AU6667694A

AU6667694A - Method of enhancing thrombolysis

Info

Publication number: AU6667694A
Application number: AU66676/94A
Authority: AU
Inventors: Leonard Garfinkel; Marian Gorecki; Amos Panet
Original assignee: Savient Pharmaceuticals Inc
Current assignee: Savient Pharmaceuticals Inc
Priority date: 1993-04-23
Filing date: 1994-04-22
Publication date: 1994-11-21
Anticipated expiration: 2014-04-22
Also published as: CA2159508A1; WO1994025582A1; ZA942818B; IL109383A0; JPH08512291A; AU677659B2; EP0701606A4; EP0701606A1

Description

METHOD OF ENHANCING THROMBOLYSIS

BACKGROUND OF THE INVENTION

The present application is directed towards the use of a fragment of von Willebrand Factor (vWF) comprising the platelet glycoprotein lb (GPIb) binding domain in conjunction with known therapies to obtain enhanced protection against thrombosis, improved thrombolysis, and a decreased likelihood of reocclusion following thrombolytic treatment.

The references referred to by Arabic numerals in parentheses are found at the end of the specification immediately prior to the claims.

Von Willebrand Factor (vWF) is a large plasma protein which is synthesized in the endothelial cells which form the inner surface lining of the blood vessel wall, and by megakarocytes, the precursor of platelets.

Mature vWF is a multivalent molecule comprising domains which constitute binding sites for several proteins. One of the domains constitutes a binding site for the platelet glycoprotein lb (GPIb) . Using proteolytic digests this site has been .localized to the region between amino acid residues 449 and 7?8 of mature vWF. In addition, vWF has at least two collagen binding sites, at least two heparin binding sites, a Factor VIII binding site, and a RGD site which binds to the platelet GP Ilb/I Ia receptor.

Von Willebrand factor plays an important role in hemostasis

⁽24) , and its absence results in von Willebrand disease (VWD) , an inherited bleeding disorder. Recent studies have found that von Willebrand factor is essential for the formation of platelet thrombi, especially under flow conditions characterized by high shear stress, such as occur in stenosed coronary arteries (24) . Von Willebrand factor interacts with glycoprotein lb receptors on the platelet membrane to initiate platelet adhesion (13-15) and to activate platelet release of ADP, thromboxane, and serotonin, which cause platelet aggregation and thrombus formation (16-20) . Therefore, efforts have been made to prevent platelet adhesion and thrombus formation by blocking the platelet glycoprotein lb receptors. For example, aurin tricarboxylic acid blocks the platelet glycoprotein lb recognition site on von Willebrand factor and may be useful in antithrombotic therapy (25) .

Evidence that vWF, and specifically the binding of vWF to the platelet GPIb receptor, is essential for normal platelet adhesion, is based on both clinical observations and in vitro studies. Patients with the bleeding disorder von Willebrand Disease (vWD) have reduced levels of vWF or are completely lacking in vWF. Alternatively, they may have defective vWF. Another disorder, Bernard-Soulier Syndrome (BSS) , is characterized by platelets lacking GPIb receptors.

Hemostasis is a dynamic and ongoing process. It includes on one hand formation of clots to prevent bleeding and on the other hand dissolution and lysis of unwanted fibrin deposits and platelet aggregates in order to maintain blood flow throughout the body.

Studies have revealed that most acute transmural myocardial infarctions are caused by thrombus formation in atherosclerotic coronary arteries (1-4) . .An interruption of the protective endothelium covering the vessel wall results in platelet adhesion and aggregation and thrombosis, which blocks the blood supply to the myocardium (5-7) . Thrombolytic agents such as tissue plasminogen activator (t-PA) and streptokinase have been effective in the treatment of thrombosis in patients with acute transmural myocardial infarction ("Q-wave infarcts") (8-10), and are often administered with adjuncts such as anticoagulants, e.g. heparin and aspirin. However, 15-20% of patients do not achieve reperfusion. In addition, reocclusion of coronary arteries has limited the efficacy of thrombolytic therapy in some patients (11-12) .

Therefore, enhancing thrombolytic treatment by shortening the time to thrombolysis and reducing the incidence of reocclusion following thrombolysis would be a significant advance in the field of thrombolytic therapy.

In both thrombosis and reocclusion of coronary artery, platelet adhesion is the initiating event. In platelet adhesion, von Willebrand factor (vWF) , a multivalent protein, forms a bridge between the subendothelial extracellular matrix and glycoprotein lb on the surface of circulating platelets (13-15) . The adhesion of platelets to the subendothelium results in the activation of platelets and the release of adenosine diphosphate (.ADP) , thromboxane, and serotonin. These in turn activate additional platelets. Ultimately, a platelet plug forms (16-20) . Therefore, preventing platelet adhesion may be useful in preventing thrombosis and reocclusion in coronary arteries (21) .

Several peptides isolated from von Willebrand factor have been shown to bind to the platelet membrane glycoprotein lb receptor and inhibit the interaction of von Willebrand factor with glycoprotein lb receptors. This inhibition has led to the inhibition of platelet adhesion and aggregation (22,23) .

One such peptide, designated VCL, is a recombinaπt fragment of von Willebrand factor, Leu⁵⁰⁴ - Lys⁷²⁸, with a single intrachain disulfide bond linking residues Cyε⁵⁰⁹ and Cys⁶⁹⁵ (23) .

VCL is disclosed in coassigned International Patent Application Publication No. WO91/13903 which also discloses a method of inhibiting platelet aggregation in a subject prior to or after the subject has undergone angioplasty, thrombolytic treatment or coronary bypass surgery by use of the vWF fragment alone to achieve the desired effect.

It has now been surprisingly and unexpectedly discovered that administration of a vWF polypeptide fragment comprising the GPIb binding domain in conjunction with thrombolytic therapy provides greatly improved therapeutic results. In one aspect, the time required for thrombolysis is considerably lessened as evidenced by the shortened time to reperfusion. In another aspect there is a much lower incidence of reocclusion following the thrombolytic therapy. If occurrence of thrombosis is foreseen, such as may occur during surgery e.g. cardiovascular surgery, pretreatment with the vWF polypeptide fragment may reduce occurrence of thrombosis.

Additionally, it has been discovered that the vWF GPIb binding domain polypeptide and aspirin synergistically prevent complications following traumatic vascular damage.

SUMMARY OF THE INVENTION

The present invention provides a method of enhancing thrombolysis comprising administering a vWF GPIb binding domain polypeptide in conjunction with a thrombolytic agent and an anticoagulant which results in the shortening of the time to thrombolysis and reducing the incidence of reocclusion following thrombolysis.

In an additional embodiment, the invention provides a method for preventing complications following traumatic vascular damage in a subject comprising administering to the subject an amount of a vWF GPIb binding domain polypeptide in conjunction with an amount of aspirin, which together are effective to prevent the complications.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Plasmid PvWF-VC3

This figure shows plasmid pvWF-VC3 which expresses a vWF GPIb binding domain polypeptide under the control of the deo P_tP₂ promoter. This plasmid has been deposited in E. coli Sφ930 under ATCC Accession No. 68241.

Figure 2: Plasmid pyWF-VCL

This figure shows plasmid pvWF-VCL which is under control of the λP_L promoter and the deo ribosomal binding site and expresses the same vWF GPIb binding domain polypeptide as pvWF-VC3. This plasmid has been deposited in E.coli 4300 (F") under ATCC Accession No. 68242.

