Kinetics of blood coagulation factor Xa alpha autoproteolytic conversion to factor Xa beta - Effect on inhibition by antithrombin, prothrombinase assembly, and enzyme activity

Autoproteolysis of blood coagulation factor Xa (FXa) results in the excision of a 4-kDa fragment (beta-peptide) from the intact subform, factor Xa alpha (FXa alpha), to yield factor Xa beta (FXa beta) In the preceding paper, we showed that generation of FXa beta leads to expression of a plasminogen binding site, FXa beta may consequently participate in fibrinolysis; therefore, the timing of subform conversion compared with thrombin production is important. In the current study we evaluated the kinetics of FXa beta generation, which showed that autoproteolysis of FXa alpha followed a second order mechanism where FXa alpha and FXa beta behaved as identical enzymes, Rate constants of 9 and 172 M(-1) s(-1) were derived, respectively, in the absence and presence of FXa alpha binding to procoagulant phospholipid. Under identical conditions the latter is estimated to be 6 orders of magnitude slower than thrombin generation by prothrombinase. Since heparin binding and prothrombin recognition have been previously attributed to a region of FXa alpha proximal to the beta-peptide, functional comparisons were conducted using homogeneous and stabilized preparations of FXa alpha and FXa beta. Comparisons included 1) the recognition of small substrates; 2) the rate of interaction with antithrombin/heparin; 3) the assembly of prothrombinase; and 4) the activation of prothrombin by prothrombinase, Although the beta-peptide neighbors a probable functional region in FXa alpha, conversion to FXa beta was not observed to influence these functions, The data support a model where FXa alpha is predominantly responsible for thrombin generation and where slow conversion to FXa beta coordinates coagulation and the initiation of fibrinolysis at sites of prothrombinase assembly.