WO1999047070A1

WO1999047070A1 - Improved implantable biomaterials, compositions and methods for their preparation and uses thereof

Info

Publication number: WO1999047070A1
Application number: PCT/US1999/005967
Authority: WO
Inventors: David C. Sane
Original assignee: Wake Forest University
Priority date: 1998-03-18
Filing date: 1999-03-18
Publication date: 1999-09-23
Also published as: AU3192299A

Abstract

Implantable biomaterials adapted for use in contact with blood or blood components containing platelets have applied to the surface thereof a recombinant platelet-binding, adhesive protein, such as vitronectin, which is mutated at the platelet receptor binding site. The resultant biomaterials exhibit reduced thrombosis due to platelet adhesion and aggregation.

Description

IMPROVED IMPLANTABLE BIOMATERIALS, COMPOSITIONS AND METHODS FOR THEIR PREPARATION AND USES THEREOF

FIELD OF THE INVENTION

The present invention relates to improved implantable biomaterials which are used in contact with blood or blood components containing platelets, and which are characterized, inter alia, by reduced thrombogenicity and incidence of infection as a result of applying to at least a portion of the surface thereof a modified form of an adhesive, platelet - binding protein, which is altered at the platelet receptor binding site.

DESCRIPTION OF THE PRIOR ART

One of the more active areas in biomedical engineering today is in the development of implantable biomaterials. Illustrative examples of such materials include stents, catheters, guide wires, arterial grafts, cardiac pacer leads, automatic implantable cardiodefibrilator (AICD) leads, ventricular assist devices, artificial hearts and the like. Although these biomaterials provide many notable health benefits, they are not without certain limitations. A limitation of particular concern is the tendency of implantable biomaterials to give rise to thrombosis. Thrombus formation on biomaterials has the potential to cause embolization, which may result in strokes, myocardial infarctions and other end-organ damage. In addition, thrombus formation on the biomaterial surface may interfere with its proper function and possibly cause it to fail altogether. Another limitation is the incidence of infection attributable to thrombus formation on biomaterials, the thrombus serving as a nidus for infection.

There have been various proposals to reduce the thrombogenesis of implantable biomaterials. One approach has been to modify the biomaterial surface so as to prevent endogenous protein adhesion and aggregation, thereby avoiding thrombus formation. Surface modification techniques that have been investigated for improving biocompatability and functionality of implantable biomaterials have included utilization of materials having low polarity surfaces, e.g., silicone polymers, negatively charged surfaces, e.g., hydrogels, as well as surface conjugation to biological substances such as heparin, hirudin, thrombomodulin, albumin and streptokinase . None of these approaches has proved to be entirely satisfactory for eliminating the occurrence of thrombosis .

Accordingly, there continues to be a need for an effective way of reducing the occurrence of thrombosis resulting from the implantation of biomaterials of the type described above.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, a composition is provided for imparting reduced thrombogenicity and incidence of infection to implantable biomaterials adapted for use in contact with blood or blood components containing platelets. The composition comprises a modified, platelet -binding adhesive plasma protein which is altered at the platelet receptor binding site preferably by site- directed mutagenesis. This mutation effectively provides an anti-stick coating on the biomaterial surface, thus rendering it resistant to platelet binding.

According to another aspect of the present invention, there is provided a method for reducing the thrombogenicity of an implantable biomaterial adapted for use in contact with blood or blood components containing plasma. The method involves applying the composition of the invention, described above, to at least a portion of the surface of the biomaterial. Such implantable materials include but are not limited to stents, catheters, guide wires, arterial grafts, cardiac pacer leads, prosthetic heart valves, cardiopulmonary bypass membranes, automatic implantable cardiodefibrilator leads, ventricular assist devices or artificial hearts.

The resulting implantable biomaterial constitutes another aspect of the present invention.

According to a further aspect of this invention, a method is provided for reducing the occurrence of thrombosis and infection following implantation of a biomaterial in a patient, which comprises the steps of :

(a) applying the composition of the invention to a least a part of the biomaterial; and (b) implanting the resulting biomaterial into the patient.

