3' is constructed from pML, SV40, and HBV. pML is digested with EcoRI, blunted with Klenow, and then digested with Hindlll. The vector fragment containing the E. coli origin of replication and the ampicillin resistance gene is isolated and ligated to the isolated 540 bp fragment containing the early and late promoters and origin of replication of SV40, obtained by digestion of SV40 DNA by Hindlll and Hindi. The resulting vector, designated pSVori, is then digested with BamHI for acceptance of a 585 bp fragment isolated from a BamHI/ Bglll digest of HBV DNA which contains the 3' termination sequences of the surface antigen gene. Correct orientation is confirmed by restriction analysis — digestion with Hindlll and BamHI yields a 350 bp fragment from the correct vector. The resulting ligated vector, pSVoriHBV3', thus contains the SV40 promoter and origin sequences upstream of the HBV terminator and permits a coding sequence to be inserted conveniently between them.

Also prepared was ptPA-BAL17, which contains the tailored upstream portion of the tPA gene in a bacterial replication vector. The tPA cDNA is furnished by the vector pMON-1068, which is a bacterial vector containing an insert of the entire cDNA sequence obtained for tPA as described in Pennica, D., et al. Nature (1983) 301:214- 221. Of course, any bacterial replication vector containing this coding sequence could just as well have been used, and the restriction sites designated below fall within the disclosed sequence of the tPA cDNA set forth in the Nature reference. pMON-1068 is first digested with BamHI to excise the tPA encoding cDNA and then with BAL-31 to chew back at each end of the gene. Digestion with BAL- 31 was continued until analysis of the lengths and sequence of linear fragments indicated that the 5' end of the fragment was within 17 bp of the ATG start codon. The precise distance of chew-back is not critical so long as it is within sufficiently short distance to permit the ATG to be placed an operable distance from the promoter in the expression cassette. A separation in this fragment of the 5' terminus from the ATG of about 10 bp is, in fact, preferred. The selected linear fragment was then digested with Sad, which cuts inside the coding sequence of the tPA gene, and the resulting blunt/SacI fragment was isolated. This contains the suitably tailored 5' end of the gene and was ligated into SacI/HincII-digested pUC13 to give the intermediate plasmid ptPA-BAL17. pUC-DHFR was used as a cloning vector for the

DHFR-encoding sequences, absent their associated control sequences. pUC-DHFR was constructed by digesting pDHFR-11 (Simonsen, C.C., et al, Proc Natl Acad Sci USA (1983) 8JJ:2495-2499) with Fnu4HI, blunting with Klenow and then digesting with Bglll to isolated the 660 bp fragment as there described, and ligating this fragment into pUC13 which had been digested with Hindi and BamHI. Thus, pUC- DHFR represents a straightforward cloning vector for DHFR analogous to the ptPA-BAL17 vector described for the 5' portion of the tPA gene above.

Finally, a separate cloning vector for the termination sequences derived from the hepatitis B surface antigen gene, pUC-HBV3', was constructed by digesting HBV DNA with BamHI and Bglll and isolating the 585 bp fragment, as described above, and ligating this fragment into BamHI-digested pUC13. pSV-tPA17, which contains the full-length tPA coding sequence under control of SV40 promoter and HBV terminating sequences was prepared as a three-way ligation of the vector fragment from pSVoriHBV3' digested with Hindlll and BamHI, which thus provides the promoter and terminator along with vector sequences; the 3' portion of tPA obtained by Sacl/Bglll digestion of pMON-1068_? and the tailored 5^r portion of the tPA coding sequence, which was obtained as a Hindlll/SacI digest of ptPA-BAL17. The resulting ligation mixture was transfected into E. coli, the transformants selected for ampicillin resistance, and plasmid DNA containing the desired pSV-tPA 17 isolated. The counterpart vector for DHFR expression, designated pSV-DHFR, was also obtained in a three-way ligation. Again, the vector fragment obtained from Hindlll/BamHI digestion of pSVoriHBV3 ' was used to provide the control sequences, and the 5' and 3' portions of the DHFR coding sequence were obtained by digestion of pUC- DHFR with Hindlll and Sad (partial) and with Bglll and TaqI (partial), respectively. The ligation mixture was used to transform E. coli, ampicillin resistant transformants were selected, and plasmid DNA, designated pSV-DHFR, was isolated. A single plasmid containing a weak expression system for the DHFR coding sequence was also prepared. This plasmid, pMDH, was obtained in a 3-way ligation using the 1 kb fragment obtained by EcoRI/TaqI (partial) digestion of pDR34, the vector fragment from EcoRI/Sall- digested pML, and the 3' end of the gene isolated from

Sad (partial)/Sail digested pSV-DHFR. (The pDR34 vector is described by Gasser, C.S., et al, Proc Natl Acad Sci USA (1 &2) 29*6522-6526, supra) and contains the mouse DHFR gene linked to its own promoter.) The resulting vector, pMDH, is analogous to pSV-DHFR, except that the DHFR gene is under control of the urine DHFR promoter. The weak expression cassette residing on pMDH and strong expression cassette residing on pSV-tPA17, when used in admixture to transfect suitable DHFR-deficient cells, thus constitute one embodiment of the expression system of the invention.

Finally, pSTH-MDH, which contains the expression cassettes for tPA and for DHFR on a single vector, was constructed as a three-way ligation of the appropriate isolated fragments of pSV-tPA17, pMDH, and pUC-HBV3' . pSV-tPA18 is digested with SacII and Sail, pMDH with EcoRI and Smal, and pUC-HBV3' with SacII and EcoRI.

Substitution of the DNA encoding the tPA variants of the invention is effected by excising a suitable sized fragment of the native form from pSTH-MDH and replacing it with the corresponding fragment of the variant.

