Proc. Nadl. Acad. Sci. USA
Medical gpp. ciences
Vol. 89,
7880-7884, September 1992
A monomeric von Willebrand factor fragment, Leu-504-Ser-728, inhibits von Willebrand factor interaction with glycoprotein lb-IX (glycoprotein Ib/trombosls)
HARVEY R. GRALNICK*t, SYBIL WILLIAMS*, LAURIE MCKEOWN*, WENDY KRAMER*, HENRY KRUTZSCH*, MARIAN GORECKI§, AMOS PINET§, AND LEONARD I. GARFINKEL§ *Hematology Service, National Institutes of Health, Building 10, Room 2C390, and tLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and §Bio-Technology General (Israel) Ltd., Rehovot, Israel
Communicated by Oscar D. Ratnoff, March 23, 1992
aggregation, particularly at high shear rates (11, 12). von Willebrand factor binding to GPIb. results in the contact of platelets to the subendothelial surface, whereas binding of von Willebrand factor to GPIIb/IIIa results in spreading of these platelets and the formation of a platelet thrombus (13-15). In porcine von Willebrand disease, von Willebrand factor has been shown to be important not only in hemostasis but also in the development of atherosclerosis and occlusive thrombus formation in partially stenosed or injured arteries (16-21). We have produced and studied a recombinant fragment of von Willebrand factor that encompasses amino acids Leu504-Lys-728 of plasma von Willebrand factor. This fragment contains one disulfide bond linking residues Cys-509-Cys695. We report the studies of this fiagment in the interaction of von Willebrand factor to GPIba under static and flow conditions at high shear forces.
ABSTRACT von Willebrand factor interaction with glycoprotein Ib. (GPIb.) plays a critical role in the initial phase of platelet adhesion at hih shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474-Pro-488 (peptide 153) and Ser-692-Pro-708 (peptide 154), inhibit von Willebrand factor-GPIba interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504-Ser-728, with a single intrachain disulfide bond linking residues Cys-509-Cys-695 and examined its ability to inhibit von Willebrand factor-GPIb. interactions and platelet adhesion at hg shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetininduced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an ICso = 0.011-0.260 FM, sigicantly iower than the IC5. of peptide 153 or 154, ICs% = 86-700 saM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (ICso > 500 pM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 AM and at 7.6 pM inhibited >80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.
MATERIALS AND METHODS The recombinant von Willebrand factor fragment, Leu-504Lys-728, was expressed in Escherichia coli with an inducible APL promoter (22, 23). The fragment, named VCL, was purified on carboxymethyl-Sepharose. The isolated VCL was analyzed on a Superose 12 column by FPLC. VCL was analyzed nonreduced and reduced (10 mM dithioerythritol) in the presence of SDS on 7.5% and 10%0 polyacrylamide gel electrophoresis. N-terminal amino acid analysis of VCL was performed on an ABI475A protein sequenator. The VCL was diluted either in distilled H20 or 0.154 M NaCl for all experiments. A recombinant fragment of fibronectin, the 33-kDa cell-binding domain, was used as a control in the platelet adhesion studies (24) (see below). Two peptides, Cys-474-Pro-488 (CQEPGGLVVPPTDAP), and Ser-692-Pro-708 (SYLCDLAPEAPPPTLPP), derived from von Willebrand factor, were synthesized on a Biosearch model 9600 peptide synthesizer using standard Merrifield solid-phase synthesis protocols and t-butoxycarbonyl chemistry. The Cys-474-Pro-488 peptide is designated peptide 153, and the Ser-692-Pro-708 peptide is designated peptide 154. The peptides were analyzed by reverse-phase HPLC, found to be substantially free of impurities, and used without further purification. One peptide was also characterized by sequence analysis and gave the expected sequence. Botrocetin (Sigma), the venom coagglutinin, was prepared from Bothropsjararaca venom with modification of the methods of Read et al. and Fujimura et al. (25, 26). Plasma von Willebrand factor was purified from human cryoprecipitate by Sepharose 4B chromatography after sequential polyethylene glycol precipitations as described (10).
The initial studies of von Willebrand disease indicated a defect in platelet function (1-3). Subsequent studies of patients with von Willebrand disease have identified decreases in platelet retention in glass bead columns and ristocetininduced platelet aggregation. Ex vivo studies examining platelet adhesion to the subendothelial surface or extracellular matrix proteins at high shear rates show reduced platelet adhesion in von Willebrand disease. The addition of von Willebrand factor corrects the adhesion and the other platelet defects. The initial role defined for von Willebrand factor in platelet adhesion was its interaction with the platelet receptor, glycoprotein Ibex (GPIbj). Ristocetin or botrocetin induces binding of native von Willebrand factor to the platelet GPIba, and von Willebrand factor that has had the terminal sialic acid residues removed binds directly to GPIba (4-6). Previous studies have indicated that the interaction of von Willebrand factor with GPIb. is critical in the initiation of platelet adhesion (7, 8). Platelets stimulated by thrombin, collagen, or ADP bind von Willebrand factor and other adhesive proteins to the platelet receptor, GPIIb/IIIa complex (9, 10). Subsequent studies have identified von Willebrand factor binding to the GPIIb/IIIa complex as important in platelet adhesion and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: GP, glycoprotein. tTo whom reprint requests should be addressed.
