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

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Abstract
Factors Va and Xa (FVa and FXa, respectively) assemble on phosphatidylserine (PS)-containing platelet membranes to form the essential “prothrombinase” complex of blood coagulation. The C-terminal domain (C2) of FVa (residues 2037−2196 in human FVa) contains a soluble phosphatidylserine (C6PS) binding pocket flanked by a pair of tryptophan residues, Trp2063 and Trp2064. Mutating these tryptophans abolishes FVa 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 FVa cofactor activity, we expressed W(2063,2064)A mutants of the recombinant C2 domain (rFVa2-C2) and of a B domain-deleted factor V light isoform (rFVa2) in Hi-5 and COS cells, respectively. Intrinsic fluorescence showed that wild-type rFVa2-C2 binds to C6PS and to 20% PS/PC membranes with apparent Kd values of 2.8 μM and 9 nM, respectively, while mutant rFVa2-C2 does not. Equilibrium dialysis confirmed that mutant rFVa2-C2 does not bind to C6PS. Mutant rFVa2 binds to C6PS (Kd ∼ 37 μM) with an affinity comparable to that of wild-type rFVa2 (Kd ∼ 20 μM), although it does not bind to PS/PC membranes to which wild-type rFVa2 binds with native affinity (Kd ∼ 3 nM). Both wild-type and mutant rFVa2 bind to active site-labeled FXa (DEGR-Xa) in the presence of 400 μM C6PS with native affinity (Kd ∼ 3−4 nM) to produce a solution rFVa2−FXa complex of native activity. We conclude that (1) the C2 domain PS site provides all but ∼1 kT of the free energy of FVa 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 FXa−FVa complex or its activity, but (4) another PS site on FVa does have a regulatory role.

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