The Essential Covalent Structure of Human Fibrinogen Evinced by Analysis of Derivatives Formed during Plasmic Hydrolysis (1973)

Abstract In an effort to determine the essential structural features of human fibrinogen, plasmic hydrolysis was carried out and degradation products were identified and analyzed. In agreement with previous work, the initial attack was shown to occur in the Aα chains with the release of portions of their COOH-terminal regions (designated /α chains in the terminology developed to describe the present findings), whereas simultaneous, but slower, degradation occurs in the NH2-terminal region of the Bβ/ chains) and results in the removal of remnants (designated Bβ/ chains) containing peptide B. These changes, plus internal cleavages leading eventually to the separation of Fragments D and E (terminology of Nussenzweig et al.), produce a series of high molecular weight derivatives which can be separated by polyacrylamide gel electrophoresis of unreduced samples in sodium dodecyl sulfate. (These were designated Bands II through VII in order of decreasing molecular size; Band VII was identified as Fragment X in the terminology of Marder et al.) Upon further hydrolysis derivative Band VII can lose the COOH-terminal portion of one of its Bβ chains (as remnant /β6, molecular weight 32,000) to form derivative Band VIII (Fragment Y in the terminology of Marder et al.) or, alternatively, can undergo cleavage to yield early forms of Fragments D and E (designated D1 and E1, respectively). Derivative Band VIII, in turn, is degraded to Fragment E1 plus a later form of Fragment D. Once cleaved from the parent molecule, these Fragments D continue to release /β chains. These degradative pathways lead to a series of Fragments D (D1 through D5) which differ in molecular weight. Additional cleavages occur in Fragment E1 to form a smaller fragment (E2) which, unlike E1, lacks peptide A. Immunochemical studies of derivatives obtained at successive phases of digestion demonstrated the presence of at least five antigenic determinants on the fibrinogen molecule. One of these is located on /α chains; another, on Fragment E; three others, on Fragment D. Of the latter, one is associated with /β6 chains; a second (designated F) is associated with Fragments F, which arise by further degradation of Fragments D; the third (designated D) is that lost when Fragment F is evolved. Electrophoretic behavior of plasmic derivatives before and after reduction revealed at least one intrachain disulfide bridge in each /α15 and /β6 chain. Studies of Fragment D subunit composition and recovery at various phases of digestion indicated that it is a dimeric structure containing substantial portions of the COOH-terminal region of both γ chains (as /γ1 remnant chains, each having a molecular weight of 42,000) and hence that only one such fragment (linked by at least five interchain disulfide bridges) can be generated by each fibrinogen molecule. These results, plus those reported by others, led to the conclusion that fibrinogen itself has a dimeric structure, the backbone of which consists of a pair of γ chains linked directly to each other by disulfide bridging in the NH2-terminal region and covalently linked (enter directly or indirectly) with the Aα and Bβ chains in at least two regions of the molecule.