Figures 3A-3C; Translated Sequence of the vWF GPIb

Binding Domain Polypeptide Expressed by Plasmids pyWF-VC3 and PVWF-VCL

This figure shows the translated sequence of the von Willebrand Factor GPIb binding domain polypeptide expressed by plasmids pvWF-VC3 (ATCC Accession No. 68241) and pvWF-VCL (ATCC Accession No. 68242) . The sequence consists of 226 amino acids including an N-terminal methionine.

The nucleotide and amino acid numbering starting from 1 are shown in the margins. Met¹ is the initiator methionine. The sequence Leu² - Lys²²⁶ is identical to the sequence Leu³⁰⁴ - Lys⁷²⁸ of von Willebrand factor as shown in Figure 12 of International Application Publication No. WO91/13093.

Figure 4: Occlusion Time in Pretreated Dogs

Elapsed times from electrical injury to the formation of occlusive thrombi in coronary arteries of animals pretreated with saline, VCL,and aspirin (ASA) intravenously (protocol 1). Compared to saline, *p < 0.05; **p <0.001.

Figure 5: Thrombolysis Time in Pretreated Dogs

Elapsed times from t-PA administration to thrombolysis in coronary arteries of animals pretreated with saline, VCL, and aspirin (ASA) intravenously (protocol 1) . Compared to saline, *p < 0.05.

Figure 6. Thrombolysis Time in Non-pretreated Dogs

Elapsed times from intravenous administration of thrombolytic agents to thrombolysis in coronary arteries of animals that were not pretreated (protocol 2) .

Figure 7: Reocclusion Time in Pretreated Dogs

Elapsed times from thrombolysis to reocclusion of coronary arteries in animals pretreated with saline, VCL, and aspirin (protocol 1) . Compared to saline plus t-PA and aspirin plus t-PA, *p < 0.05.

Figure 8: Reocclusion Time in Non-pretreated Dogs

Elapsed times from thrombolysis to reocclusion of coronary arteries of animals treated with t-PA, heparin, VCL, and aspirin (protocol 2) . Compared to t-PA plus heparin and t-PA plus heparin plus aspirin, *p < 0.05; ** p < 0.001.

Figure 9: Hematocrit Following Thrombolytic Treatment

Changes in hematocrit before and after treatments with t-PA, heparin, VCL, and aspirin (protocol 2) . Figure 10: Clotting Time Following Thrombolytic Treatment

Activated clotting times (ACT) before (time 0) and 5, 60,

120, and 180 min after treatments with t-PA, heparin, VCL, and aspirin (ASA) (protocol 2) . Compared to control levels before each treatment, *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 11: Platelet Aggregation Following VCL Treatment

Changes in ex vivo ADP induced platelet aggregation (panel A) and botrocetin induced platelet aggregation (panel B) before (control) and after the treatment with intravenous VCL. Compared to control, *p < 0.001.

Figure 12: Platelet Aggregation Following Aspirin

Treatment

Changes in ex vivo ADP induced platelet aggregation (panel A) and arachidonic acid induced platelet aggregation (panel B) before (control) and after treatment with intravenous aspirin. Compared to control, *p < 0.001

Figure 13 : Synergy of GPIb Binding Domain Polypeptide With Aspirin

This figure shows the results of the experiments described in Example 3 as the amount of platelet deposition over time. ■ - aspirin + VCL @ lmg/kg + 2mg/kg/hr; • - VCL alone @

4mg/kg + 8mg/kg/hr,* - control; T - VCL alone @ lmg/kg

+ 2mg/kg/hr,* ♦ - aspirin alone. DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of enhancing a thrombolytic treatment in a subject comprising administering to the subject, in conjunction with a thrombolytic agent and an anticoagulant, an amount of a von Willebrand factor glycoprotein lb binding domain polypeptide effective to enhance the thrombolytic treatment.

Enhancing a thrombolytic treatment is hereby defined as shortening the time to thrombolysis and reperfusion, and reducing the incidence of reocclusion following thrombolysis.

The term "in conjunction" is defined as meaning before, during or after.

The thrombolytic agent may be any thrombolytic agent known to those skilled in the art. Examples of thrombolytic agents include tissue plasminogen activator (tPA) and streptokinase. It is also envisaged that the combination treatments of the invention may also be used in conjunction with other known methods (e.g. angioplasty) of obtaining revascularization of blocked blood vessels and of maintaining blood vessel patency.

The thrombolytic treatment includes, in addition to the thrombolytic agent, other pharmaceutical substances e.g. anticoagulants such as heparin and aspirin separately or in conjunction. Other anticoagulants known to the skilled artisan administering the thrombolytic treatment may also be used.

In a particular embodiment of the invention, the polypeptide is administered intravenously. In preferred embodiments of the invention, the intravenous administration is a bolus, continuous infusion, or bolus followed by continuous infusion.

In a particular embodiment of the invention, the polypeptide is administered intravenously as a bolus of 0.4-40 mg/kg body weight.

In a more preferred embodiment of the invention, the polypeptide is administered intravenously as a bolus of 1-20 mg/kg body weight.

In an especially preferred embodiment of the invention, the polypeptide is administered intravenously as a bolus of 2-10 mg/kg body weight.

In a particular embodiment of the invention, the polypeptide is administered intravenously by continuous infusion at a rate of 0.2-20 mg/kg body weight per hour.

In an especially preferred embodiment of the invention, the polypeptide is administered intravenously by continuous infusion at a rate of 1-10 mg/kg body weight per hour.

In the most preferred embodiment of the invention, the polypeptide is administered intravenously as a bolus of 0.4- 4 mg/kg body weight followed by continuous infusion of 0.2- 20 mg/kg body weight per hour.

In yet another aspect, the invention relates to situations in which occurrence of thrombosis may be foreseen, such as during surgery, for example cardiovascular surgery. In such situations, pretreatment with the vWF GPIb binding domain polypeptide may reduce the occurrence of thrombosis.

In another aspect, the invention provides a method for preventing complications following traumatic vascular damage in a subject comprising administering to the subject an amount of a vWF GPIb binding domain polypeptide in conjunction with an amount of aspirin, which together are effective to prevent the complications.

The cause of the traumatic vascular damage is exemplified by but not limited to coronary artery bypass surgery, angioplasty, thrombolysis, or unstable angina. Traumatic vascular damage caused by other therapeutic or clinical occurrences is also encompassed. The complications resulting from the traumatic vascular damage are exemplified by but not limited to myocardial infarction, ischemia, coronary bypass surgery, repeat angioplasty, or death. Other complications resulting from traumatic vascular damage are also encompassed.

The effective amount of the vWF GPIb binding domain polypeptide is in the range of about 0.1-20 mg/kg bolus and 0.1-20 mg/kg/hr continuous infusion of and the effective amount of aspirin is in the range of about 0.1-50 mg/kg. The precise dosages will be readily determined by one skilled in the art based on the details of the case in treatment.

The vWF GPIb binding domain polypeptide may be administered by any clinically appropriate means known to one skilled in the art; intravenous administration is a presently preferred embodiment.

The aspirin may be administered by any clinically acceptable means such as orally or parenterally and may be administered in a single dose, in multiple doses or by continuous administration. Parenteral adminisration refers to intravenous, intraperitoneal, subcutaneous or intramuscular administration. The glycoprotein lb (GPIb) binding domain of von Willebrand factor may be obtained from a variety of sources such as from naturally occurring von Willebrand factor or by recombinant protein production, e.g. in bacteria, fungi, plant, insect or mammalian cells.

The term "von Willebrand factor glycoprotein lb binding domain polypeptide" also encompasses a homolog of the GPIb binding domain which may be used in the methods of the invention with the proviso that the homolog has GPIb binding activity.

