Kinetics of blood coagulation factor Xaalpha autoproteolytic conversion to factor Xabeta. 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 Xaalpha (FXaalpha), to yield factor Xabeta (FXabeta). In the preceding paper, we showed that generation of FXabeta leads to expression of a plasminogen binding site. FXabeta 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 FXabeta generation, which showed that autoproteolysis of FXaalpha followed a second order mechanism where FXaalpha and FXabeta behaved as identical enzymes. Rate constants of 9 and 172 M-1 s-1 were derived, respectively, in the absence and presence of FXaalpha 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 FXaalpha proximal to the beta-peptide, functional comparisons were conducted using homogeneous and stabilized preparations of FXaalpha and FXabeta. 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 FXaalpha, conversion to FXabeta was not observed to influence these functions. The data support a model where FXaalpha is predominantly responsible for thrombin generation and where slow conversion to FXabeta coordinates coagulation and the initiation of fibrinolysis at sites of prothrombinase assembly.
Pryzdial
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Research Department, The Canadian Red Cross Society, Ottawa, Ontario K1G 4J5, Canada.
Kessler
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GE
D000990:Antithrombin III / Q000494:pharmacology*; D015951:Factor Xa / Q000096:biosynthesis / Q000378:metabolism*; D065427:Factor Xa Inhibitors; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D013379:Substrate Specificity; D013925:Thromboplastin / Q000378:metabolism*
D065427:Factor Xa Inhibitors; D000990:Antithrombin III; D013925:Thromboplastin; D015951:Factor Xa