A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex.
Tightly associated factor V(a) (FVa) and factor X(a) (FXa) serve as the essential prothrombin-activating complex that assembles on phosphatidylserine (PS)-containing platelet membranes during blood coagulation. We have previously shown that (1) a soluble form of PS (C6PS) triggers assembly of a fully active FVa-FXa complex in solution and (2) that 2 molecules of C6PS bind to FVa light chain with one occupying a site in the C2 domain. We expressed human factor V(a) (rFVa) with mutations in either the C1 domain (Y1956,L1957)A, the C2 domain (W2063,W2064)A, or both C domains (Y1956,L1957,W2063,W2064)A. Mutations in the C1 and C1-C2 domains of rFVa reduced the rate of activation of prothrombin to thrombin by FXa in the presence of 400 muM C6PS by 14 000- to 15 000-fold relative to either wild-type or C2 mutant factor rFVa. The K(d')s of FXa binding with rFVa (wild-type, C2 mutant, C1 mutant, and C1-C2 mutant) were 3, 4, 564, and 624 nM, respectively. Equilibrium dialysis experiments detected binding of 4, 3, and 2 molecules of C6PS to wild-type rFVa, C1-mutated, and C1,C2-mutated rFVa, respectively. Because FVa heavy chain binds 2 molecules of C6PS, we conclude that both C2 and C1 domains bind one C6PS, with binding to the C1 domain regulating prothrombinase complex assembly.
Majumder
Rinku
R
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, USA.
Quinn-Allen
Mary Ann
MA
Kane
William H
WH
Lentz
Barry R
BR
D000596:Amino Acids / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002522:Chlorocebus aethiops; D004252:DNA Mutational Analysis; D005165:Factor V / Q000378:metabolism*; D015943:Factor Va / Q000737:chemistry* / Q000378:metabolism*; D015951:Factor Xa / Q000378:metabolism*; D006801:Humans; D008954:Models, Biological; D050505:Mutant Proteins / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011516:Prothrombin / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility; D013329:Structure-Activity Relationship
D000596:Amino Acids; D050505:Mutant Proteins; D010718:Phosphatidylserines; D011994:Recombinant Proteins; C022475:prothrombinase complex; D015943:Factor Va; D005165:Factor V; D011516:Prothrombin; D015951:Factor Xa