As used herein, a homolog of the polypeptide is a polypeptide which has substantially the same amino acid sequence and substantially the same biological activity as such polypeptide. Thus, a homolog may differ from the polypeptide of the invention by the addition, deletion, or substitution of one or more non-essential amino acid residues, provided that the resulting polypeptide retains the biological activity of the polypeptide. Persons skilled in the art can readily determine which amino acids residues may be added, deleted, or substituted (including with which amino acids such substitutions may be made) using established and well known procedures, including, for example, conventional methods for the design and manufacture of DNA sequences coding for bacterial expression of polypeptide homologs of the subject polypeptide, the modification of cDNA and genomic sequences by site-directed mutagenesis techniques, the construction of recombinant proteins and expression vectors, the bacterial expression of the polypeptides, and the measurement of the biochemical activity of the polypeptides using conventional biochemical assays.

Examples of homologs of the polypeptide of the subject invention are deletion homologs containing less than all the residues specified in the subject polypeptide, substitution homologs wherein one or more residues specified are replaced by other residues, and addition homologs wherein one or more amino acids residues is added to a terminal or medial portion of the polypeptide, all of which homologs share the biological activity of the polypeptide of the subject invention.

Substantially the same amino acid sequence is herein defined as encompassing the addition or deletion of fewer than four amino acids at the N-terminus of the amino acid sequence of the polypeptide. Furthermore, there may be substitutions and/or deletions in the sequence which do not eliminate the biological activity of the protein. Examples of substitutions are ser for cys and ala for gly. Other substitutions are known to those skilled in the art. Substitutions may encompass up to 10 residues in accordance with the homologous or equivalence groups described by e.g. Lehninger, Biochemistry. 2nd ed. Worth Pub., N.Y. (1975); Creighton, Protein Structure, a Practical Approach. IRL Press at Oxford Univ. Press, Oxford, England (1989) ; and Dayhoff, Atlas of Protein Sequence and Structure 1972, National Biomedical Research Foundation, Maryland (1972) .

Substantially the same biological activity refers to biological activity the same as that of the polypeptide possibly differing slightly in degree or level which would still be known by the skilled artisan to be the same biological activity.

The term "von Willebrand factor glycoprotein lb binding domain polypeptide" also encompasses various mutants and variants of the vWF GPIb binding domain having different amino acid sequences, but having substantially the same biological activity. The biological activity of the polypeptides encompassed by the invention is the ability to bind to glycoprotein lb of platelets or to prevent platelet adhesion to subendothelial matrix.

Examples of plasmids and hosts for production of recombinant polypeptides comprising the vWF GPIb binding domain are plasmids pvWF-VC3 in Escherichia coli Sφ930 and pvWF-VCL in Escherichia coli 4300(F) which were deposited on February 26, 1990, pursuant to, and in satisfaction of, the re¬ quirements of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852 under ATCC Accession Nos. 68241 and 68242 respectively.

EXAMPLES

The Examples which follow are set forth to aid in understanding the invention but are not intended to, and should not be so construed as to, limit its scope in any way. The Examples do not include detailed descriptions for conventional methods employed in the construction of vectors, the insertion of genes encoding polypeptides of interest into such vectors or the introduction of the resulting plasmids into bacterial hosts. Such methods are well-known to those skilled in the art and are described in numerous publications including Sambrook, Fritsch and Maniatis, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, USA, (1989) .

Various methods and other aspects relating to vWF in general and to the GPIb binding domain polypeptide in particular have been disclosed in coassigned, copending application U.S.S.N. 753,815, filed September 3, 1991, and coassigned PCT Publication No. WO91/13093, both of which are hereby incorporated by reference.

Methods involving expression of recombinant proteins by E. coli transformed with plasmids controlled by the λ P_L promoter are disclosed inter alia in coassigned U.S. Patent No. 5,126,252, granted June 30, 1992 which is hereby incorporated by reference.

Example 1: Production and Purification of Oxidized. Folded and Biologically Active vWF GPIb Binding Domain Polypeptide

Plasmid pvWF-VCL (Figure 2) was constructed as described in coassigned, copending patent application U.S.S.N. 753,815, filed September 3, 1991 and is maintained in E.coli strain A4300(F^") under ATCC Accession No. 68242. This host/plasmid system was fermented essentially as known in the art for vectors harboring a gene expressed under control of the λP_L promoter, i.e. growth at 30°C until mid-log stage followed by induction at 42°C for about two hours, essentially as described in U.S. Patent No. 5,126,252. Following fermentation and induction, the growth medium was centrifuged and a cell cake obtained which was stored frozen until further processing.

The general scheme of the downstream process consists of steps A through H as follows:

A. Isolation of Inclusion Bodies

Following fermentation as described above, the cell cake is resuspended in 50mM Tris-HCl pH=8, 50mM NaCl, ImM EDTA, ImM DTT, ImM PMSF, and 10% glycerol. The suspension is then sonicated to disrupt the cells and centrifuged to obtain a pellet containing the insoluble inclusion bodies.

The resulting pellet containing the inclusion bodies is dissolved at about 10% w/v in a solution such that the final concentrations are 8M urea, 20mM DTT, 20mM HEPES pH 8, and lOOmM NaCl. The solution may be further purified by ion exchange ch.romatography as described below. Alternatively, the inclusion bodies may be solubilized in a buffer containing 6M guanidine hydrochloride followed by buffer exchange to urea. The inclusion bodies may also be dissolved at different concentrations of urea, guanidine hydrochloride or any other denaturant or in the absence of denaturants, for example, at extremes of pH.

B. Cation exchange chromatography

This step eliminates most of the contaminants and produces the vWF GPIb binding domain polypeptide at greater than 90% purity. .Any cation exchange method (e.g. carboxymethyl) may be used in this step such as CM-Sepharose fast flow (Pharmacia) chromatography. The functional group may be carboxymethyl, phospho group or sulphonic groups such as sulphopropyl. The matrix may be based on inorganic compounds, synthetic resins, polysaccharides, or organic polymers; possible matrices are agarose, cellulose, trisacryl, dextran, glass beads, oxirane acrylic beads, acrylamide, agarose/polyacrylamide copolymer (Ultrogel) or hydrophilic vinyl polymer (Fractogel) . In a specific embodiment, the polypeptide is loaded onto a CM-Sepharose FF column equilibrated with 8M urea, ImM DTT, 20mM HEPES pH 8, lOO M NaCl. Pure polypeptide elutes in 8M urea, ImM DTT, 20mM HEPES pH 8 and 200mM NaCl. Up to about 30 OD₂₈₀ units of solubilized inclusion bodies may be loaded per ml CM-Sepharose FF. At this ratio the eluted polypeptide typically has a concentration of 4-5 OD₂₈₀/ml.