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Proc. Natl. Acad. Sci. USA 89 (1992)
Medical Sciences: Gralnick et al. Table 1. Inhibition of von Willebrand factor-induced platelet aggregation ICso, ,uM Asialo-von Fragment Ristocetin Willebrand factor Botrocetin Peptide 153 302 + 30 150 + 19 >700 Peptide 154 86 + 12 87 ± 10 >600 VCL 0.24 + 0.09 0.29 + 0.08 0.04 ± 0.02 Values are expressed as mean ± SD (n = 5-9). von Willebrand factor-platelet interactions were examined by several methods in the presence and absence of the recombinant von Willebrand factor fragment, VCL, and the two peptides, 153 and 154. Ristocetin-induced platelet aggregation was performed utilizing arabinogalactan-separated platelets and platelet-rich plasma (10). Various concentrations of VCL or peptide were incubated with the platelets (2 x 108 platelets per ml) for 5-15 min in a platelet aggregometer prior to the addition of purified von Willebrand factor (1 ,g/ml) and ristocetin (0.55 mg/ml) (Chrono-Log, Havertown, PA). In the platelet-rich plasma experiments, ristocetin was added at a concentration of 1 mg/ml. In all of the aggregation experiments the platelets were stirred at 900 rpm at 37°C. The diluent for VCL was employed as a control. Asialo-von Willebrand factor was prepared using purified neuraminidase, and its ability to induce platelet aggregation in platelet-rich plasma was tested as described (27). Botrocetin-induced platelet aggregation was performed with purified platelets and von Willebrand factor as described above for ristocetin-induced platelet aggregation, except botrocetin was added to the platelet von Willebrand factor mixture or platelet-rich plasma at a concentration of 10 ,g/ml. von Willebrand factor and asialo-von Willebrand factor were iodinated using Iodo-Beads (Pierce) as described (27). Ristocetin- and thrombin-induced 125I-labeled von Willebrand factor binding to platelets was studied as described (10). The ability of VCL to inhibit ristocetin-, botrocetin-, or asialo-induced 125I-labeled von Willebrand factor binding to arabinogalactan platelets was studied. The method(s) for botrocetin-induced von Willebrand factor binding was the same as described for ristocetin-induced von Willebrand factor binding, except that botrocetin (2.5 ,ug/ml) was added to the von Willebrand factor platelet mixture. Binding studies were incubated for 30 min at 24°C. Platelet adhesion to human umbilical artery subendothelium was performed at a shear force of 2600 s-1 as described (15, 28). In these studies VCL was added to citrate anticoagulated whole blood at concentrations varying from 0.46 to 10 AM, the peptides 153 and 154 were at concentrations ranging from 25 to 500 ,uM, and the 33-kDa recombinant fibronectin cell-binding domain fragment was at concentrations varying from 0.2 to 12.2 ,uM.
RESULTS The recombinant von Willebrand factor fragment, VCL, had a retention time on FPLC typical of a globular monomeric 25-kDa protein. When VCL was analyzed under reduced and unreduced conditions, the reduced VCL migrated slightly
slower than the unreduced sample, indicating a transformation from a more to a less compact configuration after reduction (data not shown). The N-terminal amino acid sequence of VCL was Met-Leu-His-Asp-Phe. The Leu-His-Asp-Phe sequence corresponds to amino acids 504-507 of native von Willebrand factor. The recombinant fibronectin 33-kDa cellbinding fragment has been described and characterized (24). VCL and peptides 153 and 154 were employed in studies of aggregation of purified platelets and platelet-rich plasma. Ristocetin-induced platelet aggregation of purified platelets was inhibited by all three peptides (Table 1). IC50 values were as follows: peptide 153, 302 + 30 AuM; peptide 154, 86 12 /xM; VCL, 0.24 0.09 uM. VCL also inhibited ristocetininduced platelet aggregation in platelet-rich plasma with an IC50 of 0.17 0.04 ,tM (n = 3). The inhibition of asialo-von Willebrand factor-induced aggregation was studied only in platelet-rich plasma. The IC50 for VCL was significantly lower than that for either peptide 153 or peptide 154 (P < 0.001) (Table 1). The IC50 of peptide 153 was significantly lower with asialo-von Willebrand factor-induced platelet aggregation than ristocetin-induced platelet aggregation (P < 0.005). The IC50 of VCL in botrocetin-induced platelet aggregation of purified platelets was 0.04 0.02 pM (Table 1). The IC50 with platelet-rich plasma was 0.08 0.03 ,uM (n = 4). Peptide 153 or 154 had no effects on botrocetin-induced platelet aggregation. The inhibition of ristocetin-induced binding of von Willebrand factor to platelets mirrored the IC50 observed with ristocetin-induced platelet aggregation (Table 2, Fig. 1). IC50 values were as follows: peptide 153,435 50,uM; peptide 154, 110 19 ,M; VCL, 0.26 0.12 ,M. The differences observed between peptides 154 and 153 in IC50 values in binding and aggregation studies were significant (P < 0.005). The VCL IC50 was significantly lower than that of either peptide 153 or peptide 154 (P < 0.001; Tables 1 and 2 and Fig. 1). Neither VCL nor peptides 153 and 154 inhibited thrombin-induced binding of von Willebrand factor to platelets (Table 2). In studies examining the binding of asialo-von Willebrand factor to purified platelets the VCL IC50 was significantly lower than that of either peptide 153 or peptide 154 (Table 2). The IC50 values of VCL and peptides 153 and 154 were significantly lower in asialo-von Willebrand factor binding to platelets than in the ristocetin-induced von Willebrand factor binding (P < 0.01; Table 2). Similar to the platelet aggregation results, peptide 153 IC50 of asialo-von Willebrand factor binding to platelets is significantly lower than the IC50 ob±
±
±
±
±
±
±
±
served with the ristocetin-induced von Willebrand factor binding (P < 0.005; Table 2, Fig. 1). VCL inhibited botrocetin-induced von Willebrand factor binding with an IC50 of0.022 0.002 tM (Table 2, Fig. 1). The IC50 values of peptides 153 and 154 were >700 ,uM and >600 ,uM, respectively. The VCL IC50 of botrocetin-induced von Willebrand factor binding was significantly lower than the VCL ICso observed in the asialo- or ristocetin-induced von Willebrand factor binding to platelets (P < 0.01; Table 2, Fig. 1). When VCL and peptides 153 and 154 were tested for inhibition of platelet adhesion of whole blood to umbilical ±
Table 2. Inhibition of von Willebrand factor binding to platelets
IC50, AM Fragment
Peptide 153 Peptide 154
Ristocetin 435 ± 50 110 ± 19 0.260 ± 0.120
Asialo-von Willebrand factor 180 + 40 77 ± 14 0.040 ± 0.020
VCL Values are expressed as means ± SD (n
=
5).
