COMPARATIVE STRUCTURAL AND FUNCTIONAL FEATURES OF THE HUMAN FIBRINOGEN ALPHA-C DOMAIN AND THE ISOLATED ALPHA-C FRAGMENT - CHARACTERIZATION USING MONOCLONAL-ANTIBODIES TO DEFINED COOH-TERMINAL A-ALPHA CHAIN REGIONS

The alpha C domain of fibrinogen (A alpha-(220-610)) plays a central r ole in maintaining hemostasis by serving as a substrate for factor XII I(a) 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 d omain of intact fibrinogen and in purified alpha C fragments, and 3) e xplore the degree to which the alpha C domain's role as a factor XIII( a) 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 ag ainst larger cyanogen bromide (A)alpha chain derivatives with each epi tope subsequently localized to discrete A alpha chain sequences (A alp ha-(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 resi due of alpha C fragments was either A alpha-220 or A alpha-231 and tha t, although the extreme COOH-terminal region, A alpha-(603-610), was a bsent, all molecules were intact at least through A alpha-(563-578). S olution phase competitive assays indicated that the release of the alp ha C domain from intact fibrinogen was associated with several conform ational changes, e.g. in the vicinity of A alpha-(220-230), A alpha-(2 59-276), A alpha-(487-498), and A alpha-(529-539), but that the relati ve 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 fragment s could serve as a substrate for factor XIII(a). Immunoblotting studie s of the A alpha chain proteolysis that occurs during thrombolytic the rapy indicated that alpha C fragments, similar in size and epitope con tent 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 alp ha C domain and its functional implications and also draw attention to the as yet unexplored role of alpha C fragments in the pathophysiolog y of thrombosis and hemostasis.