CA2176874C - Fibrin d-domain multimer coated prostheses and methods for their production - Google Patents

Abstract

Methods for coating a prosthetic surface with anti-thrombogenic, or anti--coagulant, proteins are disclosed. The methods involve contacting a surface of a prosthetic material with a composition containing multimers of fibrin degradation products. The fibrin multimers, preferably D-dimers, have cross-linked D- domains. The methods of the invention are useful for providing an anti-thrombogenic coating on prosthetic implants which are exposed to a patient's blood after implantation, for example vascular grafts and artificial heart valves.

Description

05/16/1996 18:06 2066820446 STRATTONBALLEW-PALEV PAGE 09 FIBRIN D-DOMAIN MULTIMER COATED PROSTI-IRlSESI AND METHODS
FOR TIiEIR PRODUCTION

T~ni~it~!si The invention relates to methods for coating prosthetic surfacea with unti-thrombogenic proteina, and to prosthetic devices prepared by those methods.

Racitgrot!nd of the Invention A major limitation in developing prosthetic implants for medical uso is that most prosthetic materials thus far developed tend to be excessively thrornboQenie, i.e.
they trigger excessive blood clot formation, or thrombosis, at surl'aces of the implant exposed to the patient's blood. This problern can lead to severe complications, t s particularly in the case of prosthetic vascular grafts used to corndct coranary artety disease, lower limb ischemia, arterial aneurysms and othercirculatory problems. About 20% to 30% of patients undergoing vascular bypass or replacement surgery require prosthetic grafts, because autologous veins are unavailable due to previous surgery or other roasons. In these patients, there is a high rate of graft failure due to the high tlirombogenicity of synthetic materials used to produce the prosthetic graits.
One multi-center study demonstrated that the cumulative 4-year patency of prosthetic grafts used for distal arterial reconstruction was only 12% for polytetrafluoroethylene (PTFE) $rafls; far lower than the long-term patency obscrved for autologous gratts. Vieth et al.
(J_ Vasc.
h.1tCg. 3; 104-114, 1996).
The mechanism by which thrombosis occurs in prosthetic vascular grafts is generally well u.nderstood. Shortly affter implantation of a synthatic graft, adsorption and accumulation of blood proteins on the lumenal surface of the graft begins. ]n particular, a surface coating of polymorized flbrin, referred to as the pseudointims, appears first, followed by an accumulation of thrombin bound to the fibrin. The thrombin contributes to platelet activation and fortnation of a platelet rich thrombus. Bound thrombin may also contribute to further fibrin accretion that in turn leads to distal embolism.

_, _ _ _ .

05/16/1996 18:06 2066820446 STRATTONBALLEW-PALEV PAGE 10 217 6 8 7 4~-An important diPPettimce between prosthetic and autologous vascular grafts is that prosthetic grafts lack a lining of endothelirl cells which cover the lumenal surface of natural vessels. It is widely believed that the endothclial lining provides an anti-thrombogenic effect and is among the most important factors in tneintaining long term patency in autologous grafts. Accordingly, numerous atternpts have been made to artiticially seed endothelial cells onto surfaces of prosthetic gratts, (reviewcd by Mosquera and Goldman, Br. J. Surg= 71: 656-660, 1991). Most of these efforts involved coating the prosthetic surface with a "biological glue," comprised of adhesive proteins and other materials which enhance endothclial cell adhesion and growth. These to investigations have used a variety of extracellular matrix proteins and other matcrials, including fibrin, fibrin gels cross-linked with factor XiII, fibronectin,laminin, various forms of collagen, aibumen and blood. Although endothelial seeding of gratb has shown benef3cial results in experimental settings, the technology has not progressed sufficiently that seeded grafts suitable for routine use can be produced.
ts Another primary focus of research concerning prosthetic vascular grafts has involved efforts to develop non-cellular, anti-thrombogenic eoating:i, such as protein coatings, which may reducc the thrombogenicity of implanted grafts. One such study involved coating prosthetic surfaces with the anticoagulant protein heparin.
Nagaoka et al. (A,rLifici Organs 17: 598-601, 1983). Other studies have used natural, extracellular 2o matrix materials to coat vascular grafts. b'or example, European Patent No.

