Efficient Thrombin Generation Requires Molecular Phosphatidylserine, Not a Membrane Surface

• Published in: Biochemistry (Easton) 2005-12, Vol.44 (51), p.16998-17006
• Main Authors: Majumder, Rinku, Weinreb, Gabriel, Lentz, Barry R
• Format: Article
• Language: English
• Subjects: Arginine - analogs & derivatives; Arginine - chemistry; Blood Platelets - physiology; Calcium - metabolism; Cell Membrane - physiology; Dansyl Compounds - chemistry; Enzyme Activation; Factor V - chemistry; Factor V - metabolism; Factor Va - metabolism; Factor Xa - chemistry; Factor Xa - metabolism; Humans; Kinetics; Micelles; Molecular Weight; Phosphatidylserines - metabolism; Phosphatidylserines - physiology; Prothrombin - metabolism; Spectrometry, Fluorescence; Thermodynamics; Thrombin - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi051469f

Activation of prothrombin to thrombin is catalyzed by a “prothrombinase” complex, traditionally viewed as factor Xa (FXa) in complex with factor Va (FVa) 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 the release of an intermediate). We ask here whether the PS molecule itself and not the membrane surface is sufficient to produce the fully active human “prothrombinase” complex in solution. Both FXa and FVa bind soluble dicaproyl-phosphatidylserine (C6PS). In the presence of sufficient C6PS to saturate both FXa and FVa2 (light isoform of FVa), 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 shows that the complex migrates with an apparent molecular mass only slightly larger than that expected for one FXa and one FVa2, further ruling out complex assembly on C6PS micelles. Human prothrombin activation by this complex occurs at nearly the same overall rate (2.2 × 108 M-1 s-1) and via the same reaction pathway (50−60% channeling, with the rest via the meizothrombin intermediate) as the activation catalyzed by a complex assembled on PS-containing membranes (4.4 × 108 M-1 s-1). These results question the accepted role of PS membranes as providing “dimensionality reduction” and favor a regulatory role for platelet-membrane-exposed PS.