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