issued to Sawamoto et al. discloses polymeric materials coated with a hydrolyzed fibrin layer optionally cross-linked with factor XIII. In addition, U.S. Patent No.
5,324,647 issued to Rubens et al. discloses polymeric rnaterials coated with either a layer of ribrinogen, thermally denaturcd fwbrinogen or factor XIII croas-linked fibrin.
While 2s various of these natural coatings are reported to have anti-thrombogenic eflf'ects, prosthetio materials treated with such methods have not yct been widely employed in a clinical setting.
In view of the above, there is a clear need in the art for prosthetic materials having improved biological compatibility over currently availabie materials.
In 3o particular, there is a need for prosthetic materials which hava reduced thrombogenicity compared to available synthetic materials used for prostheses. Such materiala should be 05/16/1996 18:06 2066920446 STRATTONBALLEW-PALEV PAGE 11 resistant to the deposition of blood proteins and to platelet adherence. These materials would be useful for producing vascular grafts, synthetic heart valves, a.rtificial organs and in any other application where a surface of the prosthetic material will be exposed to a patient's blood, so as to create a potential for thrombogenesis at the exposed prosthetic S surface. The preserit invention provides such materials, as well as methods for preparing those materials. Materials prepared in accordance with the methods of the invention are useful for providing biocompatible implants, medical treatment methods employing such implants, as well as useful materials for experimental modeling of thrombogenic and fibrinolytic proccsses.
D'~1osq,re o the Inventinn The present Invention provides methods for coating prosthetic surfaces with anti-thrombogenic proteins, and prosthetic devices prepared according to those methods. The methods of the invention generally comprise contacting a prosthetic f s surface with a composition containing multimers of fibrin dcgradation products which have cross-linked D-domains, to produce an anti-thrombogenic coating of the multimers on the prosthetic surface. Fibrin degradation products useftil within the invention are limited to fibrin degradation products posscssing fibrin D-domains, preferably in the form of D-monomers or D-multimers generated by plasmin lysis. The multimers are also 2o preferably formed by cross-linking the D-domains with Factor Xi1I, before or after the fibrin degradation products arc generated.
It is preferred within the invention to contact tha prosthetic surface with a composition that consists essentially of the multimers of fibrin degradation products in a suitable carrier. It is also preferred to use a liquid carrier to form a solution that is 25 substantially free of intact fibrin, and of fibrin degradation products laeking D-domains.
In alternate ernbodimcnts of the invention, one or more additional agents are added to the composition of multimers to enhanca endothelial cell growth on the anti-thrombogenic coating after the prosthetic surface has been contacted with the composition. Useful proteins for this purpose include a variety of extra.ceilular matrix 30 materials as well as chemotactic and/or cell growth factors. Specific agents contemplated for use in this manner include basic fibroblast growth factor, endothelial cell growth factor, u2 maeroglobulin, vittoneetin, Qbronectin, and oell-bindiag fra:gtnenta of fibronectin. In a related embodiment, coated prosthetic surfhce.a treated according to one of the above methods are seeded with endothelial cells to Ruther enhance biocompatibility of the coated eurface.
With1n Another aspect of the invention, the composition of multimps Is a solution, and the prosthetic surfaca Is incubated In the solution within a aelected temperature nnge. Within one embodimient, the incubation is oartied out between approximately 20 C and 25 C. Within another embodiment, tbo incubation I.
conducted at a tcmperature aumcicnt to thermally denature the multimers, between about 56 C and 1000 C, preferably at about 70 C. in order to enhance adheronco of the multimers onto the prosthetic surfacx.
Preferred prosthetic materials for use withln the invention include a broad range of biologically compatible polymers known to have rnedically useful structures and prosthetic surface characteristics. Useful polymera witbin this context Include polyethyieneterephthaiate, polytetrafluoroethylene, cxpanded polytdratluoroethyiene, polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethanes, polypropylenes and polyesters. Other usetW materials with suitnble p[osthetic surfaces include stainless 9teel, titanium. cobalt chrome alloys and silivon-beaod materials.
Within another aspect of the invention, a variety of prosthetic devicea are provided, which are made according to the coating methods of the invention. In alternate embodiments, the prosthetic surface to be eoated is a surface of an artiflcial vascular implant, duct implant, heart valve, bone implant, patch or other artiticial impluit.

wthin yet other aspects of the invention are prosthetic members comprising a coated prosthetic surface of this invention as well as the use of such a member for treatment of a manunalian patient by implantation of the member.
These and other aspects of the invention will become apparent upon reference to the following detailed description.