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Botrocetin >700 >600 0.022 + 0.002
Thrombin >700 >600 >25
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Proc. Natl. Acad. Sci. USA 89 (1992)
100 90
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.075
.100
FIG. 1. Inhibition of binding of von Willebrand factor to platelets. Purified platelets (8 x 107 platelets in 0.4 ml) were incubated with the recombinant von Willebrand fragment, VCL, for 15 min prior to addition ofthe purified radiolabeled von Willebrand factor (0.5 g). (A) Inhibition of ristocetin-induced von Willebrand factor binding (ristocetin, 0.55 mg/ml). (B) Inhibition of asialo-von Willebrand factor binding (0.5 mg). (C) Inhibition of botrocetin-induced von Willebrand factor binding (botrocetin, 2.5 ug/ml) by the VCL fragment. Note the different scale on the abscissa in C.
artery subendothelium at shear rates of 2600 s-1, a doseresponse curve was observed with VCL. The IC5o was 0.94 + 0.21 jLM, and at 7.6 ,AM maximum inhibition of adhesion (83% + 4%) occurred (Fig. 2). In contrast, peptides 153 and 154 at concentrations varying from 25 to 500 AuM had no effect on platelet adhesion at high shear rates. The 33-kDa recombinant fibronectin fragment (0.2-12.2 pLM) did not reduce platelet adhesion or thrombus formation under identical
experimental conditions. The VCL inhibition of platelet adhesion reduced the number of contact and spread platelets and the number of thrombi formed (Table 3). DISCUSSION The interaction of von Willebrand factor with GPIba is the initial mechanism by which platelets make contact with subendothelium or with extracellular matrix proteins (7, 8).
Medical Sciences: Gralnick et al.
Proc. Natl. Acad. Sci. USA 89 (1992) CO
CD3
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* VCL o 153
o 154
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's
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FIG. 2. Effect of peptides 153 and 154 and the VCL fragment on platelet adhesion of normal blood to deendothelialized umbilical artery segments at shear rates of 2600 s-1. No inhibition was observed with peptides 153 and 154 at concentrations of 25-500 ,uM. The VCL IC5o for platelet adhesion (contact and spread platelet) was 0.94 ,uM and maximum inhibition of adhesion (83%) was observed at 7.6 ,uM. The 33-kDa recombinant fibronectin fragment did not reduce platelet adhesion.
Studies of limited proteolytic digestion of von Willebrand factor, the use of monoclonal antibodies directed against specific epitopes of von Willebrand factor, and synthetic peptides derived from the von Willebrand factor sequence have identified the GPIba binding domain on von Willebrand factor in the Al domain between residues 449 and 728 (29-34). Heparin and one collagen binding domain are also present within the Al domain (35, 36). Deletion of the Al domain of von Willebrand factor by oligonucleotide-directed mutagenesis resulted in a protein that did not interact with platelet GPIba and further supported the location of the von Willebrand factor GPIba binding domain (37). Studies of recombinant fragments of von Willebrand factor expressed in E. coli have also identified the GPLbC. binding domain of von Willebrand factor (38, 39). The two fragments encompass amino acids Val-449-Asn-730 and Ser-445-Asn-733, respectively. Although these recombinant fragments do not have the native conformation of the intact molecule and require chemical modification or reducing agents for solubilization, they inhibit von Willebrand factor-platelet GPIb,, interaction (38, 39). VCL is a smaller monomeric fragment of von Willebrand factor, is water-soluble, and is about 1 or 2 orders of magnitude more potent than the previously reported fragments. VCL inhibited von Willebrand factor binding to platelets in the presence of ristocetin, botrocetin, or asialo-von Willebrand factor; however, the IC50 values for VCL ofbotrocetininduced von Willebrand factor binding and platelet aggregation were significantly lower than those observed with either asialo- or ristocetin-induced platelet aggregation and von Willebrand factor binding. This suggests that VCL recognizes and binds with greater affinity to the botrocetin-induced von Willebrand factor domain on the GPIba than either the ristocetin-induced or the asialo-induced von Willebrand factor binding sites. The presence of plasma proteins did not Table 3. Platelet adhesion n Contact, % Fragment Control 10 1.68 ± 0.14 VCL (7.6 1uM) 5 0.86 ± 0.32* *P < 0.025. tp < 0.001.
