Gorkun O V, Veklich Y I, Medved L V, Henschen A H, Weisel J W
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104.
Biochemistry. 1994 Jun 7;33(22):6986-97. doi: 10.1021/bi00188a031.
The role of the carboxyl-terminal portion of the alpha chains of fibrin (alpha C domains) in clot formation was investigated by transmission and scanning electron microscopy and turbidity studies of clots made from preparations of molecules missing one or both of these domains. Highly purified and entirely clottable preparations of bovine fragment X monomer, one containing primarily molecules missing a single alpha C domain (fragment X1) and the other consisting of molecules missing both alpha C domains (fragment X2), were used for these experiments. These preparations were characterized by various methods, including the complete determination of the amino- and carboxyl-termini of all peptides and fragments. These preparations formed clots on dilution to neutral pH. In all cases, clots observed by either scanning or transmission electron microscopy were made up of a branched network of fibers, similar to those formed by thrombin treatment of intact fibrinogen, suggesting that the alpha C domains are not necessary for protofibril and fiber formation or branching. However, both the fiber and clot structure varied with the different fractions, indicating that the alpha C domains do participate in polymerization. The rate of assembly, as indicated by the lag period and maximum rate of turbidity increase, as well as the final turbidity, was decreased with removal of the alpha C domains, suggesting that they accelerate polymerization. preparations of isolated alpha C fragment added to fibrin monomer have striking effects on the turbidity curves, showing a decrease in the rate of polymerization in a dose-dependent manner but not complete inhibition. Electron microscopy of fibrin monomer desA molecules at neutral pH showed that most of the alpha C domains, like those in fibrinogen, remain associated with the central region. Thus, it appears that normally with thrombin cleavage of fibrinogen the effects of the interactions of alpha C domains observed here will be most significant for lateral aggregation.
通过透射电子显微镜和扫描电子显微镜以及对缺失一个或两个这些结构域的分子制剂所形成凝块的浊度研究，来探究纤维蛋白α链羧基末端部分（αC结构域）在凝块形成中的作用。这些实验使用了高度纯化且完全可凝的牛X片段单体制剂，一种主要包含缺失单个αC结构域的分子（X1片段），另一种由缺失两个αC结构域的分子组成（X2片段）。这些制剂通过各种方法进行了表征，包括对所有肽和片段的氨基末端和羧基末端的完整测定。这些制剂在稀释至中性pH时形成凝块。在所有情况下，通过扫描或透射电子显微镜观察到的凝块均由纤维分支网络组成，类似于凝血酶处理完整纤维蛋白原形成的凝块，这表明αC结构域对于原纤维和纤维的形成或分支并非必需。然而，纤维和凝块结构因不同组分而异，表明αC结构域确实参与了聚合。随着αC结构域的去除，由延迟期和浊度增加的最大速率以及最终浊度所表明的组装速率降低，这表明它们加速了聚合。添加到纤维蛋白单体中的分离αC片段制剂对浊度曲线有显著影响，以剂量依赖方式显示聚合速率降低但并非完全抑制。在中性pH下对纤维蛋白单体desA分子进行电子显微镜观察表明，大多数αC结构域，与纤维蛋白原中的那些结构域一样，仍与中心区域相关联。因此，似乎正常情况下，随着纤维蛋白原被凝血酶切割，此处观察到的αC结构域相互作用的影响对于横向聚集将最为显著。
