Comparative Structural and Functional Features of the Human Fibrinogen αC Domain and the Isolated αC Fragment CHARACTERIZATION USING MONOCLONAL ANTIBODIES TO DEFINED COOH-TERMINAL Aα CHAIN REGIONS

Abstract The αC domain of fibrinogen (Aα-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIIIa and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the Aα chain were employed as structural probes to: 1) isolate the human αC domain, 2) compare the topography of the eight epitopes within the αC domain of intact fibrinogen and in purified αC fragments, and 3) explore the degree to which the αC domain's role as a factor XIIIa substrate in intact fibrinogen is preserved within the structure of isolated αC fragments. Five antibodies were raised against small, synthetic peptide immunogens (Aα-(220-230), Aα-(425-442), Aα-(487-498), and Aα-(603-610)), and three were generated against larger cyanogen bromide (A)α chain derivatives with each epitope subsequently localized to discrete Aα chain sequences (Aα-(259-276), Aα-(529-539), and Aα-(563-578)). Human αC preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-Aα-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH2-terminal residue of αC fragments was either Aα-220 or Aα-231 and that, although the extreme COOH-terminal region, Aα-(603-610), was absent, all molecules were intact at least through Aα-(563-578). Solution phase competitive assays indicated that the release of the αC domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of Aα-(220-230), Aα-(259-276), Aα-(487-498), and Aα-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. Aα-(425-442) and Aα-(563-578). Immunoblotting analysis of αC cross-linking in vitro revealed that isolated αC fragments could serve as a substrate for factor XIIIa. Immunoblotting studies of the Aα chain proteolysis that occurs during thrombolytic therapy indicated that αC fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen αC domain and its functional implications and also draw attention to the as yet unexplored role of αC fragments in the pathophysiology of thrombosis and hemostasis.