Early alpha chain crosslinking in human fibrin preparations.
Purified fibrinogen preparations were clotted under crosslinking conditions and the evolution of alpha polymers was examined by Western blotting (SDS-PAGE). Monoclonal antibodies that bind to two localized regions within the COOH-terminal portion of the (A) alpha chains of fibrinogen (F-103, A alpha #259-276; F-102, A alpha #540-554) were used for immunodetection. Three crosslinked components (100K, 168K, 210K) that each exhibited coincident F-102 and F-103 immunoreactivities were evident as early as gamma dimer formation under the in vitro conditions employed. Initial events in the alpha chain crosslinking process appeared to include interactions between relatively intact chains and a variety of degraded ones that shared the structure A alpha #1-276, but differed in the extent to which regions between residues approximately #276 and approximately #539 were preserved. Degraded fibrinogen molecules whose A alpha chains all terminated before A alpha #540-554 were isolated from purified fibrinogen preparations by immunoaffinity chromatography on F-102 Sepharose. Immunoblotting data obtained for the crosslinking capacity of these degraded molecules indicated that early crosslinked components (95 K, 205 K) could form even in the absence of intact alpha chain partners. This crosslinking, moreover, could progress to the polymer stage. These findings demonstrate that some early crosslinking activity is localized exclusively within regions NH2-terminal to A alpha approximately #540-554 and suggest that fibrinogen molecules with partially degraded A alpha chains, which are likely to be circulating under many pathophysiologic conditions, can undergo fibrin stabilization through crosslinking despite a loss of at least 70 COOH-terminal A alpha chain residues.
Sobel
J H
JH
Department of Medicine, College of Physicians & Surgeons, Columbia University, New York, NY.
Thibodeau
C A
CA
Canfield
R E
RE
D015153:Blotting, Western; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000032:analysis*; D005340:Fibrinogen / Q000302:isolation & purification*; D006801:Humans
D003432:Cross-Linking Reagents; D005337:Fibrin; D005340:Fibrinogen