In yet another aspect of the invention, kits are provided which contain reagents and materials which may be used to advantage in practicing the methods described herein. Such kits include for example, a composition of the invention in lyophilized form, a suitable sterile buffer for reconstitution of the composition and an implantable material or device to which the composition is to be applied. Optionally, the kits may include protocols for preparing the biomaterial of reduced thrombogenicity according to the present invention and an information sheet pertaining to the storage, handling and/or reconstitution of the lyophilized composition. The implantable biomaterials of the invention exhibit a good biocompatability with blood and blood components including platelets, and serve to maintain the proper functioning of the biomaterial.

Insofar as is known a mutant, recombinant protein has not previously been used to attenuate the thrombogenicity of biomaterial surfaces .

DETAILED DESCRIPTION OF THE INVENTION

The expression "adhesive, platelet-binding protein", as used herein, refers to such proteins as vitronectin, fibronectin, thrombospondin, fibrinogen, von Willebrand's factor, collagen and the like, which exhibit platelet binding activity. All of these proteins also contain at least one Arg-Gly-Asp platelet binding sequence. In addition, fibrinogen contains another platelet binding site (HHLGGAKQAGDV) on the C-terminus of the gamma chain. This binding site on fibrinogen could also be mutated using the methods of the present invention to abrogate platelet binding mediated by this molecule.

Although various mutated adhesive, platelet- binding proteins are suitable for use in the compositions, biomaterials and methods of this invention, the invention is described below with specific reference to modified vitronectin as a preferred embodiment of the invention. Vitronectin - 5 -

was chosen for prototype testing because it is highly adsorbed from plasma onto a wide variety of biomaterials, and once adsorbed, remains attached to the surface to a greater extent than other proteins. It should be understood, however, that other adhesive, platelet-binding proteins, such as those mentioned above may be used if desired.

As is well known, vitronectin (VN) is an adhesive glycoprotein present in the plasma and extracellular matrix that is involved in cell adhesion, spreading and migration (Tomasini, B.R. et al . , (1990) Prog. Haemostasis Thromb . 10:269-305). Vitronectin also has important roles in the regulation of the complement, coagulation and fibrinolytic systems (Tomasini et al . , supra; Preissner, K.T and D. Jenne (1991) Thromb. Haemostas 66:123-132). Vitronectin regulates fibrinolysis by binding and stabilizing plasminogen activator inhibitor-I (PAI-I) (Tomasini et al . , supra). While not wishing to be bound to any particular theory, it is believed that mutant forms of vitronectin that attach to a biomaterial surface will prevent the adsorption of plasma vitronectin. Because the platelet receptor binding site, Arg-Gly-Asp (RGD) , has been altered, the occurrence of platelets becoming bound to the treated biomaterial surface is decreased. Consequently, the thrombogenicity of the biomaterial will be reduced. A surface with less platelet-fibrin deposition will also be less prone to infection. Protein modification, as described herein, may also have a direct effect on reducing infections. This is because certain bacteria, e.g., Staphylococcus Aureus, are known to bind to proteins such as vitronecin; - 6 -

however, modification of the protein may impair its capacity to become bound by the microorganism. In addition, cells, such as smooth muscle cells and fibroblasts, will not migrate into the modified vitronectin-bearing surface.

In order to test the foregoing hypothesis, mutants of vitronectin were prepared in which the platelet receptor binding site was mutated, the resulting mutants having the sequences Arg-Ala-Asp and Arg-Gly-Glu. These two mutants are hereafter referred to as RAD- rVN and RGE-rVΝ, respectively. This mutation converted the platelet binding sequence to inactive forms.

A similar effect may be achieved by chemically modifying adhesive, platelet-binding proteins to abrogate platelet adhesion activity. The use of such chemically modified protein in the manner described herein is contemplated to be within the scope of the invention. As described in further detail below, these mutant forms of vitronectin have been expressed and purified using a baculovirus-based system.

The mutated form of vitronectin (or other adhesive, platelet -binding plasma proteins) may be applied to a biomaterial surface in various ways, including, without limitation, chemical coupling or cross-linking, or surface adsorption via immersion, padding, brushing, roller coating, spray coating or the like. The protein may be applied using a suitable carrier vehicle such as phosphate-buffered saline (PBS) . Procedures for chemically coupling proteinaceous substances to biomaterial surfaces are well known to those skilled in the art. See, for example, U.S. Patents Nos . 5,061,750, and 5,098,977. The benefit of chemical coupling is the development of a longer lasting anti-thrombogenic effect.

