Optimization of a Recombinant von Willebrand Factor Fragment as an Antagonist of the Platelet Glycoprotein Ib Receptor

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser⁴⁴⁵-Val⁷³³), can inhibit binding of native vWF to GP Ib and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF^508−704 is close to the minimal sequence with optimal RG12986-like activity (IC₅₀ for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 M0.01, n=3) and that it too binds directly to GP Ib. Under in vitro conditions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF^508−704 (containing a disulfide bond between Cys⁵⁰⁸ and Cys⁶⁵⁹) is approximately 5-fold less active than reduced and alkylated rvWF^508−704; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF^508−704 fragment offers distinct advantages over RG 12986. In particular, removal of non-active NH₂ and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mostly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.

1. Ruggeri, Z.M. and Zimmerman, T. 1985. Platelets and von Willebrand disease. Semin. Hematol. 22: 230−218.
2. Bellinger, D., Nichols, T., Read, M., Reddick, R., Lamb, M., Brinkhous, K., Evatt, B. and Grigg, T. Proc. Natl. Acad. Sci. USA 84: 8100−8104. | PubMed  | ChemPort |
3. DeMarco, L., Girolami, A., Zimmerman, T.S. and Ruggeri, Z.M. 1985. Interaction of purified type IIB von Willebrand factor with the platelet membrane glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and initiates aggregation. Proc. Natl. Acad. Sci. USA 82: 7424−7428. | PubMed  | ChemPort |
4. DeMarco, L., Girolami, A., Russell, S. and Ruggeri, Z.M. 1985. Interaction of Asialo von Willebrand Factor with glycoprotein Ib induces fibrinogen binding to the glycoprotein IIb/IIIa complex and mediates platelet aggregation. J. Clin. Invest. 75: 1198−1203. | PubMed  | ISI | ChemPort |
5. Ruggeri, Z.M., Bader, R. and DeMarco, >L. 1982. Glanzmann thrombasthenia: deficient binding of von Willebrand factor to thrombin stimulated platelets. Proc. Natl. Acad. Sci. USA 79: 6038−6041. | PubMed  | ChemPort |
6. Ruggeri, Z.M., DeMarco, L., Gatti, L., Bader, R. and Montgomery, R.R. 1983. Platelets have more than one binding site for von Willebrand factor. J. Clin. Invest. 72: 1−12. | PubMed  | ISI | ChemPort |
7. Ikeda, Y., Handa, M., Kawano, K., Kamata, T., Murata, M., Araki, Y., Anbo, H., Kawai, >Y., Watanabe, K., Itagaki, I., Sakai, K. and Ruggeri, Z.M. 1991. The role of von Willebrand factor and fibrinogen in platelet aggregation under varying shear stress. J. Clin. Invest. 87: 1234−1240. | PubMed  | ISI | ChemPort |
8. Weiss, H.J., Turitto, V.T. and Baumgartner, H.R. 1978. Effect of shear rate on platelet interaction with subendothelium in citrated and native blood. Shear-rate dependent decrease of adhesion in von-Willebrand's disease and the Bernard-Soulier syndrome. J. Lab. Clin. Med. 92: 750−764. | PubMed  | ISI | ChemPort |
9. Fujimura, >Y., Titani, K., Holland, L.Z., Russel, S.R., Roberts, R.B., Elder, J.H., Ruggeri, Z.M. and Zimmerman, T.S. 1986. A reduced and alkylated 52/48-kDa fragment beginning at amino acid residue 449 contains the domain interacting with platelet glycoprotein Ib. J. Biol. Chem. 261: 381−385. | PubMed  | ISI |
10. Mohri, H., Yashioka, A., Zimmerman, T. and Ruggeri, Z. 1989. Isolation of the von Willebrand factor domain interacting with platelet glycoprotein Ib, heparin and collagen and characterization of its three distinct functional sites. J. Biol. Chem. 264: 17361−17367. | PubMed  | ISI | ChemPort |
11. Sugimoto, M., Ricca, G., Hrinda, M.E., Schriebcr, A.B., Scarfoss, G.H., Bottini, E. and Ruggeri, Z.M. 1991. Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in E. coli. Biochemistry 30: 5202−5209. | PubMed  | ISI | ChemPort |