C. Oxidation/Refolding

The polypeptide solution eluted from the cation exchange step above is treated with 6M guanidine hydrochloride (GuCl) to disrupt any aggregates. The polypeptide is then diluted to 0.05 OD₂g₀/ml in 2M GuCl, pH 5-11, preferably 20mM HEPES pH 8, O.lmM GSSG (glutathione, oxidized form) . This mixture is allowed to stand overnight at room temperature. The phproducts are analyzed by gel filtration on fast protein liquid chromatography (FPLC) such as Superose 12 before proceeding. Analysis shows that this protein concentration reproducibly yields about 30% correctly oxidized monomers, and 70% S-linked dimers and multimers, as well as reduced and incorrectly oxidized monomers. A higher protein concentration gives a higher absolute yield of correctly oxidized monomers but a lower percentage yield due to increased formation of S-linked dimers and multimers. For example, a protein concentration of 0.1 OD₂₈₀/ml yields only 20% correctly oxidized monomers. Reducing the concentration to 0.025 OD₂₈₀/ml yields 35-40% correctly oxidized monomers but a lower absolute yield per liter oxidation. Oxidations may also be performed in urea instead of in GuCl, or in any other denaturant or in the absence of denaturants under appropriate buffer conditions in which, for example, pH, ionic strength, and hydrophobicity are varied. The preferred concentration of urea is in the range 0.5M to 10M, preferably 4M, and the preferred oxidant is GSSG in the range O.OlmM to 5mM preferably O.lmM. Other oxidants such as Cud, may be used or alternatively no oxidant may be added, thereby utilizing air oxidation only. For scale-up, 4M urea is the presently preferred solution for the oxidation step.

D. Concentration

The oxidation products are concentrated, preferably to about OD_2g0=i by a tangential flow ultrafiltration system with a 30K cutoff membrane, such as a "MINIT.AN" or "PELLICON" system of Millipore. The filtrate is quite clear as the material is relatively clean and most of the contaminants are large enough not to pass through the 30K membrane. It is thus possible to reuse the filtrate for performing oxidations. No difference in the oxidation products of oxidations performed in reused versus freshly prepared 2M GuCl was detectable by FPLC analysis. E . Dialysis

To reduce the GuCl or urea concentration to less than lOmM, dialysis against 20mM HEPES pH8, lOOmM NaCl is performed in dialysis tubing with 2-3 changes of buffer, or alternatively by diafiltration against the same buffer in a tangential flow ultrafiltration system with a 10K MW cutoff membrane.

During dialysis, as the concentration of GuCl (or urea or other denaturant) decreases, a white precipitate forms. This precipitate contains disulfide - linked dimers, reduced and incorrectly oxidized monomer and some contaminants which coeluted from the cation exchange step. The supernatant is nearly 100% correctly oxidized and refolded monomer at a concentration of 0.2 OD₂₈₀/ml, which is about 20% of the protein yield of step D.

F. Recovery from pellet

The yield of correctly oxidized monomer can be greatly increased by further recovery of correctly oxidized monomer from the precipitate. The solution containing the correctly oxidized monomer is clarified by centrifugation. The supernatant containing the correctly oxidized monomer is saved. The pellet containing disulfide - linked dimers and reduced and incorrectly oxidized monomer is treated with DTT to reduce disulfide bonds. Accordingly, the pellet is dissolved in a minimal volume of 6M GuCl, 20ml HEPES pH 8, 150mM NaCl, 20mM DTT. The solution is passed through Sephadex G25 in a buffer similar to the dissolution buffer but containing only ImM DTT (instead of 20mM) . The eluate is then diluted to OD_2g0=0.05 and treated as in steps C, D and E. This procedure may be repeated more than once as long as additional purified monomer is obtained. All of the supernatants may then be combined. G. Cation exchange

The combined supernatant of the dialysate of steps E and F is concentrated by binding to CM Sepharose in 20mM HEPES pH8, lOO M NaCl. Elution is with 20mM HEPES pH8, 400mM NaCl . The eluate is exclusively monomeric despite the high salt concentration. Concentrations of up to 3 mg/ml have been achieved by this method and that is not the upper limit. This step can alternatively be performed with Heparin-Sepharose which also binds the purified monomer in lOmM Tris pH 7.4, 150mM NaCl. Elution from Heparin- Sepharose is performed using lOmM Tris-HCl pH 7.4, 500mM NaCl.

H. Dialysis

The product of the previous step is dialyzed against 20mM HEPES pH8, 150mM NaCl.

I. Storage

At this stage the purified vWF GPIb binding domain polypeptide may be lyophilized. Upon reconstitution in a volume of water equal to the volume before lyophilization, the resultant solution contains exclusively monomeric protein showing no traces of dimers or other multimers on FPLC.

In a specific embodiment of the above method, the following procedure was performed:

a) 10 gm inclusion bodies (comprising 0.43 g net dry weight) were dissolved in a final volume of 100ml 8M urea, 20mM DTT, 20mM HEPES pH 8, 100 mM NaCl.

b) The protein was loaded onto a CM Sepharose column equilibrated with 8M urea, ImM DTT, 20mM HEPES pH 8, lOOmM NaCl. The protein eluted at 200mM NaCL in 8M urea, 20mM HEPES pH 8, ImM DTT, and was saved.

c) The saved eluate of the previous step was treated with 6M GuCl to eliminate any aggregates, and was then diluted to 0.05 OD₂₈₀/ml in 2M GuCl, 20mM HEPES pH 8, 0.ImM GSSG.

Oxidation was performed overnight at room temperature.

(Note that the oxidation step can be performed in the presence of urea instead of GuCl.)

d) The oxidation products were concentrated to OD₂₈₀=l by ultrafiltration on a "MINITAN" unit containing a 30K membrane.

e) The concentrate of the previous step was dialyzed with three buffer changes against 20mM HEPES pH 8, lOOmM NaCl. During dialysis, as the GuCl concentration decreased, a white precipitate formed which was removed by centrifugation and reprocessed once as described above. The supernatants were combined.

f) The combined supernatants were concentrated by binding to CM Sepharose in 20mM HEPES pH 8, lOOmM NaCl. The polypeptide was eluted in 20mM HEPES, pH 8, 400mM NaCl and stored at 4°C.

g) The saved eluate from the previous step was dialyzed against 20mM HEPES pH 8, 150mM NaCl at 4°C.

h) After dialysis, the purified vWF GPIb binding domain polypeptide, designated VCL, was lyophilized.

Analysis of VCL

1. Amino acid sequence analysis of VCL purified as described above revealed that the N- erminal sequence is Me -Leu-His-Asp-Phe which is the expected sequence according to Figures 3A-3C.

2. Examination of VCL on polyacrylamide gels revealed that the VCL migrated under non-reducing conditions at a lower apparent molecular weight than under reducing conditions (?- mercaptoethanol) . This shift from compact to less compact configuration is consistent with the reduction of a disulfide bond. Such an intramolecular bond is formed between the cysteines at positions 509 and 695. (The shift in molecular weight is not large enough to be consistent with the reduction of an intermolecular bond.)

Example 2: Use of the vWF GPIb Binding Domain in Conjunction with Thrombolytic Treatment to Prevent Reocclusion

SUMMARY

The ability of a GPIb binding domain polypeptide fragment of vWF to enhance thrombolysis by shortening the time to reperfusion and to prevent reocclusion following thrombolysis was studied. The GPIb binding domain fragment tested was the VCL polypeptide described in Example 1. A canine model was evaluated for the effect of VCL on (l) the formation of intracoronary thrombosis, (2) the duration of thrombolysis, and (3) the incidence of reocclusion of coronary arteries after thrombolysis with t-PA. The results suggest that VCL delays thrombus formation, shortens the duration of thrombolysis, and reduces the incidence reocculsion of coronary arteries following thrombolysis.

In this canine model, thrombus formation is induced in a coronary artery by electrical stimulation. Following thrombus formation, thrombolysis is induced by infusion of tissue plasminogen activator (tPA) . The effect of the GPIb binding domain polypeptide in preventing thrombus formation and in reducing the incidence of reocclusion following thrombolysis was tested. The GPIb binding domain polypeptide proved effective in delaying onset of electrically stimulated thrombus formation. In addition, the GPIb binding domain polypeptide shortened the time to reperfusion during thrombolysis and reduced the incidence of reocclusion when used in the thrombolytic therapy in conjunction with heparin and aspirin.