Preparation of the biomaterials of the invention can be facilitated by providing the constituents in kit form. A preferred kit includes lyophilized recombinant mutant or chemically modified protein and a suitable sterile buffer for reconstitution. Such a kit would facilitate the wide application of the invention to any blood-contacting biomaterial that may potentially be implanted into a test subject.

Experiments have been performed, as described below, in which guide wires were coated with RAD-rVN and RGE-rVN, and then placed in the arterial circulation of a test animal. Scanning electron microscopy showed reduced platelet adherence and fibrin, in comparison to uncoated and RGD-VN coated wires. The mutant forms of vitronectin were also found to lack the ability to stimulate smooth muscle cell migration.

A particular benefit of the present invention is derived from an improvement in arterial stents . Specifically, stents which are coated with the modified forms of VN described herein are expected to be less prone to restenosis, since thrombus formation is likely a key element in the restenotic process and since the above-described RGE and RAD mutants fail to support (or do so only weakly) the haptotaxis, or migration of smooth muscle cells.

Since smooth muscle cell migration is a key element in the restenotic process, the modified protein-coated stents may have an attentuating effect on the process. Restenosis has been called the "Achilles' heel" of interventional cardiology, occurring in 30-50% of all procedures, i.e., angioplasties, atherectomies, and the like. Although stent implantation has reduced the incidence of restenosis to perhaps 20%, this is still a serious problem. Another added benefit of the present invention is that biomaterials, e.g., stents, coated with the compositions of the invention would be less likely to induce platelet-fibrin thrombus formation. Accordingly, treated or coated stents or the like may reduce the need for the administration of platelet inhibiting agents (e.g., ticlopidine, abciximab, tirofiban, eptifibitide) to patients receiving implants. While the foregoing agents have shown benefit in the setting of stents, they are quite expensive. Coating such implantable biomaterials may effectively reduce medical costs associated with such procedures .

The following examples are provided to describe the invention in further detail. These examples are intended to illustrate and not to limit the invention.

Of the materials referred to in the examples, plasmid pMel Bac transfer vector, Sf9 cells and Hi -5 cells were purchased from Invitrogen, San Diego, CA. pFast Bac Dual expression vector, recombinant hTGF-βl, the synthetic RGD peptide GRGDSP, monoclonal antibody to human integrin o.vβ5 (clone P1F6) , E. coli competent cells DH 10 Bac and cell culture medium SF 900II SF were from GIBOO BR Life

Technologies, Gaithersburg, MD . A monoclonal antibody to vitronectin (clone VIT-2), and protease inhibitor cocktail were from Sigma Chemicals, St. Louis, MO. The humanized form of monoclonal antibody LM609 to human vβ3 (Vitaxin) was provided by IXSYS, Inc., San Diego, CA. Sheep anti-mouse IgG horseradish peroxidase conjugate was purchased from Amersham. Human aortic smooth muscle cells (CRL 1999) were purchased from ATCC, Rockville, MD. Rabbit aortic smooth muscle cells were a gift from Dr. Maurice Nachtigal, University of South Carolina School of Medicine, Columbia, SC. All chemicals were of analytical or molecular biological grade.

EXAMPLE I CONSTRUCTION OF RECOMBINANT BACULOVIRUS

The plasmid pGEMHVN containing a full length cDNA for human vitronectin is available from the ATCC. Expression vectors or recombinant hVN and hVN mutants were constructed as described previously (Zhao, Y, and D.C. Sane (1993) Biochem. Biophys . Res. Comm. 192:575-582) . The cDNA for human vitronectin and its RGD mutants (RAD-rVN and RGE-rVΝ) were cloned into the Bam HI site of plasmid pMelBac B. The recombinant plasmids (pMelbac B rVΝ, pMelbac E RGE-rVΝ or pMelbac B RAD-rVΝ) were co-transfected into Sf9 cells with linear AcMΝPV DΝA. Recombinant virus was isolated and plaque-purified on X-gal plates using the plaque assay, in accordance with the manufacturer' s recommended protocol .

