Veklich Y I, Gorkun O V, Medved L V, Nieuwenhuizen W, Weisel J W
Department of Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia 19104-6058.
J Biol Chem. 1993 Jun 25;268(18):13577-85.
The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.
通过对旋转阴影制备物的电子显微镜观察，确定了纤维蛋白原及其一些衍生物中Aα链羧基末端三分之二部分（即αC结构域）的位置。一种针对Aα链羧基末端150个氨基酸的单克隆抗体G8，结合在纤维蛋白原的中央区域附近，这表明大多数分子的αC结构域通常不可见，因为它们位于氨基末端二硫键结上或附近。在pH 3.5时，纤维蛋白原和纤维蛋白单体看起来相似，大多数分子的两端都有一个终止于小球形结构域的突起。相比之下，通过纤溶酶从纤维蛋白原上切割αC结构域产生的片段X单体则没有这种突起。当在聚合反应延迟的条件下将纤维蛋白单体调至中性pH时，仍可看到单个分子，其αC结构域表现为中央区域附近的一个额外的单个结节。此外，对分子簇的分析揭示了一些通过αC结构域的分子间关联。从纤维蛋白原的纤溶酶消化物中分离出了一个包含αC结构域的40 kDa片段，并通过SDS-聚丙烯酰胺凝胶电泳和氨基末端氨基酸序列测定对其进行了表征。αC片段的电子显微镜观察显示出单个球形结构以及寡聚聚集体。将αC片段添加到纤维蛋白单体中，然后稀释至中性pH以引发聚合反应，会导致较低的浊度、较长的延迟期和较慢的最大浊度增加速率。此外，电子显微镜观察揭示了在中性pH下αC片段与纤维蛋白单体的复合物。看来游离的αC片段可以与纤维蛋白的αC结构域结合，与聚合反应中涉及的正常αC结构域相互作用竞争。