The resulting expression vectors containing the coding sequence for the variant under control of the SV40 promoter are transfected into CHO cells and amplified in the presence of methotrexate. The CHO cells are grown in suitable media supplemented with fetal bovine serum, with the addition of aprotinin to prevent cleavage into the two chain form, or in serum-free medium.

The cells are scaled up in selected media by growing to a density of 10 cells/ml and subculturing to obtain practical amounts of secreted tPA.

After purification, the tPA is labeled with iodine-125 or technetium 99m and verified to have fibrin- binding and thrombus-binding activities.

Example 4 In Vitro Assays Fibrin binding is tested using a modification of the method of Tran Thang, C, et al, J Clin Invest (1984) 7_4_:2009. Fibrinogen solution (500 ul, 2 g/ul fibrinogen and 1% BSA) is mixed with plasminogen (0.2 mg/ml), thrombin and labeled 1-125 tPA variant (or 1-125 native tPA as control) at test concentrations in imidazole buffer, pH 4, and incubated at 37 C. After incubation, the clots are centrifuged at 1000 x g for 10 min at 4°C, and an aliquot of the supernatant removed for gamma counting. The clots are washed twice with buffer and the radioactivity of the clots measured in a gamma counter. Binding is the percentage of radioactivity bound to the clot compared to the amount added. Nonspecific binding is determined as the extent of binding of labeled tPA in the presence of a 100-fold excess of cold tPA, and is subtracted from the total percentage binding found.

Thrombus binding is tested using thrombi formed from human blood, according to the method of Chandler (supra) which yields thrombi approximately 2 mm in diameter, 4-8 mm long and 5-10 g dry weight with morphological similarity to thrombi observed in vivo. The thrombi, contained in Tygon tubing, are then tested with saline and 1-125 labeled tPA variant. The labeled variant and control are added and the tubes closed and rotated at 30 rpm for 30 minutes at 37°C. The contents of the tube were then poured into a preweighed mesh filter and washed with saline containing 0.01% v/v Tween 80. The radioactivity of the washed thrombi is measured and the amount of variant bound per mg of thrombus calculated.

Example 5

In Vivo Tests A rabbit jugular vein thrombosis model of Colleπy D., et al, J Clin Invest (1983) 7_1^{!368_376 uses} New Zealand white rabbits anesthetized with Innovarvet and ketamine. A standard femoral cut down is performed followed by catheterization of the artery and vein, and additional anesthesia using pentabarbitol is administered as needed. The jugular vein is exposed and a thread introduced into the vein and the vein clamped. A thrombus is formed by injection of bovine thrombin and fibrinogen into the vessel, and the clot allowed to mature for 30 minutes. The animals are then injected with heparin (500 units/kg) subcutaneously and the clamps removed. Iodine- 125 labeled tPA variant is then injected as a bolus or by infusion, and the number of counts bound to the clot determined by removal of the clot followed by detection of radioactivity in a gamma counter.

Similarly, binding to coronary thrombi can be evaluated in dogs injected subcutaneously with morphine sulfate and anaesthetized with sodium thiopental and

L-chloralose. Coronary thrombi are induced by advancing a copper coil under fluoroscopic control into the left anterior descending artery and the induced coronary thrombi detected by typical electrocardiographic signs of ischemia and confirmed by angiography. 1-2 hours after induction, the dogs are given 99 mTc-tPA variant intravenously and the dogs are imaged using a gamma camera to detect the presence of 99 mTc at the location of the clot. In an additional rabbit pulmonary emboli model,

New Zealand white rabbits are anaesthetized and the external jugular vein exposed on one side by blunt dissection and the vessel catheterized with Silastic tubing which has a three-way stop cock attached. 1 ml of blood is withdrawn and used to form an autologous ex vivo thrombus which is then washed with saline and suspended in a syringe filled with saline. The thrombus is injected directly into the catheterized blood vessel through the three-way stop cock and the vessel is flushed with saline. The catheter is removed and the incision closed. The animal is allowed to awaken, and the pulmonary embolism is allowed to mature for 24 hrs. The animal is then anaesthetized and the femoral cut down performed. Both artery and vein are catheterized with separate catheters and the thrombus labeling agent, Tc99 tPA variant, is injected via venous catheter. The animal is sacrificed using 10 ml saturated solution of potassium chloride and the heart and lungs removed. The pulmonary emboli are dissected and the amount of bound radioactivity is determined by gamma counting.

Claims

Clai s

1. A method to locate the position of one or more thrombi in an animal subject which method comprises administering to said subject an effective amount of tPA labeled with a radioisotope detectable by ex vivo scintographic means, which tPA is modified so as to be enzymatically inactive.

2. The method of claim 1 wherein said tPA is modified by deletion of the serine protease domain.

3. The method of claim 1 wherein the tPA is modified by deletion or substitution of the serine residue at position 478.

4. The method of claim 1 wherein the scintographic label is selected from technetium 99m, indium-III and iodine-125.

5. The method of claim 1 wherein the labeled tPA is administered by intravenous injection.

6. A pharmaceutical composition suitable for use in detecting thrombi in animal subjects, which composition comprises an effective amount of a tPA labeled with a radioisotope detectable by ex vivo scintographic means,- said tPA modified so as to lack enzymatic activity.

7. A modified protein wherein said protein is substantially equivalent to tissue plasminogen activator prior to said modification, wherein said modification consists essentially of elimination of the enzymatic activity of said tPA.

8. The modified protein of claim 7 wherein the modification comprises deletion of the serine protease binding domain.

9. The protein of claim 8 wherein the modification comprises deletion or substitution of the serine at position 478.

10. The protein of claim 9 wherein said serine is substituted by threonine.

11. The protein of claim 9 conjugated to a radioactive nuclide label.