METHODS

All procedures used in this study were conducted according to the principles of the American Physiological Society and were approved by the Institutional Animal Care and Use Committee at the Texas Heart Institute, Houston, Texas.

Surgical Preparation

Mongrel dogs (n=57 ) weighing 25 to 35 kg were anesthetized with sodium pentobarbital (30 mg/kg intravenously) , intubated, and connected to mechanical respirators (Harvard Model 60, Natick, MA) . Plastic catheters were placed in a carotid artery for monitoring aortic pressure and in a jugular and a peripheral vein for drug and fluid administration. A left fifth intercostal space thoracotomy was performed, and the heart suspended in a pericardial cradle. A 1- to 2-cm segment of left anterior descending coronary artery was carefully exposed and nearby branches ligated. An ultrasonic Doppler flow probe (Hartley Instruments, Houston, TX) was placed around the proximal portion of the exposed left anterior descending coronary artery to measure the velocity of blood flow. Baseline hemodynamics, including heart rate, systolic and diastolic aortic pressures and phasic and mean coronary blood flow velocities were recorded on an eight-channel recorder (Gould, Model 3000, Cleveland, OH) .

A needle electrode (the 8-mm tip of a 25-gauge needle crimped on the end of a l0-cm length of 30-gauge Teflon- insulated silver wire) was inserted obliquely approximately 4 mm into the lumen of the exposed left anterior descending coronary artery at a site distal to the Doppler flow probe. The needle was stabilized on the vessel with 6-0 silk suture. To prevent the electric current from injuring the surrounding tissue, heat-shrink tubing was applied to the needle/wire and soldered connection. A ground wire was connected to the subcutaneous tissue to complete the electrical circuit. To induce thrombosis, a current of 150μA was applied through the electrode, which was connected in series with the positive terminal of a 9-V battery, a 50kΩ potentiometer, a multimeter, and the ground wire. Thrombus formation was determined by the reduction of coronary blood flow velocity, which was monitored by the externally positioned Doppler flow probe. The electric current was maintained until 30 min after persistent thrombotic occlusion had occurred.

Experimental Procedures

Two separate protocols were employed in this study of VCL; one examined its effect on coronary arterial thrombus formation, the other its effect on thrombolysis and reocclusion.

Protocol l. To evaluate the effect of VCL on intracoronary thrombus formation, treatment began on the dogs 30 min before electrical stimulation of their coronary arteries. Three different groups of animals were studied. In the control group (n = 12) , saline was given intravenously at 1 ml/min. In one experimental group (n = 7) , VCL was given intravenously at 4 mg/kg body weight as a bolus dose and at 2 mg/(kg-h) as a continuous infusion until the end of study. In the other experimental group (n = 8) , aspirin was given intravenously at 5 mg/kg body weight as a bolus dose. The change in coronary blood flow before and after electrical stimulation was carefully monitored. The amount of time elapsed from the beginning of electrical stimulation to the total occlusion of the coronary arteries was recorded. Three hours after the total occlusion of coronary arteries, all animals were given t-PA (Genentech, Inc., San Francisco, CA) intravenously at 80μg/kg body weight as a bolus dose and at 8μg/(kg-min) as a continuous infusion for 90 min. This treatment was intended to induce lysis of the thrombi formed in the coronary arteries. A thrombus was considered to be lysed (and the artery reperfused) when the flow velocity of the coronary artery returned to at least 70% of the value that existed before thrombus formation. The amount of time elapsed from t-PA administration to reperfusion was recorded as thrombolysis or reperfusion time. Dogs in whom reperfusion had not occurred after 90 min of t-PA infusion were excluded from further study. Dogs in whom reperfusion did occur were further monitored until the coronary arteries reoccluded or until 180 min had elapsed without reocclusion. The time from reperfusion to reocclusion was recorded as reocclusion time. Dogs in whom coronary arteries had not reoccluded after 180 min of reperfusion were considered not to have reoccluded. Dogs in whom reocclusion did occur were monitored for 30 min to verify persistent reocclusion.

Protocol 2. To further explore the effect of VCL on thrombolysis and reocclusion, additional animals were studied and treated three hours after the occlusion of coronary arteries. These animals were assigned to one of four additional groups: t-PA and heparin (n=7) ,* t-PA, heparin, and VCL (n=7) ; t-PA, heparin, and aspirin (n=8) ; and t-PA, heparin, VCL, and aspirin (n=8) . Heparin was given at 200 U/kg as an intravenous bolus. t-PA, VCL and aspirin were given at the same dose as in protocol 1. The follow-up for thrombolysis and reocclusion was also the same as in protocol 1.

Hematocrit. Coagulation, and Platelet Aggregation Studies

Hematocrit was checked before and at the end of t-PA administration in all dogs in protocol 2. Activated whole blood clotting time was measured in these dogs before and 5, 60, 120, and 180 min after the administration of t-PA on an automated blood coagulation timing device (HemoTec 2001370, Englewood, CO) . Ex vivo platelet aggregation was analyzed before and 10 min after the administration of VCL and aspirin in dogs in protocol 1. Blood samples were collected in plastic tubes containing a 3.8% solution of sodium citrate (9 volumes blood:1 volume sodium citrate). Platelet-rich plasma was obtained by centrifuging blood samples at 200 x g for 20 min and platelet-poor plasma was obtained by centrifuging the residual blood at 3000 x g for 10 min. The platelet count in platelet-rich plasma was adjusted to 300,000/mm³. A four-channel platelet aggregometer (Bio-Data, Model PAP-4, Horsham, PA) was used for the assay. Agonists and their final concentrations were ADP (Sigma, St. Louis, MO) at 5, 10, and 20μM; botrocetin (produced as described by Fujimura et al., Biochemistry !_:1957-1964 (1991)) at 1.1, 2.2, and 4.4 μg/ml; and arachidonic acid (Sigma, St. Louis, MO) at 12.5, 25, and 50 μg/ml. Because canine platelets do not aggregate in response to arachidonic acid, epinephrine was added to the platelet suspension at 10 μmol/1 before arachidonic acid. The degree of platelet aggregation was reported as the maximal percentage that light transmission increased in platelet-rich plasma over light transmission in platelet-poor plasma.

Statistical .Analyses

All values are expressed as a mean ± standard error of mean (SEM) . Fisher's exact test was used to compare the frequency of reperfusion and reocclusion in different groups of animals. A one-way analysis of variance (ANOVA) with repeated measurements was used to compare the hemodynamic values and activated clotting times obtained at different time periods and the duration of time to reperfusion and reocclusion in different groups of animals. Student's t test was used to compare the percentage of platelet aggregation and hematocrit values before and after treatment in each group of animals. A p value less than 0.05 was considered significant.

RESULTS

Intracoronary Thrombus Formation

Insertion of the electrode needle into the coronary artery caused some stenosis in the arteries of all animals, as determined by a reduction of blood flow velocity to approximately 65% of the baseline level (Tables 1 and 2 ) . After electrical stimulation, all animals developed total occlusion of the affected coronary arteries. In protocol 1, the elapsed time from electrical stimulation to total occlusion of the coronary arteries was significantly longer in dogs treated with VCL (p < 0.001) and aspirin (p < 0.05) than in dogs treated with saline (Figure 4) . In all animals, aortic blood pressure and heart rate changed slightly after the occlusion of coronary arteries. In aspirin-treated animals, however, heart rate and mean aortic pressure increased at each time-point (Table 1) .