Detailed Description of the Invention The present invention provides methods for coating prosthetic surfaces with anti-thrombogenic proteins, and also provides prosthetic devices prepared according s to those methods. The methods of the invention generally comprise contacting a prosthetic aurface with a composition containing multGncts of fibrin degradation products which have cross-linked D-domains, to produce an anti-thrombogenic coating of the multimers on the prosthetic surface. As used hercin, "anti-ttuombogenic costing" refers s to a surface layer, preferably a continuous surface coatin& which confers a medicaily significant reduced thrombogenicity. or blood clot Inducing affict, when the costed prosthetic surface Is exposed to a platelet preparation, or to an actual patient's blood after itnplamtation, cornpared to thrombogenicity of an unc,oated surfaco. To determine a medieally significant reduction In thrombogenictty, uneoated and coated prostheaes eire to preferably implanted into a mammal fot a suHicient period of tUne for adsorption and accumulation of blood proteins on a lumcnal surface of the uncoated prosthetie surface.
Subsequently, the uncoated and coated proAeses arc rcmoved and comparatively analyzed microscopically, or by other conventional methods, to deterroine whetber the coating of multirners on the prosthetic surface confers an anti-thrombogenic etFect. i.e.
I s whether thrombus formation is significantly reduced due to the presence of tha coating.
Altereatively, several in vitro models may be used to determine the anti-thrombogenic effect of the multimer coating. for example models which compare protein adsflrption andlor platelet adhasion on native fibrinogen coated surfficm to adsorption and/or adhesion on rnultimer coated surrtiaces.
20 A variety of fibrin degradstion products me usotltl within the invention, but they are limited to degradation produets which include substantially eomplete fibrin U-domains. As used herein, "D-domains" of fibrin Qenerally refea to globular portions of the fibrin molecule disposed near opposite ends of the molecule. T'tK D-domsin includes roughly two-thirds of the carboxy termini of ttie fibrin and y cbains, and a 25 small portion of ihc carboxy termimts of the fibrin a chain (see iis~mta8i3~ud Thrninhnsis, Ba:ic Prin ts and Clinical P ic= 3d edition, eds. R.W. Cobnan et al., J.B. L.ippincott, Co., Philadelphia, !'994, and sec in particular Chapter 14, Fibrinogen Siructwe and Physiology, p. 271).
The fibrin degradation products useful in the invention are preferably D-30 monomers or D-multimers generated by plasmin lysif. Plaat-in digestion of ctoas-linked fibrin in the presence of calcium gives ri:e to a series of croa-tinked interqAediate fragments Including D dimers, trimers and tetramers, with D-0imen predominating (Slebeniist et al., J. iol. Chem. 2A2: 2841429419, 1994; Robson et al., $pi.l.
tlaemato1. M: 322-326, 1994). A
suitable method for preparing D-domain from purified human fibrinogen is described in Delvos et al. (bleClri0S18~s18: 99-103.19$0).
Briefly, Fibrinogen is incubated with plasrnin in the pnesence of CaCI= and, after the digestion has progressed an apprapriate amount of time to yield the desired end products, the reaction is terminated by the addition of aprotinin. The digest is then passed over a Sepharose-lysine column, as described in Haverlcate et al.
(T1upmbJRem.1II: 803-to 812, 1977). The effiuent is then dialyzed against a sodium bicarbonato buffer and eluted fYom a DEAE-Sephadex column with a linear pH/salt gradient betvreea the dialysis butyer and bicarbonate buffer. Samples ftom each protein-containing fraction are then asacsaed by SDS-PAt3E. Fractions with the highest purity of D-domain arc eoncentrated 10-fold by ultrahltration. To prepare D-dimers, buman fibrinogen is Incubated in the preseme of is rFXIII, as described in Haverkate et al. (Fur_ J. Clin. Invest. Q: 2S3-2SS, 1979). The cross-linked fibrin clot is squeezed with filter paper to remove entrapped liquid, frozen, lyophilized, ground to a fine powder, and resuspended (approx. 10 mg/mL) in an appropriate buffer containing plasmin. After plasmin digestion, aprotinin is added and the solution is chromatographed on Sepharose-Iysine. D-dimer Is Isolated by passage of the 2o e#liuent through a Sephacryl S-300 HR column. Fractions identified as containing D-dimer are then concentrated by ultrnfiltmtion.
Although D-dimers are the preferred flbrin degradation products for usa in the invention, other fibrin desradation products having substantially complete D-domains are also contemplated. Such alteraate degradation products can be generated by a variety 23 of convcntional methods, auch as limited trypsin digestion.
In a prefcrred embodiment of the invention, the prosthetic surface is contactcd with a composition that consists essentially of the muftimers of fibrin degradation products in a suitable earrier. A composition is defined as "consisting essentially of multimers of fibrin degradation products" if there aro no additional 30 substances preaent In the composition that materially alter the anti-thrombogenic effect of the multimer coating. It is also prefeered to use a composition that Is substantially free of 05/16l1996 18:0E 2066820446 STRATTONBALLEW-PALEV PAGE 15 intact fibrin, and of ftbrin degradation products lacking D-domains. A
solution is considered substantially free of intact flbrin and of ilbrin degradation products lacking D-domains when it contains about 90% pure, m.ultirnerie fibrin dcgradation products that include D-domains. The composition which contains the multimcrs can be in the form of a liquid solution, a solid, such as a fine powder, a paste or a colloidal suspension.
Suitable carriers include non-corrosive, biocompatible, solid or liquid carriers suitable for application to prosthetic materiais, and in which the multimors remain intact and cross-linked.
Within a preferred embodiment of the invention, the composition of to multimers is a solution, and the prosthetic surface is incubated in the solution within a selected temperaturc rangc. Preferred solutions include standard biological or medical buffers, such as low ionic strength aqueous buffers at near neutral pH, for example tris buffered saline (TBS). The incubation is carried out at a temperature at which the solution is a liquid and the prosthetic surface is not degraded. Within one embodiment of 13 the invention, the incubation is carried out at less than 56 C, preferably less than 37 C, most preferabiy at about 20-25 C, so that the multimers remain in an undenatured state.
tfowever, it may be preferable in certain applications to carry out the incubation at a tcmperature sufficient to thermally denature the multimers, betwcen about 56 C and 1000 C, and preferably at about 70 C, to enhance adsorption of the muitimers onto the 20 prosthetic surfacc.
Whcn a solution of multimers is used to coat the prosthetic sttrfaee, it is generally desired to provide a eoncentration of multimers in the solution of between about 0.05 mg/mt and 100 mg/ml. Preferably, the solution has a multimer concentration of betwesn about 0.1 mg/ml and 10 mglml. Higher concentrations may be employed 25 however, depending on solubility considerations, which may Improve the rate or extent of adsorption of the multimers onto the prosthetic surface, Preferred prosthetic materials for use within the invention include a broad range of biologically compatible polymers known to have generally useful structures and surface characteristics for medical or experimetttal, prosthetic purposes.
Useful polymers 30 within this context include polyethyleneterephthalate (eg. Dacron ), polytctrafluorocthylene, cxpandcd polytetrafluorocthylcne (cg. (3ore-Tcx%, W.L. Gore, 05/16/1996 18:06 2066820446 STRATTONBALLEW-PALEV PAGE 16 :

Flagstaff, Ariz.), polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethanes, polypropylenes and polyesters. Other useful materials with suitable prosthetic surfaces include stainless steel, titanium, cobalt chrome alloys and silicon-based materials.

