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 XIIIa-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