Spread, % 31.1 ± 2.79 7.71 ± 2.05t
Thrombi, % 0.4 ± 0.1 0 ± 0*
interfere with the VCL inhibition of ristocetin- or botrocetininduced platelet aggregation. Peptides 153 and 154 did not inhibit botrocetin-induced von Willebrand factor binding and platelet aggregation. In contrast, peptides 153 and 154 did inhibit the ristocetin- and asialo-induced von Willebrand factor binding and platelet aggregation. These studies suggest that von Willebrand factor binding to GPIba by ristocetin and asialo-von Willebrand factor is mediated, in part, by different mechanisms than is botrocetin. The aggregation and binding studies that were performed in a static system could not address the issue of whether VCL would be effective in inhibiting platelet adhesion to the subendothelium surface at high shear rates. We studied the platelet adhesion in whole blood to the subendothelial surface of umbilical arteries. At high shear forces in this flowing system VCL inhibited the percentage of contact and spread platelets on the subendothelial surface and decreased thrombus formation. These studies add further support to the localization of the GPIbcx binding domain of von Willebrand factor between amino acid residues 449-728 and indicate that VCL, a soluble recombinant fragment of von Willebrand factor, Leu-504Lys-728, with a single disulfide bond at residues Cys-509Cys-695 almost totally inhibits von Willebrand factor-GPIb< interaction(s) (37-42). VCL, like anti-von Willebrand factor antibodies, has the potential of being an effective antithrombotic agent (21, 43). Previous studies indicate that subendothelial and plasma von Willebrand factors are crucial in the initiation of platelet adhesion to subendothelial or matrix protein surfaces. Our data indicate that the monomeric von Willebrand factor fragment, Leu-504-Lys-728, recognizes the von Willebrand factor GPIba binding site(s) induced by ristocetin and/or botrocetin and the Willebrand factor binding site(s) exposed on GPIb, by high shear forces. In this study, we have shown that VCL can inhibit platelet adhesion to and thrombus formation on the subendothelium at high shear forces. These results suggest that in a setting of partially stenosed coronary or cerebral arteries that VCL would be an effective antithrombotic agent. We thank Cynthia Williams and Candyce McLean for their excellent secretarial assistance, Tamar Richter for her dedicated technical support, Tikva Vogel for her generous gift of the 33-kDa fibronectin
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fragment, and Margaret Rick for her valuable suggestions and critical review of the manuscript. 1. von Willebrand, E. A. & Jurgens, R. (1933) Klin. Wochenschr. 12, 414. 2. von Willebrand, E. A. & Jurgens, R. (1933) Dtsch. Arch. Klin. Med. 175, 453-483. 3. von Willebrand, E. A., Jurgens, R. & Dahlberg, U. (1934) Fin. Laekaresaelisk. Handl. 76, 193. 4. Coller, B. S., Peerschke, E. I., Scudder, L. E. & Sullivan, E. A. (1983) Blood 61, 99-110. 5. Brinkhous, K. M., Read, M. S. & Reddick, R. L. (1981) Ann. N. Y. Acad. Sci. 370, 191-204. 6. DeMarco, L. & Shapiro, S. S. (1981) J. Clin. Invest. 75, 1198-1203. 7. Weiss, H. J., Tschoop, T. B., Baumgartner, H. R., Sussman, I. I., Johnson, M. M. & Egan, J. J. (1974) Am. J. Med. 57, 920-925. 8. Weiss, H. J., Turitto, V. T. & Baumgartner, H. R. (1978) J. Lab. Clin. Med. 92, 750-764. 9. Ruggeri, Z. M., Bader, R. & DeMarco, L. (1982) Proc. Natl. Acad. Sci. USA 79, 6038-6041. 10. Gralnick, H. R., Williams, S. B. & Coller, B. S. (1984) Blood 64, 797-800. 11. Moake, J. L., Turner, N. A., Stathopoulos, N. A., Nolasco, L. A. & Hellums, J. D. (1986) J. Clin. Invest. 78, 1456-1461. 12. Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, Y., Watanabe, K., Itagaki, I., Sakai, K. & Ruggeri, Z. (1991) J. Clin. Invest. 87, 1234-1240. 13. Sakariassen, K. S., Nievelstein, P. F., Coller, B. S. & Sixma, J. J. (1986) Br. J. Haematol. 63, 681-691. 14. Weiss, H. J., Turitto, V. T. & Baumgartner, H. R. (1986) Blood 68, 322-330. 15. Lawrence, J. B. & Gralnick, H. R. (1987) J. Lab. Clin. Med. 109, 495-503. 16. Reddick, R. L., Griggs, T. R., Lamb, M. A. & Brinkhous, K. M. (1982) Proc. Natl. Acad. Sci. USA 79, 5076-5079. 17. Fuster, V., Bowie, E. J. W., Lewis, J. C., Fass, D. N., Owen, C. A., Jr., & Brown, A. L. (1978) J. Clin. Invest. 61, 722-730. 18. Fuster, V., Fass, D. N., Kaye, M. P., Josa, M., Zinsmeister, A. R. & Bowie, E. J. W. (1982) Circ. Res. 51, 587-593. 19. Nichols, T. C., Bellinger, D. A., Tate, D. A., Reddick, R. L., Read, M. S., Koch, G. G., Brinkhous, K. M. & Griggs, T. R. (1990) Arteriosclerosis 10, 449-461. 20. Nichols, T. C., Bellinger, D. A., Johnson, T. A., Lamb, M. A. & Griggs, T. R. (1986) Circ. Res. 59, 15-26. 21. Bellinger, D. A., Nichols, T. C., Read, M. S., Reddick, R. L., Lamb, M. A., Brinkhous, K. M., Evatt, B. L. & Griggs, T. R. (1987) Proc. Natl. Acad. Sci. USA 84, 8100-8104. 22. Beck, Y., Bartfeld, D., Yavin, Z., Levanon, A., Gorecki, M. & Hartman, J. (1988) BioTechnology 6, 930-935.