Table 1. Hemodynamics Before and After Intercoronary Thrombosis and Thrombolysis in Animals in the 3 Pretreatment Groups (Protocol 1) .

HR AOM PHFLO MNFLO (beats/min) (mmHg) (% Of (% Of baseline) baseline)

Saline and t-PA (n = = 12)

Baseline 149 ± 7 78 ± 5 100 100

Needle-in 149 ± 6 77 ± 5 65 ± 7 73 ± 11

3-h 135 ± 10 96 ± 4* 0 0 occlusion

Reperfusion 133 ± 9 89 ± 5 68 ± 7 86 ± 9 (n = 4)

1-h 138 ± 7 86 ± 6 0 0 reperfusion

Pre-VCL and t-PA (n ^**■ 7)

Baseline 139 ± 8 81 ± 3 100 100

Needle-in 146 ± 10 82 ± 4 67 ± 7 68 ± 8

3-h 128 ± 11 115 ± 4*** 0 0 occlusion

Reperfusion 120 ± 11 116 ± 3*** 95 ± 15 117 ± 22 (n=5)

1-h 130 ± 19 96 ± 6 79 ± 18 88 ± 22 reperfusion

(n = 4)

Pre-aspirin and t-PA (n = 8) Baseline 144 ± 8 87 ± 3 100 100

Needle-in 159 ± 7 93 ± 3 63 ± 6 68 ± 8

3-h 172 ± 5** 94 ± 2 0 0 occlusion

Reperfusion 174 ± 4*** 98 ± 7 76 ± 8 87 ± 11 (n = 4)

1-h 178 ± 8 99 ± 6 96 ± 26 131 ± 41 reperfusion

Values are mean ± SEM. Compared to baseline level, *p 0.05; **p < 0.01; ***p < 0.001. AOM, mean aortic pressure; HR, , heart rate; MNFLO, mean flow velocity in the coronary artery; PHFLO, phasic flow velocity in the coronary artery; t-PA, tissue-type plasminogen activator. Table 2. Hemodynamics Before and After Intracoronary Thrombosis and Thrombolysis in Animals in the 4 Treatment Groups (Protocol 2) .

HR AOM PHFLO MNFLO (beats/ (m Hg) (% of (% of min) baseline) baseline) t-PA and Heparin (n = 7)

Baseline 138 ± 5 83 ± 1 100 100

Needle-in 136 ± 6 81 ± 2 61 ± 8 76 ± 7 3-h 125 ± 9 91 ± 4 0 0 occlusion

Reperfusion .132 ± 14 92 ± 3 57 ± 9 72 ± 15 (n = 5)

1-h 114 ± 6* 88 ± 4 60 ± 16 60 ± 19 reperfusion (n=4) t-PA, Heparin, and VCL ( N -> 7)

Baseline 135 ± 6 89 ± 3 10 100

Needle-in 132 ± 60 88 ± 4 69 ± 8 74 ± 6 3-h 112 ± 7** 95 ± 6 0 0 occlusion

Reperfusion 107 ± 6** 98 ± 5 83 ± 12 100 ± 17 (n=6)

1-h 133 ± 4 81 ± 8 133 ± 34 169 ± 59 reperfusion

3-h 128 ± 12 69 ± 10 40 ± 13 53 ± 24 reperfusion

(n=4) t-PA, Heparin, and Aspirin (n » 8) Baseline 140 ± 8 79 ± 3 100 100

Needle-in 140 ± 8 83 ± 5 65 ± 8 73 ± 9

3-h 137 ± 5 95 ± 4 0 0 occlusion

Reperfusion 157 ± 6 98 ± 2 95 ± 9 107 ± 9 (n-7)

1-h 159 ± 12 77 ± 4 59 ± 12 60 ± 13 reperfusion

(n=4) Table 2. cont'd.

HR AOM PHFLO MNFLO (beats/ (mmHg) (% of (% of min) baseline) baseline)

t-PA, Heparin, VCL, and Aspirin (n = 8)

Baseline 144 t 3 89 ± 5 100 100

Needle-in 143 ± 2 88 ± 5 67 ± 6 81 ± 6

3-h 170 ± 4*** 105 ± 4 0 0 occlusion

Reperfusion 175 ± 5*** 109 ± 5 88 ± 10 108 ± 12 (n=8)

1-h 178 ± 6*** 78 ± 5 82 ± 32 102 ± 42 reperfusion

(n=7)

3-h 174 ± 12* 65 ± 5* 41 ± 26 49 ± 28 reperfusion

(n-7)

Values are mean ± SEM. Compared to baseline level, * p < 0.05; **p < 0.01; ***p < 0.001. AOM, mean aortic pressure; HR, heart rate; MNFLO, mean flow velocity in the coronary artery; PHFLO, phasic flow velocity in the coronary artery; t-PA, tissue-type plasminogen activator.

>2 -

Thrombolvsis

In protocol 1, 3 h after the occlusion of coronary arteries, only t-PA was given to the animals. The administration of t-PA resulted in thrombolysis in 4 of 12 saline- reated dogs (33%), 5 of 7 VCL-treated dogs (71%), and 4 of 8 aspirin- treated dogs (50%) . The average elapsed time from t-PA treatment to thrombolysis (thrombolysis time) was significantly shorter in VCL-treated than in saline-treated dogs (p < 0.05, Figure 5).

In protocol 2, dogs were not pretreated before their coronary arteries were occluded, and 3 h after the occlusion of coronary arteries, they received thrombolytic treatments: t-PA and heparin induced thrombolysis in 5 of 7 dogs (71%) ; t-PA, heparin, and VCL induced thrombolysis in 6 of 7 dogs (86%) ; t-PA, heparin, and aspirin induced thrombolysis in 7 of 8 dogs (85%) ; and t-PA, heparin,VCL, and aspirin induced thrombolysis in 8 of 8 dogs (100%) . The average thrombolysis time in dogs treated with t-PA, heparin, VCL, and aspirin was slightly more than half that in dogs treated only with t-PA and heparin (23.5 ± 4 min vs 45 ± 12 min) . The difference, however, was not statistically significant (Figure 6) .

Reocclusion

After thrombolysis, many animals developed reocclusion of the coronary arteries during the 3-h monitoring period. In protocol l, the frequency of reocclusion was not significantly different among dogs pretreated with saline (4 of 4), aspirin (4 of 4), or VCL (4 of 5). However, the average time from thrombolysis to reocclusion (reocclusion time) was significantly longer in dogs pretreated with VCL than in dogs pretreated with saline and aspirin (Figure 7) .

In protocol 2, coronary artery reocclusion developed in 5 of 5 dogs treated with t-PA and heparin, and the addition of aspirin did not change the frequency of reocclusion (7 of

7) . The addition of VCL to t-PA and heparin significantly reduced the frequency of reocclusion in the reperfused coronary arteries of dogs (2 of 6; p < 0.05 compared to the t-PA and heparin group) . The addition of VCL and aspirin also significantly reduced the frequency of reocclusion (1 of 8; p < 0.01 compared to the t-PA, heparin, VCL group) .

The average reocclusion time was also significantly longer in VCL-treated dogs than in dogs who did not receive VCL

(Figure 8) .

Hematocrit. Coagulation, and Platelet Aggregation Studies

In dogs treated with t-PA alone, bleeding around the surgical area was not significant. The addition of VCL or aspirin caused mild bleeding along the incisions. The combination of t-PA, heparin, VCL, and aspirin resulted in moderate to severe bleeding around the surgical areas. However, hematocrit did not change significantly in any group after treatment with tPA, heparin, VCL and aspirin (Figure 9) .

