The Phosphatidylserine Binding Site of the Factor V_a C2 Domain Accounts for Membrane Binding but Does Not Contribute to the Assembly or Activity of a Human Factor X_a−Factor V_a Complex

Abstract

Factors V(a) and X(a) (FV(a) and FX(a), respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential "prothrombinase" complex of blood coagulation. The C-terminal domain (C2) of FV(a) (residues 2037-2196 in human FV(a)) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp(2063) and Trp(2064). Mutating these tryptophans abolishes FV(a) membrane binding. To address both the roles of these tryptophans in C6PS or membrane binding and the role of the C2 domain lipid binding site in regulation of FV(a) cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFV(a2)-C2) and of a B domain-deleted factor V light isoform (rFV(a2)) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFV(a2)-C2 binds to C6PS and to 20% PS/PC membranes with apparent K(d) values of 2.8 microM and 9 nM, respectively, while mutant rFV(a2)-C2 does not. Equilibrium dialysis confirmed that mutant rFV(a2)-C2 does not bind to C6PS. Mutant rFV(a2) binds to C6PS (K(d) approximately 37 microM) with an affinity comparable to that of wild-type rFV(a2) (K(d) approximately 20 microM), although it does not bind to PS/PC membranes to which wild-type rFV(a2) binds with native affinity (K(d) approximately 3 nM). Both wild-type and mutant rFV(a2) bind to active site-labeled FX(a) (DEGR-X(a)) in the presence of 400 microM C6PS with native affinity (K(d) approximately 3-4 nM) to produce a solution rFV(a2)-FX(a) complex of native activity. We conclude that (1) the C2 domain PS site provides all but approximately 1 kT of the free energy of FV(a) membrane binding, (2) tryptophans lining the C2 lipid binding pocket are critical to C6PS and membrane binding and insert into the bilayer interface during membrane binding, (3) occupancy of the C2 lipid binding pocket is not necessary for C6PS-induced formation of the FX(a)-FV(a) complex or its activity, but (4) another PS site on FV(a) does have a regulatory role.

Modulation of Prothrombinase Assembly and Activity by Phosphatidylethanolamine

“…C6PE bound to rHFVa 2 with a K d of 6.5 Ϯ 0.5 M based on a single-site model for binding (Fig. 4A), comparable with values previously reported for C6PS binding to rHFVa 2 (ϳ20 M) (14). Interspecies control experiments confirmed that C6PE bound with affinity comparable with that of bovine Va 2 (shown in the inset to Fig.…”

“…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?…”

“…Binding of Factors Xa and Va to Soluble Lipids-A SPEX FluoroLog-3 spectrofluorometer (Horiba Jobin Yvon Inc., Edison, NJ) was used to measure intrinsic fluorescence intensity, using an excitation wavelength of 280 nm (4-nm band pass) and an emission wavelength of 340 nm (4-nm band pass) as described previously (14). In dual-ligand experiments, human factor Xa and rHFVa were preincubated with C6PS at 23°C and then titrated with C6PE and vice versa.…”

“…In dual-ligand experiments, human factor Xa and rHFVa were preincubated with C6PS at 23°C and then titrated with C6PE and vice versa. Fluorescence data were fit to a single-site binding model using a Marquardt-Levenberg algorithm (14).…”

The factor VIII C1 domain contributes to platelet binding

“…As in FVIII, the C2 domain contains the primary membrane-binding site of FV. 31,32 The FV A3 33 and C1 34 domains also appear to interact with membranes. In FV and/or FVIII, the C domains may reorient during membrane attachment.…”