Proc. NatL Acad Sci. USA 89 (1992) 23. Fischer, M., Fytlovitch, S., Amit, B., Wortzel, A. & Beck, Y. (1990) Appl. Microbiol. Biotechnol. 33, 424-428. 24. Werber, M. M., Vogel, T., Kook, M., Greenstein, L. A., Levanon, A., Zelig, Y., Havron, A., Gorecki, M. & Panet, A. (1990) in Biologicals from Recombinant Microorganisms and Animal Cells, eds. White, M. D., Reuveny, S. & Shafferman, A. (Deerfield Beach & Balaban, Philadelphia). 25. Read, M. S., Shermer, R. & Brinkhous, K. (1978) Proc. Natl. Acad. Sci. USA 75, 4514-4518. 26. Fujimura, Y., Holland, L., Ruggeri, Z. & Zimmerman, T. (1987) Blood 70, 985-988. 27. Gralnick, H. R., Williams, S. B. & Coller, B. S. (1985) J. Clin. Invest. 75, 19-25. 28. Lawrence, J. B., Kramer, W. S., McKeown, L. P., Williams, S. B. & Gralnick, H. R. (1990) J. Clin. Invest. 86, 1715-1722. 29. Girma, J. P., Chopek, M., Titani, K. & Davie, E. (1986)
Biochemistry 25, 3156-3163. 30. Fujimura, Y., Titani, K., Holland, L., Russell, S., Roberts, J., Elder, J., Ruggeri, Z. & Zimmerman, T. (1986) J. Biol. Chem. 261, 381-385. 31. Fujimura, Y., Titani, K., Holland, L., Roberts, J., Kostel, P., Ruggeri, Z. & Zimmerman, T. (1987) J. Biol. Chem. 262, 1734-1739. 32. Girma, J.-P., Kalafatis, M., Pietu, G., Lavergne, J., Chopek, M., Edgington, T. & Meyer, D. (1986) Blood 67, 1356-1366. 33. Mohri, H., Yoshioka, A., Zimmerman, T. S. & Ruggeri, Z. M. (1989) J. Biol. Chem. 264, 17361-17367. 34. Mohri, H., Fujimura, Y., Shima, M., Yoshioka, A., Hooughten, R. A., Ruggeri, Z. M. & Zimmerman, T. S. (1988) J. Biol. Chem. 263, 17901-17904. 35. Pareti, F. I., Niiya, K., McPherson, J. & Ruggeri, Z. (1987) J. Biol. Chem. 262, 13835-13841. 36. Roth, G. J., Titani, K., Hoyer, L. W. & Hickey, M. J. (1986) Biochemistry 25, 8357-8361. 37. Sixma, J. J., Schiphorst, M. E., Verwey, C. L. & Pannekoek, H. (1991) Eur. J. Biochem. 196, 369-375. 38. Pietu, G., Meulien, P., Cherel, G., Diaz, J., Baruch, D., Courtney, M. & Meyer, D. (1989) Biochem. Biophys. Res. Commun. 164, 1339-1347. 39. Sugimoto, M., Ricca, G., Hrinda, M. E., Schreiber, A. B., Searfoss, G. H., Bottini, E. & Ruggeri, Z. M. (1991) Biochemistry 30, 5202-5209. 40. Randi, A. M., Rabinowitz, I., Mancuso, D. J., Mannucci, P. & Sadler, J. E. (1991) J. Clin. Invest. 87, 1220-1226. 41. Cooney, K. A., Nichols, W. C., Bruck, M. E., Bahou, W. F., Shapiro, A. D., Walter Bowie, E. J., Gralnick, H. R. & Ginsburg, D. (1991) J. Clin. Invest. 87, 1227-1233. 42. Ware, J., Dent, J. A., Azuma, H., Sugimoto, M., Kyrle, P. A., Yoshioka, A. & Ruggeri, Z. M. (1991) Proc. Natl. Acad. Sci. USA 88, 2946-2950. 43. Sawada, Y., Fass, D. N., Katzman, J. A., Bahn, R. C. & Bowie, E. J. W. (1986) Blood 67, 1229-1239.
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Vol. 89,
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A monomeric von Willebrand factor fragment, Leu-504-Ser-728, inhibits von Willebrand factor interaction with glycoprotein lb-IX (glycoprotein Ib/trombosls)
HARVEY R. GRALNICK*t, SYBIL WILLIAMS*, LAURIE MCKEOWN*, WENDY KRAMER*, HENRY KRUTZSCH*, MARIAN GORECKI§, AMOS PINET§, AND LEONARD I. GARFINKEL§ *Hematology Service, National Institutes of Health, Building 10, Room 2C390, and tLaboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; and §Bio-Technology General (Israel) Ltd., Rehovot, Israel
Communicated by Oscar D. Ratnoff, March 23, 1992
aggregation, particularly at high shear rates (11, 12). von Willebrand factor binding to GPIb. results in the contact of platelets to the subendothelial surface, whereas binding of von Willebrand factor to GPIIb/IIIa results in spreading of these platelets and the formation of a platelet thrombus (13-15). In porcine von Willebrand disease, von Willebrand factor has been shown to be important not only in hemostasis but also in the development of atherosclerosis and occlusive thrombus formation in partially stenosed or injured arteries (16-21). We have produced and studied a recombinant fragment of von Willebrand factor that encompasses amino acids Leu504-Lys-728 of plasma von Willebrand factor. This fragment contains one disulfide bond linking residues Cys-509-Cys695. We report the studies of this fiagment in the interaction of von Willebrand factor to GPIba under static and flow conditions at high shear forces.