Rsr II and Hind III restriction sites were generated at the 5 ' and 3 ' ends of the two domain deletion mutants of vitronectin ΔS-rVM and ΔH-rVΝ using PCR. The oligonucleotides used for PCR were: 5 ' -AAA GGG CGG TCC G AA ATG AAA TTC TTA GTC AAC G-3 ' (to generate an Rsr II site at the underlined bases) - 10 -

and 5 ' -AAA GGG AAG CTT CTA CAG ATG GCC AGG AGC T-3 ' (to generate a Hind III site at the underlined bases) . Oligonucleotide primers were synthesized on an ABI 394 DNA/RNA synthesizer using standard cyanoethyl phosphoramide procedure in the DNA synthesis Core

Laboratory of the Comprehensive Cancer Center of Wake Forest University, School of Medicine.

The modified cDNAs for ΔS-rVN and ΔH-rVN were then cloned into the Rsr II- Hind III site of the pFastBac dual vector. Recombinant plasmids with cDNAs for ΔS -rVN and ΔH-rVM were sequenced to eliminate any PCR-induced errors. The recombinant deletion mutants pFastBac dual ΔS-rVN and pFastBac dual ΔH-rVN were transformed in competent E. coli DH 10 Bac cells and the recombinant bacmids were isolated from these cells, in accordance with the manufacturer's recommended protocol. Sf9 cells were then infected to produce high titer viral stocks, which were stored at 4oc.

EXAMPLE II

EXPRESSION OF RECOMBINANT HUMAN

VITRONECTIN AND ITS MUTANTS IN HI -5 CELLS

Hi -5 insect cells were grown as a monolayer at 27oc in serum-free SF900II SFM medium. Cells were infected with baculoviruses for recombinant wild type human vitronectin or VN mutants. The medium was harvested at 48 hours post-infection, protease inhibitor cocktail was added and the harvested medium was frozen at -20°C until purification.

Infection with the recombinant baculovirus resulted in the secretion of vitronectin into the medium. The maximum expression of vitronectin was - 11 -

observed at 48-50 hours post-infection. After 50 hours of postinfection vitronectin expression decreased, and different sizes of vitronectin proteolytic products were observed by immunoblotting (data not shown) .

EXAMPLE III PURIFICATION PROCEDURES

Recombinant hVN and its mutants were purified on FPLC . Medium was harvested at 48 hours post infection and filtered through a 0.22 μm filter. A protease inhibitor cocktail for general use was added and loaded onto the anion exchange column (Mono Q 10/10 or source Q 10/10), which had been previously equilibrated in 20 mM Tris-HCl pH 7.0 (buffer A) . The column was washed with 10 column volumes of buffer A and a linear gradient of buffer B (0.75M NaCl in Buffer A) was applied to elute the bound proteins from the column. The flow rate of the column was maintained at 0.5 ml/min. Absorbance was read at 280 nm and 2.0 ml fractions were collected by an online fraction collector.

EXAMPLE IV SDS POLYACRYLAMIDE GEL ELECTROPHORESIS

10% SDS PAGE was performed according to the method of Lammeli (Laemmli, U.K. (1970) Nature 227:680-685) . Medium from non-infected Hi5 cells in serum free SF900 II SFM was used as control. The gel was run overnight at constant current, stained by Coommasie Brilliant blue and destained in 30% methanol, 7% acetic acid. - 12 -

EXAMPLE V WESTERN BLOTTING

Recombinant VN and its mutants were obtained as described in Example IV, above, and transferred to a nitrocellulose membrane, which had been equilibrated for 5-10 minutes in IX transfer buffer (25 mM Tris, 192 M glycine, 20% methanol, pH 8.3) . The proteins were transferred for 90 minutes in cold transfer buffer at 0.6 amp constant current . The membrane was blocked for 60 minutes in 5% nonfat dry milk in IX TBS (Tris-buffered saline) containing 0.1% Tween 20 (TBST) . After blocking, the membrane was washed twice in IX TBST for 15-20 minutes. The membrane was placed in a heat sealable bag with primary antibody, VN mab, clone VIT-2, at a 1:1000 dilution. The primary antibody was added in blocking solution and the membrane incubated for 60 minutes at room temperature. The membrane was then washed twice in IX TBST buffer for 15-20 minutes at room temperature, followed by the addition of the secondary antibody (anti -mouse IgG- horse radish peroxidase conjugate at a 1:5000 dilution) . The membrane was incubated at room temperature for an additional 60 minutes. The membrane was then washed twice in IX TBST for 15-20 minutes. Two different substrates were used for the detection of bound secondary antibody. For chemiluminescence, Amersham' s ECL detection system was used. The membrane was exposed to Amersham' s Hyperfilm for 10-120 seconds. For visual color development, 1 tablet (5 mg) of 4-chloro-l-napthol

(Sigma) was dissolved in 10 ml of methanol, 40 mis of IX TBS, 50 μl of H₂0₂ and the mixture poured onto the membrane in the dark with color development occurring - 13 -

over 15-20 minutes. The membrane was washed in distilled water and stored in the dark until photographed .