The inethods of the present invention are usoful to produce a variety of prosthetic devices, such as artificial vascular implants, duct implants, urological implants, internal organs, heart valves, bone implants, patches, webs, or other artificial prosthetic structures, including those that are exposed to blood flow after implantation.
More specifically, artificial duct implanta which may be coated according to the methods of the lo invention include artificial urinary ducts, artificial kidney tubules, artificial lymphatic ducts, artificial bile ducts, artificial pancreatic ducts, indwelling catheters, shunts and drains. It is also contemplated that the methods of the invantion may be effectively employed for imparting an anti-thrombogenic coating to heterograft or xenogratt tissue or organ implants. Surfaces of such devices and implants are prepared as disclosed herein, i s and the devices or implants are implanted in a patient according to standard surgical techniques. In addition, the coated surfaces of the present invention provide a usefut in vitro model for studying tibrin accretion, platelet adhesion and other phenomena related to vascular graft failure, as well as for testing of potential anti-thrombogenic agents.
Coated prosthetic surfaces prepared according to the present Invention may 2o be seeded with endothelial cells. Endothelial cell seeding of vascular grafts and othar surfacos exposed to the blood provides prosthetic materials that are actively anti-thrombogenic and exhibit increased resistance to occlusion. The stability of the muttimcr coating makes it an excellent substrate for endothelial call seeding. In aiternate embodiments of the invention, one or more additional agents arc added to the 25 composition of multimers, to enhance endothelial call growth on the prosthetic aurface after it has been coated. Useful proteins for this purpose include a variety of extracellular matrix materials, as well as chemotactic and/or cell growth factors. Specific agents contemplated for use in this manner include basic fibroblast growth factor (basic FCih') and endothelial cell growth factor (vascular endothelial cell growth factor), ai 30 maeroglobulin, vitronectin, fibronectin, and fibronecdn fragments containing binding determinsnts for endothelial cells. Freparation ofthe4o proteins and protein fragments is within the level of ordinary skill in the art. See, for example, C3ospodarowicz et al., U.S.
Pat. Nos. 4,785,079 and 4,902,782; Obam et al.. VEBSjatC 2U: 261-264, 1987;
Dufour et al., PMROt, I: 2661-2671,1988; Tischer et al., WO 91/02058; Boel et al., ?,Q: 4081-4087.1990; Ruoslabti et a1., U.S. Pat. No. 4,614,317;
s Pierschbacher et al.; U.S. Pat. No. 4,589,881; and Suzuki ct al., EMBO d.4;
2519-2524, 1985. These proteins will generally be Included in the multimer composition at concentrations between about 0.5 and 10 g/mL.
E.ndotbelial cells are obtained by standard proeedures ftom umbilical vein, to saphenous vein or other souroca. See, for example, Balconi et al., bdid, BigL 54 231-243, i986; Ryan et al., Tisna Cell jZ: 171-176, 1985: Budd et al.,M
I..$uCg.26: 1259-1261, 1989. Cells can be harvratted by mechanical or, prcferably, enzymatic methods.
Briefly, veins are flushed to remove blood and tilled with a coliagenase solution to dislodge the endothelial cells. The cells are collected and cultured in a conventional 13 medium, generally at about 370 C. in a 3 /. CO2 atmosphere. Satisfactory attachment of the cells to the coated prosthetic surface is generally obtained within one to two hours. In the alternative, cultuR+ed endothelial cells are added to the composition containing the muitimers, thus trapping them vrithin the coating. Ths coated prosthetic material is then incubated to allow the cells to reproducz.
20 Factor XIII for use within the present invention may be prepared from piasma according to known methods. such as those disclosed by Cooke and Holbrook (HWdl=,y ,14,1: 799-84, 1974) and Curtis and Lorand (MethndaEazXID41. !a: 177-191, 1976). The aj dinur form of factor X1I1 may be prepared from placenta as disclosed in U.S. Pat. Nos. 3,904,751; 3.931,399;
4,597,899 Zs and 4,283,933. It is preferred, however, to ust recombinant factor X1lI so as to avoid to the use of blood- or tissue-derived products that carry a risk of disease tranamission and avoid contamination with other protcins.
Methods for preparing recombinant factor XIII are known in the art. See, for example. Davie et al., EP 268,772 and (3rundmann et al., AU-A-69896/87.
30 Within a profcrred cmbodiment, the factor XIII a2 diiner is prapared cytoplasmieally in the ye" Saccharmnyces cerev-siae.