ABSTRACT von Willebrand factor interaction with glycoprotein Ib. (GPIb.) plays a critical role in the initial phase of platelet adhesion at hih shear rates, and it may also play a role in platelet thrombus formation in partially occluded arteries. Previous studies have indicated that two peptides, Cys-474-Pro-488 (peptide 153) and Ser-692-Pro-708 (peptide 154), inhibit von Willebrand factor-GPIba interaction. We have expressed a recombinant fragment of von Willebrand factor, Leu-504-Ser-728, with a single intrachain disulfide bond linking residues Cys-509-Cys-695 and examined its ability to inhibit von Willebrand factor-GPIb. interactions and platelet adhesion at hg shear forces. This recombinant fragment, named VCL, inhibits ristocetin-induced, botrocetininduced, and asialo-von Willebrand factor-induced platelet aggregation and binding to platelets at an ICso = 0.011-0.260 FM, sigicantly iower than the IC5. of peptide 153 or 154, ICs% = 86-700 saM. Peptides 153 and 154 did not result in any inhibition of platelet adhesion (ICso > 500 pM). In contrast, VCL inhibited 50% of platelet adhesion at 0.94 AM and at 7.6 pM inhibited >80% of platelet adhesion to human umbilical artery subendothelium at high shear forces. VCL inhibited the contact and spreading of platelets and also caused a marked decrease in thrombus formation. These studies indicate that VCL may be an effective antithrombotic agent in preventing arterial thrombus formation in areas of high shear force.
MATERIALS AND METHODS The recombinant von Willebrand factor fragment, Leu-504Lys-728, was expressed in Escherichia coli with an inducible APL promoter (22, 23). The fragment, named VCL, was purified on carboxymethyl-Sepharose. The isolated VCL was analyzed on a Superose 12 column by FPLC. VCL was analyzed nonreduced and reduced (10 mM dithioerythritol) in the presence of SDS on 7.5% and 10%0 polyacrylamide gel electrophoresis. N-terminal amino acid analysis of VCL was performed on an ABI475A protein sequenator. The VCL was diluted either in distilled H20 or 0.154 M NaCl for all experiments. A recombinant fragment of fibronectin, the 33-kDa cell-binding domain, was used as a control in the platelet adhesion studies (24) (see below). Two peptides, Cys-474-Pro-488 (CQEPGGLVVPPTDAP), and Ser-692-Pro-708 (SYLCDLAPEAPPPTLPP), derived from von Willebrand factor, were synthesized on a Biosearch model 9600 peptide synthesizer using standard Merrifield solid-phase synthesis protocols and t-butoxycarbonyl chemistry. The Cys-474-Pro-488 peptide is designated peptide 153, and the Ser-692-Pro-708 peptide is designated peptide 154. The peptides were analyzed by reverse-phase HPLC, found to be substantially free of impurities, and used without further purification. One peptide was also characterized by sequence analysis and gave the expected sequence. Botrocetin (Sigma), the venom coagglutinin, was prepared from Bothropsjararaca venom with modification of the methods of Read et al. and Fujimura et al. (25, 26). Plasma von Willebrand factor was purified from human cryoprecipitate by Sepharose 4B chromatography after sequential polyethylene glycol precipitations as described (10).
The initial studies of von Willebrand disease indicated a defect in platelet function (1-3). Subsequent studies of patients with von Willebrand disease have identified decreases in platelet retention in glass bead columns and ristocetininduced platelet aggregation. Ex vivo studies examining platelet adhesion to the subendothelial surface or extracellular matrix proteins at high shear rates show reduced platelet adhesion in von Willebrand disease. The addition of von Willebrand factor corrects the adhesion and the other platelet defects. The initial role defined for von Willebrand factor in platelet adhesion was its interaction with the platelet receptor, glycoprotein Ibex (GPIbj). Ristocetin or botrocetin induces binding of native von Willebrand factor to the platelet GPIba, and von Willebrand factor that has had the terminal sialic acid residues removed binds directly to GPIba (4-6). Previous studies have indicated that the interaction of von Willebrand factor with GPIb. is critical in the initiation of platelet adhesion (7, 8). Platelets stimulated by thrombin, collagen, or ADP bind von Willebrand factor and other adhesive proteins to the platelet receptor, GPIIb/IIIa complex (9, 10). Subsequent studies have identified von Willebrand factor binding to the GPIIb/IIIa complex as important in platelet adhesion and The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: GP, glycoprotein. tTo whom reprint requests should be addressed.
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Medical Sciences: Gralnick et al. Table 1. Inhibition of von Willebrand factor-induced platelet aggregation ICso, ,uM Asialo-von Fragment Ristocetin Willebrand factor Botrocetin Peptide 153 302 + 30 150 + 19 >700 Peptide 154 86 + 12 87 ± 10 >600 VCL 0.24 + 0.09 0.29 + 0.08 0.04 ± 0.02 Values are expressed as mean ± SD (n = 5-9). von Willebrand factor-platelet interactions were examined by several methods in the presence and absence of the recombinant von Willebrand factor fragment, VCL, and the two peptides, 153 and 154. Ristocetin-induced platelet aggregation was performed utilizing arabinogalactan-separated platelets and platelet-rich plasma (10). Various concentrations of VCL or peptide were incubated with the platelets (2 x 108 platelets per ml) for 5-15 min in a platelet aggregometer prior to the addition of purified von Willebrand factor (1 ,g/ml) and ristocetin (0.55 mg/ml) (Chrono-Log, Havertown, PA). In the platelet-rich plasma experiments, ristocetin was added at a concentration of 1 mg/ml. In all of the aggregation experiments the platelets were stirred at 900 rpm at 37°C. The diluent for VCL was employed as a control. Asialo-von Willebrand factor was prepared using purified neuraminidase, and its ability to induce platelet aggregation in platelet-rich plasma was tested as described (27). Botrocetin-induced platelet aggregation was performed with purified platelets and von Willebrand factor as described above for ristocetin-induced platelet aggregation, except botrocetin was added to the platelet von Willebrand factor mixture or platelet-rich plasma at a concentration of 10 ,g/ml. von Willebrand factor and asialo-von Willebrand factor were iodinated using Iodo-Beads (Pierce) as described (27). Ristocetin- and thrombin-induced 125I-labeled von Willebrand factor binding to platelets was studied as described (10). The ability of VCL to inhibit ristocetin-, botrocetin-, or asialo-induced 125I-labeled von Willebrand factor binding to arabinogalactan platelets was studied. The method(s) for botrocetin-induced von Willebrand factor binding was the same as described for ristocetin-induced von Willebrand factor binding, except that botrocetin (2.5 ,ug/ml) was added to the von Willebrand factor platelet mixture. Binding studies were incubated for 30 min at 24°C. Platelet adhesion to human umbilical artery subendothelium was performed at a shear force of 2600 s-1 as described (15, 28). In these studies VCL was added to citrate anticoagulated whole blood at concentrations varying from 0.46 to 10 AM, the peptides 153 and 154 were at concentrations ranging from 25 to 500 ,uM, and the 33-kDa recombinant fibronectin cell-binding domain fragment was at concentrations varying from 0.2 to 12.2 ,uM.