Activated clotting time was significantly prolonged immediately after t-PA and heparin administration (Figure 10) . It returned to 1.5 times the baseline value 1 h after treatment and to just above the baseline value 3 h after treatment. The addition of aspirin or VCL, or a combination of aspirin and VCL did not affect activated clotting time.

Ex vivo platelet aggregation induced by ADP was not affected by VCL infusion (Figure 11A) but was slightly reduced by aspirin injection (Figure 12A) . Botrocetin-induced platelet aggregation was completely inhibited by VCL treatment (Figure 11B) , and arachidonic acid-induced platelet aggregation was completely inhibited by aspirin injection (Figure 12B) . Conclusion

The data from the present study demonstrate that VCL prolongs the time of intracoronary thrombus formation, enhances t-PA-induced thrombolysis, and delays coronary artery reocclusion.

Intravenous administration of VCL before electrical injury to the coronary artery significantly increased the time required for formation of an occlusive thrombus in vivo. The ex vivo platelet aggregation induced by botrocetin was completely inhibited by the treatment. These data suggest that blocking the interaction between von Willebrand factor and platelet glycoprotein lb diminishes both platelet aggregation in the stenosed and endothelium-injured area of coronary arteries and also the formation of occlusive thrombi.

After occlusive thrombi had formed in the coronary arteries, the infusion of t-PA caused thrombolysis. The thrombolysis time was significantly shorter in VCL-pretreated animals than in control animals, which suggests that VCL pretreatment may enhance t-PA-induced thrombolysis in coronary arteries. VCL given after the formation of thrombi, however, did not enhance thrombolysis by t-PA as significantly.

Coronary artery reocclusion has limited the efficacy of thrombolytic therapy in patients with acute myocardial infarction (11, 12, 29-31). Most clinical trials have used adjunct treatment with antiplatelet agents (e.g. aspirin) to prevent reocclusion (32-33) . In the present study, upon administration of VCL with t-PA or t-PA and heparin, the time from thrombolysis to coronary artery reocclusion was significantly prolonged, and in some cases reocclusion was prevented. VCL was more effective than aspirin in the same conditions. These data suggest that VCL may be useful as an adjunct to t-PA in future thrombolytic therapy for coronary artery reocclusion.

Bleeding is a common complication of thrombolytic therapy. In the present study, treatment with VCL, plus t-PA and heparin caused mild to moderate bleeding around the surgical incisions, and the combination of VCL, aspirin, t-PA, and heparin resulted in severe bleeding in some cases.

Thus, blocking platelet glycoprotein lb receptors with VCL may be effective in diminishing the formation of thrombi in injured coronary arteries. In delaying the reocclusion of the coronary arteries after thrombolysis, VCL is comparable or superior to aspirin as an adjunctive treatment with t-PA and heparin. Treatment with VCL and aspirin, in addition to t-PA and heparin, may completely prevent reocclusion.

Example 3: Synergy of GPIb Binding Domain Polypeptide With Aspirin

The effect of GPIb binding domain polypeptide was investigated using a model of traumatic vascular damage. This model is essentially that described by Kelly et al. (34) . In this model, segments of 2 mm collagen coated polytetrafluoroethylene (e-PFTE) tubes are inserted as extension pieces into chronic arteriovenous access shunts in baboons. The baboons are injected with autologous ^••'in- labeled platelets and then administered one of the treatments being studied. At specified times, the amount of deposited platelets was determined with a scintillation camera.

A series of studies on three animals was conducted to investigate the activity of the GPIb binding domain polypeptide (VCL) alone and in the presence of aspirin. The GPIb binding domain polypeptide was produced as described in Example 1.

The first set of studies was directed to the effect of the GPIb binding domain polypeptide alone in the model. The reference for this study was the lowest dose from a dose response study previously performed, i.e. 1 mg/kg bolus and 2 mg/kg/hr continuous infusion for one hour. As seen in Figure 13, this dose delayed the occlusion of the tubes to about 60 minutes compared to about 20 minutes in controls.

The second set of studies was directed to determining the effect of aspirin alone in the model. Three animals were examined. 35 mg/kg aspirin were given orally two hours before the study. All specimens occluded less than 30 minutes into the study. Qualitatively and quantitively, the aspirin curves were found to be similar to the control curves obtained using pure non-anticoagulated blood. Platelet counts were similar in both groups. The final set of experiments included study of the activity of the GPIb binding domain polypeptide in the presence of aspirin. Three animals were studied. Animals were given 35 mg/kg aspirin orally two hours before the study. The GPIb binding domain polypeptide dose used was 1 mg/kg bolus and 2 mg/kg/hr continuous infusion for one hour.

All devices remained patent during the continuous infusion period of the GPIb binding domain polypeptide. After that period they began to occlude. Two devices occluded 80 minutes into the study, while 4 stayed patent.

An increase in platelet deposition rate was observed when the continuous infusion stopped. Thus, unexpectedly, the GPIb binding domain polypeptide and aspirin exhibit synergistic activity regarding the retardation and/or prevention of platelet deposition.

The synergistic effect of the GPIb binding domain polypeptide and aspirin on platelet deposition was found to be equivalent to a four-fold higher dose of 4 mg/kg bolus plus 8 mg/kg/hr continuous infusion of the GPIb binding domain polypeptide when administered alone.

As an additional parameter, bleeding times were measured in the experiments described above. They are presented in Table 3. Bleeding times increased in a dose dependent manner, from 6 minutes at the lowest dose of the GPIb binding domain polypeptide to 22.5 minutes at the highest dose of the GPIb binding domain polypeptide. Aspirin alone resulted in a bleeding time of 6 minutes. The combination of aspirin plus the low dose of the GPIb binding domain polypeptide resulted in a slight increase to 7 minutes. However, this combination had the anti-platelet deposition effect of a dose of the GPIb binding domain polypeptide four times higher (4 mg/kg bolus and 8 mg/kg/hr infusion) with a bleeding time of 16.5 minutes. Thus, combination of the GPIb binding domain polypeptide with aspirin resulted in a dramatic increase in efficacy without a concomitant increase in bleeding time.

Table 3 Bleeding Times

Study Bleeding Time (min)

Controls 4

Aspirin 6

1 mg/kg bolus + 2 mg/kg-hr 6 infusion

2 mg/kg bolus + 4 mg/kg-hr 7 infusion

4 mg/kg bolus + 8 mg/kg-hr 16.5 infusion

8 mg/kg bolus + 16 mg/kg-hr 22.5 infusion

Aspirin + l mg/kg bolus + 7 2 mg/kg-hr infusion

REFERENCES

1. DeWood, M.A. , J. Spores, R. Notske, L.T. Mouser, R. Burroughs, M.S. Golden, H.T. Lange. 1980. N. Engl . J. Med. 303:897-902.

2. Davies, M.J. , and A.C. Thomas. 1985. Br . Heart . J. 11:363-373.

3. Mizuno, K. , A. Miyamoto, K. Satomora, A. Kurita, T. Arai, M. Sakurada, S. Yanagida, and H. Nakamura. 1991. Lancet 337:809-812.

