Soluble phosphatidylserine triggers assembly in solution of a prothrombin-activating complex in the absence of a membrane surface

Factor X-a (FXa) binding to factor V-a (FVa) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the "prothrombinase complex" that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FVa, FVa1 and FVa2. These two isoforms differ by a 3-kDa polysaccharide chain (at Asn(2181) in human FVa1 (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999) Biochemistry 38, 11448-11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FXa, finding no significant difference. A soluble form of PS (C6PS) bound to FVa1 and FVa2 with comparable affinities (K-d = 11-12 muM) and changes in FVa intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FVa2 enhanced its affinity for FXa in solution by nearly 3 orders of magnitude (K-d(eff) = 40-2 nm over a C6PS range of 30-400 mum) but had no effect on the affinity of FVa1 for FXa (K-d = 1 mum). This results in a soluble complex between FXa and FVa2, whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparent k(cat)/K-m = 10(9) M-1 s(-1)). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.