title: A phosphatidylserine binding site in factor Va C1 domain regulates both assembly and activity of the prothrombinase complex.
abstract: 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.
authors.affiliation: Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, USA.
authors.forename: Rinku
authors.initials: R
authors.lastname: Majumder
authors.forename: Mary Ann
authors.initials: MA
authors.lastname: Quinn-Allen
authors.forename: William H
authors.initials: WH
authors.lastname: Kane
authors.forename: Barry R
authors.initials: BR
authors.lastname: Lentz
mesh_terms: 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
chemical_list: 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