RESULTS The recombinant von Willebrand factor fragment, VCL, had a retention time on FPLC typical of a globular monomeric 25-kDa protein. When VCL was analyzed under reduced and unreduced conditions, the reduced VCL migrated slightly
slower than the unreduced sample, indicating a transformation from a more to a less compact configuration after reduction (data not shown). The N-terminal amino acid sequence of VCL was Met-Leu-His-Asp-Phe. The Leu-His-Asp-Phe sequence corresponds to amino acids 504-507 of native von Willebrand factor. The recombinant fibronectin 33-kDa cellbinding fragment has been described and characterized (24). VCL and peptides 153 and 154 were employed in studies of aggregation of purified platelets and platelet-rich plasma. Ristocetin-induced platelet aggregation of purified platelets was inhibited by all three peptides (Table 1). IC50 values were as follows: peptide 153, 302 + 30 AuM; peptide 154, 86 12 /xM; VCL, 0.24 0.09 uM. VCL also inhibited ristocetininduced platelet aggregation in platelet-rich plasma with an IC50 of 0.17 0.04 ,tM (n = 3). The inhibition of asialo-von Willebrand factor-induced aggregation was studied only in platelet-rich plasma. The IC50 for VCL was significantly lower than that for either peptide 153 or peptide 154 (P < 0.001) (Table 1). The IC50 of peptide 153 was significantly lower with asialo-von Willebrand factor-induced platelet aggregation than ristocetin-induced platelet aggregation (P < 0.005). The IC50 of VCL in botrocetin-induced platelet aggregation of purified platelets was 0.04 0.02 pM (Table 1). The IC50 with platelet-rich plasma was 0.08 0.03 ,uM (n = 4). Peptide 153 or 154 had no effects on botrocetin-induced platelet aggregation. The inhibition of ristocetin-induced binding of von Willebrand factor to platelets mirrored the IC50 observed with ristocetin-induced platelet aggregation (Table 2, Fig. 1). IC50 values were as follows: peptide 153,435 50,uM; peptide 154, 110 19 ,M; VCL, 0.26 0.12 ,M. The differences observed between peptides 154 and 153 in IC50 values in binding and aggregation studies were significant (P < 0.005). The VCL IC50 was significantly lower than that of either peptide 153 or peptide 154 (P < 0.001; Tables 1 and 2 and Fig. 1). Neither VCL nor peptides 153 and 154 inhibited thrombin-induced binding of von Willebrand factor to platelets (Table 2). In studies examining the binding of asialo-von Willebrand factor to purified platelets the VCL IC50 was significantly lower than that of either peptide 153 or peptide 154 (Table 2). The IC50 values of VCL and peptides 153 and 154 were significantly lower in asialo-von Willebrand factor binding to platelets than in the ristocetin-induced von Willebrand factor binding (P < 0.01; Table 2). Similar to the platelet aggregation results, peptide 153 IC50 of asialo-von Willebrand factor binding to platelets is significantly lower than the IC50 ob±
±
±
±
±
±
±
±
served with the ristocetin-induced von Willebrand factor binding (P < 0.005; Table 2, Fig. 1). VCL inhibited botrocetin-induced von Willebrand factor binding with an IC50 of0.022 0.002 tM (Table 2, Fig. 1). The IC50 values of peptides 153 and 154 were >700 ,uM and >600 ,uM, respectively. The VCL IC50 of botrocetin-induced von Willebrand factor binding was significantly lower than the VCL ICso observed in the asialo- or ristocetin-induced von Willebrand factor binding to platelets (P < 0.01; Table 2, Fig. 1). When VCL and peptides 153 and 154 were tested for inhibition of platelet adhesion of whole blood to umbilical ±
Table 2. Inhibition of von Willebrand factor binding to platelets
IC50, AM Fragment
Peptide 153 Peptide 154
Ristocetin 435 ± 50 110 ± 19 0.260 ± 0.120
Asialo-von Willebrand factor 180 + 40 77 ± 14 0.040 ± 0.020
VCL Values are expressed as means ± SD (n
=
5).