The celia are harvested and lysed. and a cleared lysate is prepared. The lysate I.
fractionated by anion exchange chromatography at neutral to slightly alkaline pH using a column of derivatized agat+osc, such as DEAE FAST-FLOW SEPHAROSETm (Pharmacia) or the like. Factor XIII is then prccipitated from the eolumn eluate by 5 concentrating the eluate and adjusting the pH to 5.2-5.5. such as by diafiltration against ammonium succinate buffer. The preeipitate is then disaolval and further purified using conventional chromatographic teehaiques, such as gel liltrstion and hydrophobic interaction chrornatography.
Although it Is prefenrcd to use the factor XIII &2 dimer within the present io invention, other xymogen forrtms of factor XIII (e.g. e7bz tetramer) may be used. Zymogen factor XIII is activated by thrombin present on the tibrin nutbce. However, activated factor XIII (factor Xilla) may also be used.
Proteins for use within the present invention (inoluding thrombin, plasmin, fibrinogen and factor XIII) can be obtained from a variety of mammalian sources, such as ts human. bovine, porcine and ovine. As will be appreciated by those skilled in the art, in prosthetic applications it is prefenmd to use proteins syngenesious with the patient in order to reduce the risk of inducing an immtme rqponse. Non-human proteins are particularly useful in the preparation of materials for veterinary use or for use in experimental models.
The following examples are offered by way of 9llustration, not limitation.
EXAMWU
MaterimLis For use in the cxperimmts pmeated below, Hepes, Tris, and porcine Zs heparin wwe obtained front Sigma Chemical Co., St. Louis, MO; D-phenylalanyl- L-prolyl-L-arginyl chloromethylketone (FPRCH= Cl) from Glbiochem, San Diego, CA;
the chromogenic substrate 5-2238 from Helena Laboratories, Mississauga, Ont., Canada;
Nat29 I from ICN Canada Ltd., Montreal, Canada; the Enzymobead reagent from BIo-Rad Laboratories, Mississauga, Ont. Canada; Na="CrOil (200-500 Ci/g) from New England Nuclear, Dorval, Que., Canada; SP SephadexTM, Sephadex G-25T"", DEAE-Cellulose, DEAE
SephadexTM, Sephacryl S-300 HRT"" and SepharoseTM-lysine from Pharmacia Fine Chemicals, Piscataway, NJ. Polyethylene tubing (tntramedic PE 240, internal diartuter 0.17 cm) was obtained from Clay,Adamy, Parsippany, NJ. This tubing was rinsed with methanol and incubated overnight in Tris buffered saline (TBS, 0.1 M NaCI, 0.05 M Tris, pH
7.4) befort It was exposed to protein eolutions.
Hunian plasmin was obtained #irom Helena Laboratories,.Bcaunaont, TX;
bovine albumin from Bochringer Mannhcim Canada, Dorval, Que, Canada; aprotinin from Mobay Chemical Co., New York, NY; human a= thrombin was a Qenerous gift of Dr. J. Fenton II, New York State Department of Health, Albany, NY; apyraa was prepared from potatoes as described previously (Kinlough-Rathbone et al., ln; Methods 3u HSn1.a,tQlAip-, Measirentents of Pl~tetet Ft!~tian L.A. Harker et al., eds.
Churchill Livingstone, Edinburgh, 1983; pp 64-91). Recombineutt human platelet factor XiII
(rFXIII) was provided by Zymogenctics, Seattle, WA. 'ilte characteristics of this pratein have been weli-defined (Bishop et al., $i0Ch01rtixtqc 24, 1861-1869, I990).
and under reducing conditions it migrated as a single band on SDS-PA(3E. Human fibrinogen was is purified from citrated human plasma by sequential P- al,tnine precipitetion (Straughn et al. Thrombos. D'athes. l semerrh. (StuttQl 16: 198-206, 1966), and was 93-94%
clottable.
This fibr'tnogen was further puritied to remove factor XIII, fibrin degradation products, and plasminogen using DEAE-cellulose chromatography according to the method of Lawric ot ai. (Bi he . Soc. Trans. _: 693-694, 1979).

D-domain was prepared Gom purifie4 hwnan tibrinogen according to the method of Delvos et al. (HumajjasjA1$: 99-105, 1988). Fibrinogen (100 mg in S
mL
TBS) was incubated with plasmin (I S casein units/mL) for 18 h at 37'C In the presence of zs 2 mM CaCI2 . The reaction was teaainated by the addition of aprotinin (100 [UU/mL), and the digest was passed over a SepharoseT""-lysine column. Haverkate et al.
(Thromb.
Res. 10:803-812, 1977). The effluent was dialyzed against 10 mM sodium bicarbonate buffer (pH
8.9) and eluted from a DEAE-SephadexTM column with a linear pH/salt gradient between the dialysis buffer and a 10 mM sodium bicarbonate buffer, pH B.O.
eontaining 0.3 M NaCI. Samples from each of the protein-cotttatninS flractions were assessed by SDS-PAGE. Fractions with the highest purity of D-domain, veritjed as mom than pure by SDS-PAGE, were concmitntod 10-fold using an ultn-tiltration eeil (Cantriprep Concentrator, Amicon Division, W.R. Orace & Co., Danvers, MA) and stored at -70=C.
D-dimers wac preparod from htunat tibrinogen (200 mg in l0 mi. of TBS) incubated at 37'C in the presence of rFXIII (S g/mL) and 30 U/mL huznan ac-s thrombin. Haverkate et al. (F>=r_ 1_ clin_ In.reat_ S: 233-255, 1979). The crose-linked fibrin clot was removed, squeezed with filter paper to reanove entrapped liquid, frozen at -70'C, lyophilixed, ground to a tine powder, and resuipended (approx. 10 mg/mL) in a buffer at pH 7.8 containing 0. 15 M NaCI. 0.05 M 1'ris. 5 mM CsC12. ard 0.13 camin units/mG of plasmin. Brenna at ai. U. Lah. Clia.Med-1.13: 682-688,1989). After ir.cubation at 37'C for 18 h, aprotinin (100 K1Y/mL) waa added and the solution was chromatographed on SepharoseT""-lysine. D-dimer was isolated by passage of the effluent through a Sephacryl S-300 HR column cluted with TBS coetaining 0.02$ M sodium citrate, pli 7.4. The column had been calibrated with Bio-RadT"" gel filtration molecular weight standards. Fractions identified as containinQ D-dimer (195 kDa), verified as more than 90% pure by SDS-PAGE, were concentrated by ultraflltration as dawcribed tbove and stored at -701C.

