Soluble phosphatidylserine triggers assembly in solution of a prothrombin-activating complex in the absence of a membrane surface.
Factor X(a) (FX(a)) binding to factor V(a) (FV(a)) 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 FV(a), FV(a1) and FV(a2). These two isoforms differ by a 3-kDa polysaccharide chain (at Asn(2181) in human FV(a1) (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 FX(a), finding no significant difference. A soluble form of PS (C6PS) bound to FV(a1) and FV(a2) with comparable affinities (K(d) = 11-12 microm) and changes in FV(a) intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FV(a2) enhanced its affinity for FX(a) in solution by nearly 3 orders of magnitude (K(d)(eff) = 40-2 nm over a C6PS range of 30-400 microm) but had no effect on the affinity of FV(a1) for FX(a) (K(d) = 1 microm). This results in a soluble complex between FX(a) and FV(a2), 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) congruent with 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.
Majumder
Rinku
R
Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599-7260, USA.
Weinreb
Gabriel
G
Zhai
Xin
X
Lentz
Barry R
BR
D000818:Animals; D001779:Blood Coagulation Factors / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005453:Fluorescence; D006868:Hydrolysis; D010718:Phosphatidylserines / Q000378:metabolism*; D011516:Prothrombin / Q000378:metabolism*; D012995:Solubility; D012996:Solutions
D001779:Blood Coagulation Factors; D010718:Phosphatidylserines; D012996:Solutions; D011516:Prothrombin