Center for Vascular and Inflammatory Diseases and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1138, USA.
Biochemistry. 2011 Sep 20;50(37):8028-37. doi: 10.1021/bi2008189. Epub 2011 Aug 24.
Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear, branching filaments when analyzed by electron microscopy. Spectral studies revealed that the αC-domains in such oligomers were folded into compact structures of high thermal stability with a significant amount of β-sheets. These findings indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of fibrin αC polymers. The oligomers also exhibited functional properties of polymeric fibrin because, in contrast to the monomeric αC-domain, they bound tPA and plasminogen and stimulated activation of the latter by the former. Altogether, the results obtained with cross-linked Aα221-610 oligomers clarify the structure of the αC-domains in fibrin αC polymers and confirm our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for further structure-function studies of fibrin αC-domains.
我们之前的研究表明纤维蛋白原的 αC 结构域与它们的配体没有反应，这表明它们的结合位点是隐蔽的，只有在纤维蛋白原转化为纤维蛋白时才会暴露出来，此时这些结构域形成 αC 聚合物。基于这一发现，我们假设纤维蛋白中 αC 结构域的聚合导致其结合位点暴露，并且这些结构域仅在 αC 结构域聚合物中采用生理活性构象。为了验证这一假设，我们制备了包含 αC 结构域（Aα392-610）的重组 αC 区（残基 Aα221-610），证明它以浓度依赖和可逆的方式形成可溶性寡聚体，并通过与因子 XIIIa 共价交联来稳定这些寡聚体。交联的 Aα221-610 寡聚体在溶液中稳定，当通过电子显微镜分析时，它们呈现出有序的线性、分支丝状。光谱研究表明，这种寡聚体中的 αC 结构域折叠成具有高热稳定性的紧凑结构，具有大量的β-折叠。这些发现表明交联的 Aα221-610 寡聚体高度有序，模拟了纤维蛋白 αC 聚合物的结构。寡聚体还表现出聚合纤维蛋白的功能特性，因为与单体 αC 结构域相比，它们结合 tPA 和纤溶酶原，并刺激后者被前者激活。总之，用交联的 Aα221-610 寡聚体获得的结果阐明了纤维蛋白 αC 聚合物中 αC 结构域的结构，并证实了我们的假设，即它们的结合位点在聚合时暴露。这些寡聚体代表了纤维蛋白 αC 聚合物的稳定、可溶性模型，可用于进一步研究纤维蛋白 αC 结构域的结构-功能关系。
