Cross-linked alpha s gamma t-chain hybrids in plasma clots studied by 1D- and 2D electrophoresis and western blotting.
Fibrinogen and fibrin related chains in reduced human plasma as well as the bonds interlinking partially cross-linked fibrin from plasma clots have been studied by means of 1D- and 2D electrophoresis and Western blotting. Immunovisualization of reduced plasma or partially cross-linked fibrin with monoclonal antibodies specific for the alpha-chains or the gamma-chains have shown that several bands represent material belonging to both chains. In order to decide whether these bands constitute alpha gamma-chain hybrids or superimposed alpha- and gamma-chain dimers, the cross-linked material was separated according to both isoelectric point (pI) and molecular weight (MW) using Pharmacia's Multiphor II system. Western blotting of the second dimension gels revealed that partially cross-linked fibrin contains alpha s gamma t-chain hybrids and gamma-polymers, in addition to the well-known gamma-dimers and alpha-polymers. The main alpha s gamma t-chain hybrid has a pI between that of the alpha- and the gamma-chains, a MW of about 200 kDa and contains A alpha-chains with intact fibrinopeptide A (FPA). It was also observed that soluble fibrinogen/fibrin complexes as well as partially cross-linked fibrin contain degraded alpha-dimers with MWs close to the gamma-dimers. These findings demonstrate that factor XIII-catalyzed cross-linking of fibrin is a more complex phenomenon than earlier recognized.
Grøn
B
B
Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Norway.
Filion-Myklebust
C
C
Bjørnsen
S
S
Haidaris
P
P
Brosstad
F
F
D015153:Blotting, Western; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000737:chemistry*; D005340:Fibrinogen / Q000737:chemistry*; D006801:Humans; D046911:Macromolecular Substances; D010446:Peptide Fragments / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013927:Thrombosis / Q000378:metabolism*
D046911:Macromolecular Substances; D010446:Peptide Fragments; D005337:Fibrin; D005340:Fibrinogen