Molecular Mechanism of Extrinsic Blood Coagulation Pathway

外源性凝血途径的启动由血浆衍生因子VII/VIIa和细胞衍生组织因子（TF）之间形成的复合物介导。为了确定相互作用的位点，对酶原VII和VIIa进行酶促和化学修饰，并通过测量它们对形成VIIa-TF复合物后增强的酰胺分解活性的抑制作用来估计它们与TF的亲和力。我们发现VII α轻链（Ki=3.5 × 10 ~ 4<-7>）和由Gla结构域和第一表皮生长因子（EGF）样结构域组成的片段（Gla-EGF_1肽; Ki=1.0 × 10 ~ 4<-6>）与TF具有亲和力，但它们的结合能力分别被常规的胰凝乳蛋白酶切割而消失，因此VII与TF的结合位点之一可能位于Gla-EGF_1区。另一方面，经丹磺酰-Glu-Gly-Arg氯甲基酮处理的Gla-结构域缺失的VIIa（Ki=0.7 × 10 - 6<-7>）显示出与TF的高亲和力，而相应的Gla-结构域缺失的VIIa（Ki=0.7 × 10 - 6）显示出与TF的高亲和力。 ...更多信息 同样处理过的VII中没有显示出结合潜力，从而表明Gla-EGF 1区以外的结合位点存在于VIIa中，而不存在于VII中。通过对VIIa的Ile-153氨基端的α-氨基进行乙酰化或氨甲酰化，破坏了Ile-153和Asp 343之间的盐桥，使VIIa失去了与TF的结合能力，从而使VIIa转变为酶原样的非活性形式。在TF存在下，VIIa的氨甲酰化率较低，与TF不存在下相比。在形成复合物后，Ile-153的α-氨基免于氨甲酰化的保护似乎是由于在VIIa-TF复合物中Ile-153和Asp-343之间形成盐桥。因此，可以得出结论，TF与VIIa重链的结合诱导了特定的构象变化，使Ile-153的α-氨基接近Asp-343的β-羧基，形成稳定的盐桥。只有在TF存在下形成的这种盐桥对于形成VIIa的催化三联体是必不可少的。少

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