Radlnl hel nar nf PrnteirL
Fibrinogen, D-domain, and D-dimer wera labeled with 1251 using Enzymobeads according to the supplier's protocol. Enzymobeads and free Iodide were removed by filtration through Sephadex G-25. Specific nadioactivities were;
flbrinogen 2 x 10s cpm/ g; D-domain 3.0 x l0r cpm/pg; D-dimer 1.0 x 10s cpm/pg. SDS=PAGE
and autoradiography of the 13s1-labeled fibrin and fibrin degradation products showed them to migratc as single bands that were coincident with their non-radioactive counterparts.

P1+ennration of H Mn Pla elet- Red Blood uRus,eensie~
Blood was obtained from donors who, in the pt+evious 2 weeks, had not reccived drugs that affect platelet function. Platelets were isalated, labeled with "Cr (10 Ci/mL) in the fitst washing fluid, and resuspended In 'Cyrode-tlburnin solution which contained 2 mM CaCi,, 1 mM MgC12, 0.14 M NaC1. 2.7 mM KC1, 11.9 mM NaHCO3, 05/16/1996 18:06 2066820446 STRQTTONBALLEW-PALEV PAGE 21 0.42 mM NaH2PO4, I mg/mL glucosc, 5 mM Hepes, 0.35% albumin, and 50 pL/mL
apyrase (pl 17.35) (Kinlough-Rathbone et al., ln: 11+tet Qds in Hgmgl0loey.
Measurements of Platelet Function L.A. Harker et al.. eds. Churchill Livingstone, Edinburgh, 1983; pp 64-91; Mustard et al., In: MSthods in EtI27tmQ14gXa PlatClt'dSo BweRtors.
Adheaion_ SGrtSfipp, Vol_ 1ff'Part A., J.J. Hawiger, ed. Academic Press, New York, 1989, pp 3-11;
Cazenave et al., J. Lgb.Cliq-m,pgL ,$,}: 60-70, 1979). Red blood cells were Isolated as described previously (Ca2enave et al., I, i ab,s'lin_Med. 23: 60-70, 1979) and added to the platelet suspension (40% hematocrit). The platelet count wss adjustaf to 500,0001 L.

lo Fxample I
Comnarative ThrombagoWilXof Fjbrin tiDg, od FgAgL IIj CXoss-linked Fibrin Coating, of PolXgthyjgneTybipg To assess the potential and-thrombogenic cffect of cross-linking of flbrin, polyethylene tubes were incubated in a labeled fibrinogen solution, cither with or without the inclusion of 1 ug/mL of rFXIII in the solution. Segments of PE 240 tubing, 12 cm in length, were coated with thermally-denatured fibrinogen (Rubens et al., j, HiQmed.Mol.
$g,g. 2~: 1651-1663, 1992) by filling them with a I mg/mL solution of fibrinogen, with or without i}tg/mL of rFXIII, and heating the filled tube segments for 10 min. at 70'C. The segments were perfused with 40 mL of THS and then filled with a 1 g/mL
solution of a-thrombin in 0.025 M NaCI, 0.025 M Tris, pH 7.4. After 5 min. at room temperature, the tubes wcre washed with 4 mL of this buffer and then perfused with 4 mL of TBS
containing fibrinogen (I mg/mL), 11s1-fibrinogen (1 pg/mL), and 2 mM CaCI=.
After incubation for 5 min. at room temperature, the tubes were perfused with 40 mL
of TBS
and, unless indicated otherwise, were filled with 10uM FPRCH2C I in TBS and incubated for 5 min. to neutralize surface-bound thrombin. (Treatment with FPRCH2C1 was found to completely neutralize surface amidolytic activity as measured by the chromogenic substrate S-223 8). The tubes were rinsed with 20 mL of TBS and platelet accumulation was measured immediately, Representative tubes (not perfused with the platelet; red blood cell suspension) were filled with I x reducing sample buffer (Bio-Rad) and 3o incubated at 37'C for 24-48 h. This treatment removes protein from the PE
surface.