EXAMPLE VI ELECTROBLOTTING OF RECOMBINANT VN AND ITS MUTANTS FOR SEQUENCING

Electroblotting for protein sequencing was performed as previously described (LeGendre, N., and P.T. Matsudara, (1989) Purification of proteins and peptides by SDS-PAGE in "A Practical Guide to Protein and Peptide Purification for Microsequencing" pgs. 52- 66, Editor, P.T Matsudaira, Academic Press, Inc) . Purified recombinant hVN and mutants of VN (7-8 μg) were separated by 10% SDS PAGE, as described previously. A PVDF membrane (BioRAD) was washed sequentially with the following: 1) 100% methanol for 2-3 seconds and 2) water for 2-3 seconds. The membrane was then equilibrated in IX transfer buffer (10 mM CAPS, 10% methanol, pH 11.0) for 15-20 minutes at room temperature. See Current Protocols in Molecular Biology (eds. Ausubel, F.M.) 10.8.16 (John Wiley and Sons, New York, 1997) for CAPS. After electrophoresis was completed, the gel was equilibrated in IX transfer buffer for 5-10 minutes and the proteins were transferred at 0.6 amp for 90 minutes. After transfer the membrane was washed several times in deionized water (5 minutes each) at room temperature. VN and its mutants were visualized by staining with Coomassie blue R-250 (0.1% in 50% methanol) for 5 minutes, and destained in 50% methanol, 10% acetic acid. The membrane was rinsed - 14 -

with several changes of deionized water and air dried. The protein bands were cut and subjected to automated Edman degradation.

EXAMPLE VII PROTEIN SEQUENCING

Electroblotted VN and VN mutants were directly sequenced using an Applied Biosystems 477A Protein sequencer in Protein Core Laboratory of the Comprehensive Cancer Center of Wake Forest University, School of Medicine. The derivatives of phenylthiohydantoin (PTH) amino acids were analyzed by an on-line PTH analyzer. Ten cycles were sequenced for VN and VN mutants .

EXAMPLE VIII ADHESION ASSAY

The adhesion assay was performed as described (Technical Bulletin (1995) Focus on Applications, GIBCOBRL Life Technology "Attachment of cells to ECM components") with little modification. Briefly, polystyrene, non-tissue culture treated 96 well plates (Nunc) were coated for 16 hours at 4°c with 10 μg/ml vitronectin or VN mutants in phosphate buffer saline pH 7.4 (137 mM ΝaCl , 2.7 mM Kcl, 4.3 mM Νa₂HP0₄.7H₂0 and 1.4 mM KH₂P0₄) . After coating, residual proteins were removed and the plate was washed with IX PBS. After washing, the coated wells were blocked by 0.1 ml of 2% BSA in DMEM and incubated for 2 hours at 37°C. Rabbit smooth muscle cells were serum starved for 24 hours in serum- free medium (DMEM/0.5% BSA), then harvested with trypsin/EDTA (Gibco/Brl) for 2 minutes. The trypsin was inactivated with DMEM/0.5% BSA containing 0.25 mg/ml soy bean trypsin inhibitor (SBTI) (GIBCO/BRL) . The - 15 -

cells were washed with serum free DMEM/0.5% BSA, and a cell suspension (0.1 ml) containing 10,000 cells was added to each well and incubated at 37°C for 2 hours. The wells were initially washed with IX PBS. Buffered formalin (10%; 0.1 ml) was added to each well and the plates incubated for 30 minutes at room temperature. After fixing the cells, 50 μl of toluidine blue (1%) in 10% buffered formalin was added to each well and incubated for 1 hour at room temperature. After incubation, the wells were extensively washed with deionized water, then air dried overnight. SDS (0.1 ml of 2% solution) was added to each well and incubated for 15 minutes at 37°C The absorbance was read at 650 nm on automatic microtiter plate reader (Molecular Devices, CA) . All the assays were performed in triplicate.

