Position of gamma-chain carboxy-terminal regions in fibrinogen/fibrin cross-linking mixtures.
There are conflicting ideas regarding the location of the carboxyl-terminal regions of cross-linked gamma-chain dimers in double-stranded fibrin fibrils. Some investigators believe that the chains are always oriented longitudinally along each fibril strand and traverse the contacting ends of abutting fibrin D domains ("DD-long" cross-linking). Other investigations have indicated instead that the chains are situated transversely between adjacent D domains in opposing fibril strands (transverse cross-linking). To distinguish between these two possibilities, the gamma dimer composition of factor XIIIa-cross-linked fibrin/fibrinogen complexes that had been formed through noncovalent D/E interactions between fibrinogen D domains and fibrin E domains was examined. Two factor XIIIa-mediated cross-linking conditions were employed. In the first, fibrin/fibrinogen complexes were formed between (125)I-labeled fibrinogen 2 ("peak 2" fibrinogen), each heterodimeric molecule containing one gamma(A) and one larger gamma' chain, and nonlabeled fibrin 1 molecules ("peak 1" fibrin), each containing two gamma(A) chains. If DD-long cross-linking occurred, (125)I-labeled gamma(A)-gamma(A), gamma(A)-gamma', and gamma'-gamma'dimers in a 1:2:1 ratio would result. Transverse cross-linking would yield a 1:1 mixture of (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers, without any gamma'-gamma' dimers. Autoradiographic analyses of reduced SDS-PAGE gels from protocol 1 revealed (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers at a ratio of approximately 1:1. No labeled gamma'-gamma' dimers were detected. Protocol 2 used a converse mixture, (125)I-fibrin 2 and nonlabeled fibrinogen 1. DD-long cross-linking of this mixture would yield only nonradioactive gamma(A)-gamma(A) dimers, whereas transverse cross-linking would yield a 1:1 mixture of (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers. Autoradiographic analyses of this mixture yielded (125)I-labeled gamma(A)-gamma(A) and gamma(A)-gamma' dimers in a 1:1 ratio. These findings provide no evidence that longitudinal (DD-long) gamma chain positioning occurs in cross-linked fibrin and indicate instead that most, if not all, gamma-chain positioning in an assembled fibrin polymer is transverse.
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Department of Biomedical Sciences, College of Health Sciences, Marquette University, P.O. Box 1881, Milwaukee, Wisconsin 53233-1881, USA. kevin.siebenlist@marquette.edu
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D000645:Ammonium Sulfate / Q000737:chemistry; D001345:Autoradiography; D002712:Chlorides / Q000737:chemistry; D002848:Chromatography, DEAE-Cellulose; D003432:Cross-Linking Reagents / Q000737:chemistry*; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000737:chemistry; D005338:Fibrin Fibrinogen Degradation Products / Q000737:chemistry*; D005340:Fibrinogen / Q000737:chemistry*; D006801:Humans; D007454:Iodides / Q000737:chemistry; D007457:Iodine Radioisotopes; D010446:Peptide Fragments / Q000097:blood* / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D013053:Spectrophotometry; D011503:Transglutaminases / Q000737:chemistry
D002712:Chlorides; D003432:Cross-Linking Reagents; D005338:Fibrin Fibrinogen Degradation Products; D007454:Iodides; D007457:Iodine Radioisotopes; D010446:Peptide Fragments; C027539:fibrinopeptides gamma; C009813:iodine monochloride; D005337:Fibrin; D005340:Fibrinogen; D011503:Transglutaminases; D000645:Ammonium Sulfate