Aliquots of the solubilized tubc eontents, containing 20,000 cpm, wero analyzed by SD3-PAGE.
To evaiuate the eomparadve thrombogenicity attributable to the flbrin coating and ct+oss-linked fibrin coating trcatments, platelet accumulation on the differently treated tube segmer;ts was compared. The coated tube tegnents were attached to silicone tubing qnd aasernbled on a peristaltic pump (Drake-WillockT"", Mode14504, Portland, OR).
Suspensions of s'Cr-Labeled Platelets with unlabeled red blood cells at 37'C
wera perfused through the tubing for 10 min. at 10 mUmin. and a wall shear rate of 764 aec The segments were then perfused under ehe same conditions for 10 min. with the moditied Tyrode solution desaribed above. The s'Cr remmalning in the segments was measured using a PackardT"" gamma counter. The number of platelets that accumulated per mm2 was calculated from the surtltce area of the Inside of the segments and the specific radioactivity of the platelets In the platelet: red blood cell suspension.
Data from these studies were determined as a mean-t t+tandard deviation (S. D.), a d were analyzed by ts Student's t-test (two-tailed).
The above studics showcd that Including rFXIIl (1 Ng/mL) In the solution of labeled fibrinogen did not diminish the amount of labeled fibrin on the PE
tubing, and perfasion did not alter the amount of labeled fibrin on the surface. Analysis by SDS-PAGE of the surface of tubas coated with fibrin in the prewnee and absence of rFXIII
9howcd the presence of an approximately 100 kDa band, correspondinS to the y-r dimer.
in the fibrin formed in the presence of rFX1II, confimning that cross-linking had occurred.
The inclusion of rFXIII (I g/mL) in the solution used to coat the tubing with labeled librin subatantially decreased the exaent of platelet socumulation from a value of 46,9741 9702 platelets per mm1(no rF)III) to 36,111 8 17964 plateleb per mm= (with rF3QII) ( n-12, p<0.01).

F.xim~lo2 Semnerrtive Thrambogeeeai ef D-Domwin C ng. and Fector ti Cren-linlked D-I? jõcner ontina_ nf Polyet ene Tubinn To as9css the potential anti-thrombogenic effect of cxoss-linkino of D-domain portions of fibrits, polyethylene tubes were incubated in a solution of either ts labeled fibrin D-domains or D-dimers, prepared aa desadbod above. Segmenb of tubing, 12 cm in length, were filled with solutions of D-domaln or D-dirner at 1 mg/mL in T$3, containing tracer amounts of the correeponding "l-labeled fngment. Tho segments were incubated for 1 h at 23'C annd rinsed with a modified Tyrode solution (without Ca2' or Me+ , but containing 0.01 M EDTA and 0.1% glucose). Platelet accumulation was meaaured as above. BrieBy, the caded tube segments wero attached to a peristaltic pump with silicone tubing, and suspensions of s'Cr-Labeled Platelds with unlabclcd red blood ce11s at 37'C were perfimed througb the tubing for 10 min.
at 10 mUmin. and a wall shear rate of 764 see 't. The segrnrnts were then perfused unckr the io same conditions for 10 min. with the modified 1'yroda solutiop described above. 'flte "Cr rtmaining in the segments was measured using a PackardT"" gamma counter. The number of platelets that accumulated per mm3 was calculated as above.
These studies showed that platelet accumulation on D-dimer coated pofyethylene surfaces was greatly reduced compared to piatelet actamtulatlon on non is cross-linked, D-domain coatod surfaces. The surface protein eoncentration of the tubing coated with D-domain was 0.70 t 0.02 g/cm3, and the number of platelets that accumulated at high shear was 34.3$3 3905 per mm1(n-3). In contrast, the surface protein cottcentration of P4 tubing eoated with D-dimer wsu 0.13 # 0.05 g/cm=, and the number of platelets that accumulated was 563 257 per mm= (n--4). Thus, the 20 thrombogenicity of the D-dimer coated surfaces nteasured 60-fold lass than that of the D-domain coated surfaces. Even when these data are approximately normalized to account for the i'ive-fold lower protein ads,orption of the D-dimers to the tubing, eomparod to the protein adsorption of the D-domains, it is nonethess evident that the D-dimer coating ls at least ten times more anti-thmmbogenic on polyethylaro tubing then the D-dottfain 25 coating.
Although certain embodinents of the inventlon have been described In detail for purposes of illusttation, It will be readily apQmnt to those skilled in the art that the methods aad producta described herein may be modified without deviating frotn the spirit and scope of tihe invention. Accordingly, the invention Is not limited by the above 30 deycription but is to be detesmined in scope by the ciaima which follow.

Claims (36)

1. A method of coating a prosthetic surface with an anti-thrombogenic protein, comprising the step of:
contacting said prosthetic surface with a composition comprising multimers of fibrin degradation products in a suitable carrier, the multimers having cross-linked D-domains, to produce an anti-thrombogenic coating of said multimers on said prosthetic surface.

2. A method according to claim 1, wherein the composition is a solution and the prosthetic surface is incubated in the solution at a temperature of between approximately 20°C and 25°C.

3. A method according to claim 1, wherein the composition is a solution and the prosthetic surface is incubated in the solution at a temperature of at least 56°C, but less than 100°C.

4. A method according to claim 1, 2, or 3, wherein the composition is a solution and a concentration of said multimers in said solution is between approximately 0.1 mg/ml and 10 mg/ml.