Addtional experiments were performed in vitro using Gore-Tex vascular grafts. Gore-Tex vascular graft material (5 cm in length) was left uncoated or coated overnight with RGE-rVN (10 ug/ml) .

The graft tubing was then perfused with 20 mis of citrated rabbit blood at a flow rate of 5ml/min and subsequently rinsed with 100 mis of phosphate buffered saline. After rinsing, adherent platelets were fixed in 2.5% glutaraldehyde and submitted for scanning EM. Scanning EM demontsrated a 10 -fold reduction in the density of adherent platelets onto the coated graft as compared to the uncoated material .

EXAMPLE IX

ANIMAL TEST

In order to obtain in vivo shear stress conditions in the absence of anticoagulated blood, an - 16 -

animal model was used in which guide wires (0.14 inch) were coated by immersion with the two recombinant forms of vitronectin described above (10 μg/ml in PBS, overnight) then placed under fluoroscopic guidance into rabbit carotid arteries at the origin from the aorta. Guide wires coated with wt-rVN and saline were also used as a basis for comparison. After 10 minutes, the guide wires were removed and the tips of the wires were rinsed in saline, excised, fixed and visualized under scanning electronmicroscopy . There was a prominent accumulation of platelets and fibrin on the wt-rVN or saline-coated tips, which was reduced on RAD-rVN coated tips, and substantially inhibited on tips coated with RGE-rVN. The results of these experiments demonstrate that applying RGD mutants of vitronectin, especially RGE-rVN, to biomaterial surfaces can markedly reduce platelet and thrombus formation.

While certain embodiments of the present invention have been described and/or exemplified above, various other embodiments will be apparent to those skilled in the art from the foregoing disclosure. For example, other mutant forms of vitronectin lacking the PAI-I binding site(s) may reduce thrombus formation on biomaterial surfaces. The present invention is, therefore, not limited to the particular embodiments described and/or exemplified, but is capable of considerable variation and modification without departure of the scope of the following claims.

Claims

- 17 -WHAT IS CLAIMED IS:

1. An implantable biomaterial, adapted for use in contact with blood or blood components containing platelets and having reduced thrombogenicity, said biomaterial having a surface, at least a part of said surface having applied thereon a modified form of an adhesive, platelet-binding protein, said protein being modified at the platelet receptor binding site.

2. An implantable biomaterial, as claimed in claim 1, wherein said modified protein is a recombinant protein in which the protein sequence of the platelet receptor binding site is mutated.

3. An implantable biomaterial, as claimed in claim 1, wherein said protein is a recombinant form of vitronectin, in which the Arg-Gly-Asp sequence of the platelet receptor binding site is mutated.

4. An implantable biomaterial, as claimed in claim 3, wherein the resultant mutated sequence is Arg-Ala-Asp.

5. An implantable biomaterial, as claimed in claim 3, wherein the resulting mutated sequence is Arg-Gly-Glu.

6. An implantable biomaterial, as claimed in claim 1, selected from the group consisting of stents, catheters, guide wires, arterial grafts, cardiac pacer leads, prosthetic heart valves, - 18 -

cardiopulmonary bypass membranes, automatic implantable cardiodefibrilator leads, ventricular assist devices or artificial hearts.

7. A composition for imparting reduced thrombogenicity to implantable biomaterials adapted for use in contact with blood or blood components containing platelets, said composition comprising a modified, platelet-binding adhesive plasma protein, said protein being modified at the platelet receptor binding site.

8. A composition, as claimed in claim 7, wherein said modified protein is a recombinant protein in which the protein sequence of the platelet receptor binding site is mutated.

9. A composition as claimed in claim 7, wherein said protein is a recombinant form of vitronectin in which the Arg-Gly-Asp sequence of the platelet receptor binding site is mutated.

10. A composition as claimed in claim 9, wherein the resulting mutated sequence is Arg-Ala-Asp.

11. A composition as claimed in claim 9, wherein the resulting mutated sequence is Arg-Gly-Glu.

12. A method for reducing the thrombogenicity of an implantable biomaterial adapted for use in contact with blood or blood components containing platelets, said biomaterial having a surface, said method comprising affixing to at least a - 19 -

portion of said surface a modified, platelet-binding adhesive plasma protein, said protein being modified at the platelet receptor binding site.