4. Taeymans, Y. , P. Theroux, J. Lesperance, and D. Waters. 1992. Circulation £1:78-85.

5. Nahagawa, S., Y. Hanada, Y. Koiwaya, and K. Tanaka. 1988. Circulation 71:1335-1344.

6. Fuster, V., L. Badimon, J.J. Badimon, and J.H. Chesebro. 1992. N. Engl . J. Med. 326:242-318.

7. Ambrose, J.A. 1992. J. Am. Coll . Cardiol . 19: 1653-1658.

8. White, H.D., R.M. Norris, M.A. Brown, M. Takayaha, M.B. Maslowski, N.M. Bass, J.A. Ormiston, and M.B. WhitlOCk. 1987. N. Engl . J. Med. 317:850-855.

9. Midgett, A.S., G.T. O'Connor, J.A. Baron, and J. Bell. 1990. Ann. Intern. Med. 111:961-968.

10. AIMS Trial Study Group. 1990. Lancet 135:427-431.

11. Ohman, E.M., R.M. Califf, E.J. Topol, R. Candela, C. Abbottsmith, S. Ellis, K.N. Sigmon, D. Kereiakes, B. George, and R. Stack. 1990. Circulation H:781-791.

12. Lim, M.J., M.A. Gallagher, M. Ziadeh, and A.J. Buda. 1991. J. Am. Coll . Cardiol . 18:879-885.

13. Sakariassen, K.S., P.A. Bolhuis, and J.J. Sixma. 1979. Nature 279:636-638.

14. Reddick, R.L., T.R. Griggs, M.A. Lamb and K.M. Brinkhous. 1982. Proc . Natl . Acad. Sci . USA

71:5076-5079.

15. Fauvel, F., M.E. Grant, Y.J. Legrand, H. Souchon, G. Tobelem, D.S. Jackson, and J.P. Caen. 1983. Proc . Natl . Acad. Sci . USA 80:551-554.

16. Kroll, M.H. , T.S. Harris, J.L. Moake, R.I. Handin, and A.I. Schafer. 1991. J. Clin . Invest . 18:1568- 1573.

17. DeMarco, L. , A. Girola i, S. Russell, and Z.M. Ruggeri. 1985. J. Clin. Invest . 71:1198-1203.

18. Willerson, J.T. , P. Golino, J. Eidt, W.B. Campbell, and L.M. Buja. 1989. Circulation 0:198_^205

19. Yao, S.K., J.C. Ober, J. McΝatt, C.R. Benedict, M. Rosolowsky, H.V. Anderson, K. Cui, J.P. Maffrand, W.B. Campbell, L.M. Buja, and J.T. Willerson. 1992. Circ. Res . 7β:39-48.

20. Ashton, J.H., C.R. Benedict, C. Fitzgerald, A. Raheja, A. Taylor, W.B. Campbell, L.M. Buja, and J.T. Willerson. 1986. Circulation 73:572-578.

21. Ruggeri, Z.M.. 1992. Circulation 16(suppl. Ill) :III-26-29. 22. Sugimoto, M. , G. Ricca, M.E. Hrinda, A.B. Schreiber, G.H. Searfoss, E. Bottini, and Z.M. Ruggeri. 1991. Biochemistry 11:5202-5209.

23. Gralnick, H.R., S. Williams, L. McKeown, W. Kramer,

H. Krutzsch, M. Gorecki, A. Panet, and L.I.

Garfinkel. 1992. Proc. Natl . Acad. Sci . USA 11:7880-7884.

24. Ruggeri, Z.M., and T.S. Zimmerman. 1987. Blood

70:895-904.

25. Phillips, M.D. , J.L. Moake, L. Νolasco, and Ν. Turner. 1988. Blood 72:1898-1903.

26. Fujimura, Y., K. Titani, L.Z. Holland, S.R. Russell, J.R. Roberts, J.H. Elder, Z.M. Ruggeri, and T.S. Zimmerman. 1986. J. Biol . Che . 261:381- 385.

27. Fujimura, Y. , L.Z. Holland, Z.M. Ruggeri, and T.S. Zimmerman. 1987. Blood 70:985-988.

28. Pietu, G., P. Meulien, G. Cherel, J. Diaz, D. Baruch, M. Courtney, and D. Meyer. 1989. Biochem.

Biophys . Res . Commun . 164:1339-1347.

29. Chesebro, J.H., L. Badimon, and V. Fuster. 1988. Curr. Opin. Cardiol . 1:492-500.

30. Harrison, D.G., D.W. Ferguson, and S. M. Collins. 1984. Circulation 69:991-994.

31. Ellis, S.G., E.J. Topol, B.S. George, D.J. Kereiakes, D. Debowey, K.Ν. Sigmon, A. Pickel, K.L.

Lee and R. M. Califf. 1989. Circulation JK):1159-

1165. 32. ISIS-2 Collaborative Group. 1988. Lancet 2:525- 530.

33. Coller, B.S. 1990. N. Engl. J. Med. 322:33-42.

34. Kelly et al . , 1992. Proc. Natl. Acad. Sci. USA 89:6040-6044

Claims

What is claimed is:

A method for preventing complications following traumatic vascular damage in a subject comprising administering to the subject an amount of a von Willebrand factor glycoprotein lb binding domain polypeptide in conjunction with an amount of aspirin, which together are effective to prevent the complications.

A method according to claim l wherein the traumatic vascular damage is caused by coronary artery bypass surgery, angioplasty, thrombolysis, or unstable angina.

3. A method according to claim 1 wherein the complications following the traumatic vascular damage are myocardial infarction, ischemia, coronary bypass surgery, repeat angioplasty, or death.

4. A method according to claim l wherein the effective amounts are 0.1-20 mg/kg bolus and 0.1-20 mg/kg/hr continuous infusion of the vWF GPIb binding domain polypeptide and 0.1-50 mg/kg aspirin.

5. A method according to claim 4 wherein the vWF GPIb binding domain polypeptide is administered intravenously.

6. A method according to claim 5 wherein the aspirin is administered orally or parenterally.

7. A method according to claim 6 wherein the aspirin is administered in a single dose, in multiple doses or by continuous administration. 8. A method according to claim 7 wherein the parenteral adminisration is intravenous, intraperitoneal, subcutaneous or intramuscular.

9. A method of enhancing a thrombolytic treatment in a subject comprising administering to the subject in conjunction with a thrombolytic agent and an anticoagulant, an amount of a von Willebrand factor glycoprotein lb binding domain polypeptide effective to enhance the thrombolytic treatment.

10. A method of claim 9 wherein the thrombolytic agent is tPA.

11. A method of claim 10 wherein the anticoagulant is heparin.

12. A method of claim 11 wherein the thrombolytic treatment additionally comprises aspirin.

13. A method of claim 9 wherein the von Willebrand factor glycoprotein lb binding domain polypeptide is administered intravenously.

14. A method of claim 13 wherein the intravenous administration is a bolus.

15. A method of claim 13 wherein the intravenous administration is by continuous infusion.

16. A method of claim 13 wherein the intravenous administration is a bolus followed by continuous infusion.

17. A method of claim 14 wherein the bolus is an amount between 0.4-40 mg/kg body weight. 18. A method of claim 17 wherein the bolus is 1-20 mg/kg body weigh .

19. A method of claim 18 wherein the bolus is 2-10 mg/kg body weight.

20. A method of claim 15 wherein the infusion is at the rate of 0.2-20 mg/kg body weight per hour.

21. A method of claim 20 wherein the infusion is at the rate of 1-10 mg/kg body weight per hour.

22. A method of claim 16 wherein the bolus is 0.4-40 mg/kg body weight and the continuous infusion is 0.2-20 mg/kg body weight per hour.

23. A method of claim 13 wherein the polypeptide is administered prior to surgery.

24. A method of claim 23 wherein the surgery is cardiovascular surgery.