5. A method according to any one of claims 1 to 4, wherein the composition consists of a solution of said multimers in the carrier, substantially free of intact fibrin and fibrin degradation products other than said multimers.

6. A method according to any one of claims 1 to 5, wherein the composition contains one or more additional proteins selected from the group consisting of basic fibroblast growth factor, endothelial cell growth factor, .alpha.2 macroglobulin, vitronectin, fibronectin and cell-binding fragments of fibronectin.

7. A method according to any one of claims 1 to 4, wherein the composition consists essentially of said multimers of fibrin degradation products in the suitable carrier.

8. A method according to any one of claims 1 to 7, wherein the fibrin degradation products are plasmin cleavage products.

9. A method according to any one of claims 1 to 7, wherein said multimers are formed by cross-linking intact fibrin with Factor XIII and then generating the degradation products from said cross-linked intact fibrin by plasmin digestion.

10. A method according to any one of claims 1 to 9, wherein the prosthetic surface is formed of a biologically compatible polymer.

11. A method according to claim 10, wherein the polymer is selected from the group consisting of polyethyleneterephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethane, polypropylene and polyester.

12. A method according to any one of claims 1 to 9, wherein the prosthetic surface is formed of a biomedically compatible material selected from the group consisting of stainless steel, titanium, cobalt chrome alloys and silicon-based materials.

13. A method according to any one of claims 1 to 12, wherein the prosthetic surface is a surface of an artificial vascular implant.

14. A method according to any one of claims 1 to 12, wherein the prosthetic surface is a surface of an artificial duct implant.

15. A method according to claim 14, wherein the artificial duct implant is selected from the group consisting of artificial urinary ducts, artificial kidney tubules, artificial lymphatic ducts, artificial bile ducts, artificial pancreatic ducts, indwelling catheters, shunts and drains.

16. A method according to any one of claims 1 to 12, wherein the prosthetic surface is selected from the group consisting of a stent surface, an artificial urological implant surface, an artificial heart valve surface, an artificial internal organ surface, an artificial bone implant surface, a patch surface and a web surface.

17. A method according to any one of claims 1 to 16, further comprising the step of seeding the prosthetic surface with endothelial cells.

18. A method according to any one of claims 1 to 9, wherein the prosthetic surface is a surface of a heterograft or xenograft tissue or organ implant.

19. A prosthetic member comprising a coated prosthetic surface prepared according to the method of any one of claims 1 to 18.

20. Use of a prosthetic member of claim 19 for treatment of a mammalian patient by implantation of the member.

21. A use according to claim 20, wherein the prosthetic member is for replacement or bypass of one or more blood vessels of the patient.

22. A prosthetic member for use by implantation in a mammalian patient, the member comprising a prosthetic surface, the surface having an anti-thrombogenic coating comprising multimers of fibrin degradation products, wherein the multimers have cross-linked D-domains.

23. The prosthetic member of claim 22, wherein the coating is substantially free of intact fibrin and fibrin degradation products other than said multimers.

24. The prosthetic member of claim 22 or 23, wherein the coating contains one or more additional proteins selected from the group consisting of basic fibroblast growth factor, endothelial cell growth factor, .alpha.2 macroglobulin, vitronectin, fibronectin and cell-binding fragments of fibronectin.

25. The prosthetic member of claim 22, wherein the coating consists essentially of said multimers.

26. The prosthetic member of any one of claims 22 to 25, wherein said multimers are formed by cross-linking intact fibrin with Factor XIII and then generating the degradation products from said cross-linked intact fibrin by plasmin digestion.

27. The prosthetic member of any one of claims 22 to 25, wherein the fibrin degradation products are plasmin cleavage products.

28. The prosthetic member of any one of claims 22 to 27, wherein the prosthetic surface is formed of a biologically compatible polymer.

29. The prosthetic member of claim 28, wherein the polymer is selected from the group consisting of polyethyleneterephthalate, polytetrafluoroethylene, expanded polytetrafluoroethylene, polymers of lactide-glycolide, polyglactin, polydioxanone, polyurethane, polypropylene and polyester.

30. The prosthetic member of any one of claims 22 to 27, wherein the prosthetic surface is formed of a biomedically compatible material selected from the group consisting of stainless steel, titanium, cobalt chrome alloys and silicon-based materials.

31. The prosthetic member of any one of claims 22 to 30, wherein the prosthetic surface is selected from the group consisting of a stent surface, an artificial urological implant surface, an artificial heart valve surface, an artificial internal organ surface, an artificial bone implant surface, a patch surface and a web surface.

32. The prosthetic member of any one of claims 22 to 30, wherein the member is for replacement or bypass of one or more blood vessels of the patient.

33. The prosthetic member of any one of claims 22 to 30, wherein the prosthetic member is an artificial vascular implant.

34. The prosthetic member of any one of claims 22 to 30, wherein the prosthetic member is an artificial duct implant.

35. The prosthetic member of claim 34, wherein the artificial duct implant is selected from the group consisting of artificial urinary ducts, artificial kidney tubules, artificial lymphatic ducts, artificial bile ducts, artificial pancreatic ducts, indwelling catheters, shunts and drains.

36. The prosthetic member of any one of claims 22 to 35, wherein the prosthetic surface is seeded with endothelial cells.