Abstract
The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigated Xa and IXabeta production by VIIa-TF in a system reconstituted with both X and IX and the principal physiologic inhibitors of this pathway TFPI and antithrombin III (AT). Kinetic studies without inhibitors established that IX and X functioned as competitive alternate substrates for VIIa/TF with similar kinetic constants. When both IX and X were present, TFPI significantly inhibited the extent of formation of either IXabeta or Xa. In contrast, AT rapidly depleted active Xa with a small effect on IXabeta formation. When both AT and TFPI were present, active IXabeta formation significantly exceeded the formation of active Xa regardless of the VIIa/TF concentration. These findings could be quantitatively accounted for by a model encompassing the kinetics of the individual activation and inhibition steps. Active Xa formation by this pathway is regulated in a principal way by its rapid inactivation by AT. In contrast, the Xa-dependent inhibitory reactions of TFPI play a primary role in limiting zymogen consumption and the formation of active IXabeta. These regulatory phenomena yield active IXabeta as a major rather than secondary product of VIIa/TF. Our findings raise the possibility that IXabeta produced by the extrinsic pathway, and its ability to function within the intrinsic Xase complex to activate X may play a significant role in producing Xa necessary for both the initiation and sustained phases of the procoagulant response following vascular damage.
Highlights
The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation
Whereas the reactions of the Kunitzlike inhibitory domains of TFPI involve tight-binding reversible cles composed of 75% (w/w) L-␣-phosphatidylcholine and 25% (w/w) L-␣-phosphatidylserine; PfIXa, methysulfonyl-D-cyclohexylglycyl-glycyl-L-arginyl-7-amino-4-methylcoumarin; Recombinant tick anticoagulant peptide (rTAP), recombinant tick anticoagulant peptide; SpXa, methoxycarbonyl-D-cyclohexylglycyl-glycylarginyl-p-nitroanilide; SpIXa, methysulfonyl-D-cyclohexylglycyl-glycylarginyl-p-nitroanilide; TF-PC, PC vesicles containing ϳ1 effective TF/ vesicle; TF-PC and 25% (w/w) PS (PCPS), PCPS vesicles containing ϳ1 effective TF/vesicle; TFPI, recombinant full-length tissue factor pathway inhibitor; TFPIK36I, full-length TFPI with Lys at position 36 mutated to Ile; MES, 4-morpholineethanesulfonic acid
Despite a significant contribution of the substrate-membrane interaction to the perceived affinity of VIIa/TF-PCPS for factor X, the overall process can be adequately described by the Henri-Michaelis-Menten equation in a reaction in which Xa acts as a product inhibitor with affinity comparable with that of the substrate [41]
Summary
The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. These studies have been used as a formal basis to provide a global kinetic analysis and full accounting for active Xa and IXa formation by VIIa/TF at physiologic concentrations of substrates and in the presence of the inhibitors TFPI and AT.
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