13. A method as claimed in claim 12, wherein said modified protein is affixed to said biomaterial by adsorption.

14. A method as claimed in claim 12, wherein said modified protein is affixed to said biomaterial by chemical coupling.

15. A method as claimed in claim 12, wherein said modified protein is a recombinant protein in which the protein sequence of the platelet receptor binding site is mutated.

16. A method as claimed in claim 12, wherein said modified protein is mutated vitronectin.

17. A method as claimed in claim 12, wherein said biomaterial selected from the group consisting of stents, catheters, guide wires, arterial grafts, cardiac pacer leads, prosthetic heart valves, cardiopulmonary bypass membranes, automatic implantable cardiodefibrilator leads, ventricular assist devices or artificial hearts.

18. A method for reducing the occurrence of thrombosis in a patient following implantation of a biomaterial in said patient, said method comprising:

(a) affixing to at least a part of said biomaterial a modified, platelet-binding adhesive - 20 -

protein, said protein being modified at the platelet receptor binding site; and

(b) implanting the biomaterial of step (a) into said patient.

19. A method as claimed in claim 18, wherein said modified protein is a recombinant protein in which the protein sequence of the platelet receptor binding site is mutated.

20. A method as claimed in claim 18, wherein said modified protein is mutated vitronectin.

21. A method as claimed in claim 18, wherein said biomaterial selected from the group consisting of stents, catheters, guide wires, arterial grafts, cardiac pacer leads, prosthetic heart valves, cardiopulmonary bypass membranes, automatic implantable cardiodefibrilator leads, ventricular assist devices or artificial hearts.

22. A method for reducing the incidence of infection in a patient, resulting from the implantation of a biomaterial in contact with the blood of said patient, said method comprising affixing to at least a part of said biomaterial, prior to implantation in said patient, a modified form of an adhesive, platelet-binding protein, said protein being modified at the platelet receptor binding site.

23. A method as claimed in claim 22, wherein said modified protein is a recombinant protein in which the protein sequence of the platelet receptor - 21 -

binding site is mutated.

24. A method as claimed in claim 22, wherein said modified protein is mutated vitronectin.

25. A method for reducing the occurrence of restenosis after surgical intervention in a blood vessel, said method comprising implanting at least one stent in said blood vessel, said at least one stent having on at least a part of the surface thereof a modified form of an adhesive, platelet-binding protein, said protein being modified at the platelet receptor binding site.

26. A method as claimed in claim 25, wherein said modified protein is a recombinant protein in which the protein sequence of the platelet receptor binding site is mutated.

27. A method as claimed in claim 25, wherein said modified protein is mutated vitronectin.

28. A method for reducing platelet-fibrin thrombus formation of stents with subsequent myocardial infarction.

29. A method for reducing the need for anti -platelet therapy following implantation of a biomaterial, comprising: a) affixing to at least a part of said biomaterial a modified, platelet-binding adhesive protein, said protein being modified at the platelet receptor binding site; and - 22 -

(b) implanting the biomaterial of step (a) into said patient, the presence of said modified protein inhibiting thrombus formation thereby reducing the need for administration of agents that inhibit platelet function.

30. A kit for preparing implantable biomaterials of reduced thrombogenicity, said kit comprising: a) a lyophilized preparation of a modified form of an andhesive, platelet binding protein, said protein being modified at the platelet receptor binding site; b) sterile buffer for reconstitution of said lyophilized preparation; and c) a means for applying said preparation selected from the group consisting of syringes, tubing, and application chambers.

31. A kit as claimed in claim 30, wherein said lyophilized preparation comprises a recombinant form of vitronectin, in which the Arg-Gly-Asp sequence of the platelet receptor binding site is mutated.

32. A kit as claimed in claim 31, wherein said mutated sequence is Arg-Ala-Asp.

33. A kit as claimed in claim 31, wherein said mutated sequence is Arg-Gly-Glu.

34. A kit as claimed in claim 30, further including at least one implantable biomaterial for - 23 -

coating selected from the group consisting of stents, catheters, guide wires, arterial grafts, cardiac pacer leads, prosthetic heart valves, cardiopulmonary bypass membranes, automatic implantable cardiodefibrilator leads, ventricular assist devices or artificial hearts .