Fibrinogen Assembly and Crosslinking on a Fibrin Fragment E Template

Abstract

SummaryThere is an ongoing controversy concerning whether crosslinked γ chains in fibrin are oriented “transversely” between fibril strands or “end-to-end” along fibril strands. From the latter viewpoint, Veklich et al. [Proc Natl Acad Sci (USA) 95: 1438, 1998] observed that fibrinogen fibrils that had been assembled on a fibrin fragment E template, crosslinked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked γ chains in fibrin under physiological conditions were also aligned endto-end. To assess its validity we studied the assembly and organization of fibrinogen molecules on a des AB-fibrin fragment E (E-des AB) or a des A-fibrin fragment E (E-des A) template.We evaluated the roles of E polymerization sites EA and EB, and D association sites γXL, Da, Db, βC, and αC in this process. EA:Da interactions caused fibrinogen: E “DED” complexes to form, and markedly enhanced the γ chain crosslinking rates of fibrinogen or des αC-fibrinogen. Fibrinogen crosslinking without added fibrin E was slower, and that of des αC-fibrinogen was still slower. These events showed that although αC domains promote fibrinogen fibril assembly and crosslinking, they contribute little to increasing the EA:Da-dependent crosslinking rate. Electron microscopic (STEM) images of E-des AB and fibrinogen plus factor XIIIa showed single-, double-, and multistranded fibrils with interstrand DED complexes aligned side-to-side. This alignment was due to βC:βC contacts resulting from D subdomain rearrangements initiated by the EB:Db interactions, and also occurred in mixtures of des αC-fibrinogen with E-des AB.In contrast, a mixture of fibrinogen and E-des A plus XIIIa revealed double-stranded fibrils with interstrand DED complexes in a halfstaggered arrangement, an alignment that we attribute to crosslinking of γXL sites bridging between fibrils strands. These and other features of E-des A-based fibrinogen fibrils, including interstrand γ chain bridges and early and extensive lateral fibril strand associations concomitant with accelerated γ chain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned γ chains.