Factor Va-factor Xa interactions: molecular sites involved in enzyme:cofactor assembly.
The generation of thrombin by the prothrombinase complex is a key event in coagulation. In this complex, the activated form of coagulation factor V (FVa) serves as an essential cofactor to factor Xa (FXa) in the activation of prothrombin to thrombin. The enzyme FXa is virtually ineffective in the absence of its cofactor. The assembly of FXa with its cofactor FVa on negatively charged phospholipid membranes enhances its catalytic efficiency by several orders of magnitude. The non-activated procofactor factor V (FV) circulates in plasma with a domain organization of A1-A2-B-A3-C1-C2 expressing little procoagulant activity. Upon activation through limited proteolysis by either thrombin or FXa, the B-domain dissociates from FVa. After activation, the procoagulant activity of FVa is greatly enhanced. This report provides insight into the interaction of FV and FXa and the molecular events important in enzyme:cofactor assembly of the FXa:FVa complex. Furthermore, light is shed on the molecular events associated with the activation process, i.e. the release of the B-domain and exposure of binding sites for FXa. The assembly of FVa and FXa was studied using a set of recombinant FV mutants. The interaction between FVa and FXa on phospholipid was investigated with a functional prothrombin activation assay as well as in a novel direct binding assay in the absence of prothrombin. We found that all three thrombin cleavages in FV contribute to increasing the FXa affinity and that the B-domain in intact FV has an inhibitory effect on the FV-FXa interaction, which is important in prohibiting premature coagulation.
Steen
M
M
Department of Laboratory Medicine, Division of Clinical Chemistry, Lund University, The Wallenberg Laboratory, University Hospital, Malmö, Sweden. marten.steen@klkemi.mas.lu.se
D001665:Binding Sites / Q000502:physiology; D015943:Factor Va / Q000737:chemistry / Q000378:metabolism*; D015951:Factor Xa / Q000737:chemistry / Q000378:metabolism*; D006801:Humans
D015943:Factor Va; D015951:Factor Xa