Molecular Mechanism of Extrinsic Blood Coagulation Pathway

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Initiation of the extrinsic blood coagulation pathway is mediated by a complex formed between plasma-derived factor VII/VIIa and cell-derived tissue factor (TF). To identify the site(s) of interaction, zymogen VII and VIIa were enzymatically and chemically modified, and their affinities with TF were estimated by measuring their inhibitory effects on the amidolytic activity enhanced after formation of the VIIa-TF complex. We found that the VIIa-light chain(Ki=3.5 X 10^<-7>) and its fragment consisting of the Gla-domain and the first epidermal growth factor (EGF)-like domain (Gla-EGF1 peptide ; Ki=1.0 X 10^<-6>) have an affinity with TF, but their binding capacity disappeared, respectively, by conventional chymotryptic cleavage.Therefore, one of the binding sites of VII with TF probably locates in the Gla-EGF1 region. On the other hand, a dansyl-Glu-Gly-Arg chloromethyl ketone-treated Gla-domainless VIIa(Ki=0.7 X 10^<-7>) showed a high affinity with TF, whereas the corresponding Gla-doma … More inless VII similarly treated showed no binding potential, thereby indicating that binding site(s) other than in the Gla-EGF1 region is present in VIIa but not in VII. Acetylation or carbamylation of alpha-amino group of NH_2-terminal Ile-153 of VIIa resulted in the loss of binding affinity with TF ; such modifications convert VIIa into a zymogen like inactive form by destroying the salt bridge between Ile-153 and Asp343 in VIIa. The carbamylation rate of VIIa in the presence of TF was low, as compared with that in the absence of TF. The protection of alpha-amino group of Ile-153 from carbamylation after complex formation seemed to be due to a salt bridge formation between Ile-153 and Asp-343 in VIIa-TF complex. Therefore, it is concluded that the binding of TF with the heavy chain of VIIa induces a specific conformational change that brings alpha-amino group of Ile-153 close to beta-carboxyl group of Asp-343 to make a stable salt bridge. This salt bridge formed only in the presence of TF is essential for the formation of the catalytic triad of VIIa. Less