Immunochemical Characterization of Crosslinked Derivatives Isolated from Alpha Chain Oligomers Formed During Early Stages of Fibrin Crosslinking

Summary

Early crosslinked α chain oligomers (EαXL) that formed within ½ h of in vitro clot formation were isolated from preparations of reduced, carboxymethylated fibrin by gel filtration on Sepharose CL-4B. Cyanogen bromide (CNBr) digestion of EaXL released a mixture of fragments which could be partially separated by Sephadex G-150 chromatography. Crosslinked ahd noncrosslinked forms of the Aα chain peptides, CNBr VIII (Aα #241-476) and CNBr X (Aα #518-584) were identified in the column effluent both by radioimmunoassay (RIA) and by immunoblotting using polyclonal antisera (anti-CNBr VIII, anti-CNBr X) and monoclonal antibodies (F-103, binds to Aα #259-276; F-102, binds to Aα #540-554), respectively. Immunoaffinity chromatography of the crosslinked material, on F-103, followed by F-102 Sepharose, resulted in the separation of two types of fragments, each of which contained an early a chain crosslink. One of these (35-37K) exhibited coincident CNBr VIII and CNBr X immunoreactivities, while the other (54-59K) exhibited only CNBr VIII immunoreactivity, based on immunoblotting and RIA findings. NH₂-terminal sequencing and cyanoethylation data provided biochemical evidence for the occurrence of covalent CNBr VIII-X as well as covalent CNBr VIII-VIII interactions during early a chain crosslinking. These findings indicate, for the first time, that both glutamine acceptor and lysine donor activities may be localized within CNBr VIII. This information should facilitate the chemical identification of a chain residues that partner during factor XIII_a-catalyzed crosslinking and thus enable development of reagents for the generation of fibrin-specific antibodies.

Keywords

Alpha chain crosslinking - Crosslinked derivatives - Early a chain crosslinks

• References

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