A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex

Abstract

Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor V(a) (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 muM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The K(d')s of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…3A, inset; K d ϭ ϳ3 Ϯ 0.5 nM) compared with the synthetic lipids used in this study. This is consistent with the observation that factor Va binding free energy derives principally from insertion of Trp residues into the membrane (27), an event that should be promoted by the reduced interfacial packing associated with highly unsaturated lipids. Adding 1% PS to 30% DOPE membranes had little effect on factor Va binding, although increasing PS content to 5-10% did improve binding somewhat but not beyond what was observed in the absence of DOPE (Table 2).…”

“…More extensive experiments are needed to prove this hypothesis. We expect that this is not the case for factor Va 2 , because only the site in the C1 domain regulates the interaction between factors Xa and Va (27). We 2 Binding of soluble phospholipids to 0.1 M rHFVa or 0.1 M human factor Xa was monitored using intrinsic fluorescence intensity in 20 mM Tris, 150 mM NaCl, and 5 mM CaCl 2 , pH 7.5, as a function of C6PS, C6PE, C6PC, or C6(D)PS and the concentration at 23°C.…”

“…In this picture, the effect of PE on membrane interfacial packing contributes only to bringing factor Va to the membrane, but not for the reduction in K m , the increase in k cat , and the decrease in K d app , which result from PE regulation of both factors Xa and Va. The lipid-binding regions through which C6PS influences factors Va and Xa have been identified (14,27,41). Are these the same sites occupied by PE?…”

Novel Role for Galectin-8 Protein as Mediator of Coagulation Factor V Endocytosis by Megakaryocytes

“…However, we found that lactadherin, which binds PS with a high affinity and shares homology to the PS-binding domains of FV (30), completely failed to prevent FV uptake (Fig. 1C).…”

“…The reported ability of Gal8 to induce PS exposure on the surface of cells (35,36) and the known affinity of FV for PS (30) raised the possibility that the uptake mechanism may be dictated by PS exposure. However, we found that lactadherin, which binds PS with a high affinity and shares homology to the PS-binding domains of FV (30), completely failed to prevent FV uptake (Fig.…”