Efficient Thrombin Generation Requires Molecular Phosphatidylserine, Not a Membrane Surface.

Activation of prothrombin to thrombin is catalyzed by “prothrombinase” complex, traditionally viewed as factor X_a (FX_a) in complex with factor V_a (FV_a) on a phosphatidylserine (PS)-containing membrane surface, which is widely regarded as required for efficient activation. Activation involves cleavage of two peptide bonds and proceeds via one of two released intermediates or through “channeling” (activation without release of an intermediate). We ask here whether the PS molecule itself, not the membrane surface, is sufficient to produce fully active human “prothrombinase” complex in solution. Both FX_a and FV_a bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FX_a and FV_a2 (light isoform of FV_a), these proteins form a tight (K_d = 0.6 ±0.09 nM at 37°C) soluble complex. Complex assembly occurs well below the critical micelle concentration of C6PS, as established in the presence of the proteins by quasi-elastic light scattering and pyrene fluorescence. Ferguson analysis of native gels show that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FX_a and one FV_a2, further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2x10⁸ M^{− 1}sec^{− 1}) and via the same reaction pathway (50–60% channeling, rest via meizothrombin intermediate) as activation catalyzed by a complex assembled on PS-containing membranes (4.4x10⁸ M^{− 1}sec^{− 1}). These results question the accepted role of PS-membranes as providing “dimensionality-reduction” and favor a regulatory role for platelet-membrane-exposed PS.