12. Prior, C.P., Chu, V., Holt, J., Windisch, V., Lee, T., Mitschelen, J., Newman, J., Ricca, G., Tarr, C. and Hrinda, M. 1992. Production and functional characterizaion of a recombinant fragment of von Willebrand Factor (vWF): An antagonist to platelet receptor GP Ib. Bio/Technology 10: 66−73. | PubMed  | ISI | ChemPort |
13. Sugimoto, M., Dent, J., McClintock, R., Ware, J. and Ruggeri, Z.M. 1993. Analysis of structure-function relationships in the GPIb-binding domain of von Willebrand factor by expression of deletion mutants. J. Biol. Chem. In press | PubMed  |
14. Studier, F.W. and Moffet, B.A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J. Mol. Biol. 189: 113−130. | PubMed  | ISI | ChemPort |
15. Collier, H.B. 1973. A note on the molar absorptivity of reduced Ellmam's Reagent, 3-carboxylate-4-nitrothiophenolate. Anal. Biochem. 56: 310−312. | PubMed  | ISI | ChemPort |
16. Grant, R.A., Zucker, M.B. and McPherson, J. 1976. ADP-induced inhibition of von Willebrand factor-mediated platelet agglutination. Am. J. Physiol. 230: 1406−1410. | PubMed  | ISI | ChemPort |
17. Mohri, H., Fujimura, Y., Sima, M., Yoshioka, A., Houghten, R.A., Ruggeri, Z. M. and Zimmerman, T.S. 1988. Structure of the von Willebrand factor domain interacting with glycoprotein Ib. J. Biol. Chem. 263: 17901−17904. | PubMed  | ISI | ChemPort |
18. Berndt, M.C., Ward, C.M., Booth, W.J., Castaldi, P.A., Mazurov, A.V. and Andrews, R.K. 1992. Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence. Mechanisms of modulation of von Willebrand factor by ristocetin and botrocetin. Biochemistry 31: 11144−11151. | PubMed  | ISI | ChemPort |
19. Sixma, J.J., Pronk, A., Nievelstein, P.N.E.M., Zwaginga, J.-J., Hindriks, G., Tuburg, P., Banga, J.-D. and de Groot, P.G. 1991. Platelet adhesion to extracellular matrices of cultured cells. Ann. N. Y. Acad. Sci. 614: 181−192. | PubMed  | ISI | ChemPort |
20. Sixma, J.J., Nievelstein, P.N.E.M., Houdijk, W.P.M., van Breugel, J.H.F.I, Hindriks, G. and de Groot, P.G. 1987. Adhesion of blood platelets to isolated components of the vessel wall. Ann. N. Y. Acad. Sci. 509: 103−117. | PubMed  | ChemPort |
21. Titani, K., Kumar, S., Takio, K., Ericsson, L.H., Wade, R.D., Ashida, K., Walsh, K.A., Chopek, M.W., Sadler, J.E. and Fujikawa, K. 1986. Amino acid sequence of human von Willebrand factor. Biochemistry 25: 3171−3184. | PubMed  | ISI | ChemPort |
22. Ware, J., Dent, J.A., Azuma, H., Sugimoto, M., Kyrle, P.A., Yoshioka, A. and Ruggeri, Z.M. 1991. Identification of a point in type IIB von Willebrand disease illustrating the regulation of von Willebrand factor affinity for the platelet membrane glycoprotein Ib-IX receptor. Proc. Natl. Acad. Sci. USA 88: 2946−2950. | PubMed  | ChemPort |
23. Ginsburg, D. and Sadler, J.E. 1993. Von Willebrand Disease: A database of point mutations, insertions and deletions. Thrombosis and Haemostasis 69: 177−184. | PubMed  | ISI | ChemPort |
24. Laemmli, U.K. 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680−685. | PubMed  | ISI | ChemPort |
25. Bradford, M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248−254. | Article | PubMed  | ISI | ChemPort |
26. Marguerie, G.A., Plow, E.F. and Edington, T.S. 1979. Human platelets possess an inducible and saturatable receptor specific for fibrinogen. J. Biol. Chem. 254: 5357−5363. | PubMed  | ISI | ChemPort |
27. Read, M.S., Shermer, R.W. and Brinkhous, K.M. 1978. Venom coagglutinin: an activator of platelet aggregation dependent on von Willebrand factor. Proc. Natl. Acad. Sci. USA 75: 4514−4518. | PubMed  | ChemPort |

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