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Botrocetin >700 >600 0.022 + 0.002
Thrombin >700 >600 >25
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FIG. 1. Inhibition of binding of von Willebrand factor to platelets. Purified platelets (8 x 107 platelets in 0.4 ml) were incubated with the recombinant von Willebrand fragment, VCL, for 15 min prior to addition ofthe purified radiolabeled von Willebrand factor (0.5 g). (A) Inhibition of ristocetin-induced von Willebrand factor binding (ristocetin, 0.55 mg/ml). (B) Inhibition of asialo-von Willebrand factor binding (0.5 mg). (C) Inhibition of botrocetin-induced von Willebrand factor binding (botrocetin, 2.5 ug/ml) by the VCL fragment. Note the different scale on the abscissa in C.
artery subendothelium at shear rates of 2600 s-1, a doseresponse curve was observed with VCL. The IC5o was 0.94 + 0.21 jLM, and at 7.6 ,AM maximum inhibition of adhesion (83% + 4%) occurred (Fig. 2). In contrast, peptides 153 and 154 at concentrations varying from 25 to 500 AuM had no effect on platelet adhesion at high shear rates. The 33-kDa recombinant fibronectin fragment (0.2-12.2 pLM) did not reduce platelet adhesion or thrombus formation under identical
experimental conditions. The VCL inhibition of platelet adhesion reduced the number of contact and spread platelets and the number of thrombi formed (Table 3). DISCUSSION The interaction of von Willebrand factor with GPIba is the initial mechanism by which platelets make contact with subendothelium or with extracellular matrix proteins (7, 8).
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Proc. Natl. Acad. Sci. USA 89 (1992) CO
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* VCL o 153
o 154
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's
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FIG. 2. Effect of peptides 153 and 154 and the VCL fragment on platelet adhesion of normal blood to deendothelialized umbilical artery segments at shear rates of 2600 s-1. No inhibition was observed with peptides 153 and 154 at concentrations of 25-500 ,uM. The VCL IC5o for platelet adhesion (contact and spread platelet) was 0.94 ,uM and maximum inhibition of adhesion (83%) was observed at 7.6 ,uM. The 33-kDa recombinant fibronectin fragment did not reduce platelet adhesion.
Studies of limited proteolytic digestion of von Willebrand factor, the use of monoclonal antibodies directed against specific epitopes of von Willebrand factor, and synthetic peptides derived from the von Willebrand factor sequence have identified the GPIba binding domain on von Willebrand factor in the Al domain between residues 449 and 728 (29-34). Heparin and one collagen binding domain are also present within the Al domain (35, 36). Deletion of the Al domain of von Willebrand factor by oligonucleotide-directed mutagenesis resulted in a protein that did not interact with platelet GPIba and further supported the location of the von Willebrand factor GPIba binding domain (37). Studies of recombinant fragments of von Willebrand factor expressed in E. coli have also identified the GPLbC. binding domain of von Willebrand factor (38, 39). The two fragments encompass amino acids Val-449-Asn-730 and Ser-445-Asn-733, respectively. Although these recombinant fragments do not have the native conformation of the intact molecule and require chemical modification or reducing agents for solubilization, they inhibit von Willebrand factor-platelet GPIb,, interaction (38, 39). VCL is a smaller monomeric fragment of von Willebrand factor, is water-soluble, and is about 1 or 2 orders of magnitude more potent than the previously reported fragments. VCL inhibited von Willebrand factor binding to platelets in the presence of ristocetin, botrocetin, or asialo-von Willebrand factor; however, the IC50 values for VCL ofbotrocetininduced von Willebrand factor binding and platelet aggregation were significantly lower than those observed with either asialo- or ristocetin-induced platelet aggregation and von Willebrand factor binding. This suggests that VCL recognizes and binds with greater affinity to the botrocetin-induced von Willebrand factor domain on the GPIba than either the ristocetin-induced or the asialo-induced von Willebrand factor binding sites. The presence of plasma proteins did not Table 3. Platelet adhesion n Contact, % Fragment Control 10 1.68 ± 0.14 VCL (7.6 1uM) 5 0.86 ± 0.32* *P < 0.025. tp < 0.001.
Spread, % 31.1 ± 2.79 7.71 ± 2.05t
Thrombi, % 0.4 ± 0.1 0 ± 0*
interfere with the VCL inhibition of ristocetin- or botrocetininduced platelet aggregation. Peptides 153 and 154 did not inhibit botrocetin-induced von Willebrand factor binding and platelet aggregation. In contrast, peptides 153 and 154 did inhibit the ristocetin- and asialo-induced von Willebrand factor binding and platelet aggregation. These studies suggest that von Willebrand factor binding to GPIba by ristocetin and asialo-von Willebrand factor is mediated, in part, by different mechanisms than is botrocetin. The aggregation and binding studies that were performed in a static system could not address the issue of whether VCL would be effective in inhibiting platelet adhesion to the subendothelium surface at high shear rates. We studied the platelet adhesion in whole blood to the subendothelial surface of umbilical arteries. At high shear forces in this flowing system VCL inhibited the percentage of contact and spread platelets on the subendothelial surface and decreased thrombus formation. These studies add further support to the localization of the GPIbcx binding domain of von Willebrand factor between amino acid residues 449-728 and indicate that VCL, a soluble recombinant fragment of von Willebrand factor, Leu-504Lys-728, with a single disulfide bond at residues Cys-509Cys-695 almost totally inhibits von Willebrand factor-GPIb< interaction(s) (37-42). VCL, like anti-von Willebrand factor antibodies, has the potential of being an effective antithrombotic agent (21, 43). Previous studies indicate that subendothelial and plasma von Willebrand factors are crucial in the initiation of platelet adhesion to subendothelial or matrix protein surfaces. Our data indicate that the monomeric von Willebrand factor fragment, Leu-504-Lys-728, recognizes the von Willebrand factor GPIba binding site(s) induced by ristocetin and/or botrocetin and the Willebrand factor binding site(s) exposed on GPIb, by high shear forces. In this study, we have shown that VCL can inhibit platelet adhesion to and thrombus formation on the subendothelium at high shear forces. These results suggest that in a setting of partially stenosed coronary or cerebral arteries that VCL would be an effective antithrombotic agent. We thank Cynthia Williams and Candyce McLean for their excellent secretarial assistance, Tamar Richter for her dedicated technical support, Tikva Vogel for her generous gift of the 33-kDa fibronectin
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