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,Peter N Walsh

doi:10.1074/jbc.m107753200

Abstract

Previously we have determined that residues 88-109 (but not Arg(94)) 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): FIXa(FVIIEGF2) (FIX Delta 88-124,inverted Delta FVII91-127), FIXa(loop1) (FIX Delta 88-99,inverted Delta FVII91-102), FIXa(loop2) (FIX Delta 95-109,inverted Delta FVII98-112), and FIXa(loop3) (FIX Delta 111-124,inverted Delta FVII114-127) and tested their ability to bind to thrombin-activated platelets. Binding affinities (K(d) values in 10(-9) m) for the proteins were as follows in the presence and absence of FVIIIa, respectively: FIXa(N) (0.55 +/- 0.06, 2.9 +/- 0.45), FIXa(WT) (0.80 +/- 0.08, 3.5 +/- 0.5), FIXa(loop1) (19 +/- 4.0, 27 +/- 5.0), FIXa(loop2) (35 +/- 9.0, 65 +/- 12.0), and FIXa(loop3) (1.1 +/- 0.09, 5.0 +/- 0.90). These K(d) values are in good agreement with K((d)(app)) values (in 10(-9) m) determined from the activation of FX (in the presence and absence of FVIIIa, respectively): FIXa(N) (0.46 +/- 0.05, 1.40 +/- 0.14), FIXa(WT) (0.72 +/- 0.08, 3.8 +/- 0.08), FIXa(loop1) (3.2 +/- 0.72, 14.0 +/- 1.60), FIXa(loop2) (18.4 +/- 1.60, 26.3 +/- 3.40), and FIXa(loop3) (0.7 +/- 0.05, 3.0 +/- 0.15). Moreover, the stoichiometry of binding (sites/platelet) showed an agreement with V(max) 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 (Leu(84)-Val(128)) was an effective inhibitor of FIXa binding to platelets in both the presence (K(i) = 0.7 x 10(-6) m) and the absence (K(i) = 1.5 x 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.ut not Ar

Highlights

Arg145-Ala146 and Arg180-Val181 mediated either by factor XIa (FXIa) or by factor VIIa/tissue factor (FVIIa/TF)
Maximal catalytic efficiency of this reaction results from assembly of the FX activating complex (3), which consists of Factor IXa (FIXa), factor VIIIa (FVIIIa), and FX assembled on a procoagulant surface such as the activated platelet membrane
The FIX zymogen binds to a reduced number of sites (250 –300 sites per platelet, Kd ϭ 3 nM), and neither its affinity nor stoichiometry is enhanced by the presence of FVIIIa and FX

Summary

Introduction

Arg145-Ala146 and Arg180-Val181 mediated either by factor XIa (FXIa) or by factor VIIa/tissue factor (FVIIa/TF). A three-receptor model has been proposed whereby platelets expose distinct binding sites for FIXa, FVIIIa, and FX (4 –7) Occupancy of these binding sites is thought to promote the interactions among these proteins and facilitate assembly of functional FX activating complexes. FIXaFXEGF2 was deficient in its FX activating activity (Vmax ϭ 3 pM FXa per min) compared with FIXaWT (Vmax ϭ 38 pM FXa per min) (16) These results highlight the important contribution of the EGF2 domain to the surface-localization and catalytic properties of FIXa. Our interest has been to further investigate the mechanism by which FIXa binds to surfaces and to understand the functional consequences of these interactions. We have identified residues contained within the sequence Cys to Cys109 that mediate surface binding and assembly of FX activating complexes on the platelet membrane

Objectives

Results

Conclusion