Immunochemical characterization of crosslinked derivatives isolated from alpha chain oligomers formed during early stages of fibrin crosslinking.
Early crosslinked alpha chain oligomers (E alpha XL) that formed within 1/2 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 E alpha XL released a mixture of fragments which could be partially separated by Sephadex G-150 chromatography. Crosslinked and non-crosslinked forms of the A alpha chain peptides, CNBr VIII (A alpha #241-476) and CNBr X (A alpha #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 A alpha #259-276; F-102, binds to A alpha #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 alpha 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 alpha 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 alpha chain residues that partner during factor XIIIa-catalyzed cross-linking and thus enable development of reagents for the generation of fibrin-specific antibodies.
Sobel
J H
JH
Department of Medicine, College of Physicians and Surgeons, Columbia University, New York, NY.
Thibodeau
C A
CA
Gawinowicz Kolks
M A
MA
Canfield
R E
RE
D000596:Amino Acids / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents; D003488:Cyanogen Bromide; D005337:Fibrin / Q000032:analysis*; D007120:Immunochemistry; D046911:Macromolecular Substances
D000596:Amino Acids; D003432:Cross-Linking Reagents; D046911:Macromolecular Substances; D005337:Fibrin; D003488:Cyanogen Bromide