Fibrinogen Assembly and Crosslinking on a Fibrin Fragment E Template
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Thrombosis and Haemostasis
There is an ongoing controversy concerning whether crosslinked γγchains 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, cross-linked with factor XIIIa, and then dissociated in acetic acid solution, were aligned end-to-end. This led to the conclusion that crosslinked γchains in fibrin under physiological conditions were also aligned end-to- 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, C , and αC in this process. EA :Da inter-actions caused fibrinogen: E “DED” complexes to form, and markedly enhanced the γchain 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 multi-stranded 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 C-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 half-staggered 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 γchain bridges and early and extensive lateral fibril strand associations concomitant with accelerated γchain crosslinking, indicate that crosslinking of fibrin fibril strands takes place preferentially on transversely positioned γchains.