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Originally published In Press as doi:10.1074/jbc.M107753200 on November 19, 2001

J. Biol. Chem., Vol. 277, Issue 8, 5734-5741, February 22, 2002
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The Factor IXa Second Epidermal Growth Factor (EGF2) Domain Mediates Platelet Binding and Assembly of the Factor X Activating Complex*

Frank H. Wilkinson, Syed S. Ahmad, and Peter N. WalshDagger

From the Sol Sherry Thrombosis Research Center, Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Previously we have determined that residues 88-109 (but not Arg94) in the second epidermal growth factor (EGF2)-like domain of factor IXa (FIXa) are important for assembly of the factor X (FX) activating complex on phospholipid vesicles (Wilkinson, F. H., London, F. S., and Walsh, P. N. (2002) J. Biol. Chem. 277, 5725-5733). Here we report that these residues are important for platelet binding affinity, stoichiometry, and assembly of the FX activating complex. We prepared several chimeric FIXa proteins using homologous sequences from factor VII (FVII): FIXaFVIIEGF2 (FIXDelta 88-124,nabla FVII91-127), FIXaloop1 (FIXDelta 88-99,nabla FVII91-102), FIXaloop2 (FIXDelta 95-109,nabla FVII98-112), and FIXaloop3 (FIXDelta 111-124,nabla FVII114-127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (Kd values in 10-9 M) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXaN (0.55 ± 0.06, 2.9 ± 0.45), FIXaWT (0.80 ± 0.08, 3.5 ± 0.5), FIXaloop1 (19 ± 4.0, 27 ± 5.0), FIXaloop2 (35 ± 9.0, 65 ± 12.0), and FIXaloop3 (1.1 ± 0.09, 5.0 ± 0.90). These Kd values are in good agreement with Kd(app) values (in 10-9 M) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXaN (0.46 ± 0.05, 1.40 ± 0.14), FIXaWT (0.72 ± 0.08, 3.8 ± 0.08), FIXaloop1 (3.2 ± 0.72, 14.0 ± 1.60), FIXaloop2 (18.4 ± 1.60, 26.3 ± 3.40), and FIXaloop3 (0.7 ± 0.05, 3.0 ± 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with Vmax of FX activation and was reduced in those proteins that also showed a decreased platelet binding affinity. A peptide corresponding to the FIX EGF2 domain (Leu84-Val128) was an effective inhibitor of FIXa binding to platelets in both the presence (Ki = 0.7 × 10-6 M) and the absence (Ki = 1.5 × 10-6 M) of FVIIIa and FX. We conclude that residues 88-109 of the FIXa EGF2 domain mediate binding to platelets and assembly of the FX activating complex.

* This study was supported by National Institutes of Health Research Grants HL56914, HL56153, and HL46213.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: The Sol Sherry Thrombosis Research Center, Temple Univ. School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4375; Fax: 215-707-3005; E-mail: pnw@astro.temple.edu.

Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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