α-Chain Cross-Linking in Fibrin(ogen) Marburg

The fibrinogen structural variant, Marburg (A α1-460 Bβγ) 2 , is comprised of normal Bβ and γ chains but contains severely truncated Aα chains that are missing approximately one half of their factor XIII, cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined Aα-chain segment, Aα 461-610, can affect the process of fibrin stabilization, ie, the factor XIII a -mediated covalent interaction that occurs between α chains of neighboring fibrin molecules and between α chains and α 2 antiplasmin (α 2 PI). The ability of Marburg (and control) α chains to serve as a substrate for factor XIII a and undergo cross-linking was examined in an in vitro plasma clotting system. The capacity for α-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of α 2 PI and includes the factor XIII a -sensitive glutamine residue responsible for the cross-linking of α 2 PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked α-chain species. Antibodies specific for the (A)α and γ/γ-γ chains of fibrin(ogen) and for the peptide and its parent protein, α 2 PI (68 kD), were used as immunoblotting probes to visualize the various cross-linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg α-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of α-chain. In the absence of α 2 PI 1-12 peptide, the α chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included α 2 PI. Both the peptide-decorated monomers and the native cross-linked α-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg Aα chain. Collectively, the findings indicate that, although deletion of the Aα chain region no. 461-610 in fibrinogen Marburg prevents formation of an extensive α polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely,factor XIII binding and the ability of α chains to undergo limited cross-linking to one another and to α 2 PI.