Isolation and Characterization of the S-Carboxymethyl Derivatives of Crosslinked and Noncrosslinked Human Fibrin

The S-carboxymethyl derivative chains of crosslinked and noncrosslinked fibrins were prepared from purified human fibrinogen. For crosslinked fibrin, fibrinogen was clotted with thrombin in the presence of calcium and purified human factor XIII. For noncrosslinked fibrin, ethylenediaminetetraacetate was substituted for factor XIII and calcium. After reduction with dithiothrcitol and alkylation with tritiated iodoacetic acid, the derivative chains were separated on carboxymethyl cellulose in a sodium acetate-pH gradient that contained 8 M urea. Purity of the separated chains was determined by polyacrylamide gel electrophoresis at acid and at neutral pH. The derivative chains of noncrosslinked fibrin were eluted from carboxymethyl cellulose in the order: γ-chain, β-chain, and α-chain. Each of the purified derivative chains was characterized and identified by amino-terminal aminoacid analysis, aminoacid composition, tryptic peptide mapping, and molecular weight estimation by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. In like manner, the derivative components of crosslinked fibrin were eluted from carboxymethyl cellulose in the order: γ-γ-dimer, β-chain, and α-polymer. Application of the same analytical criteria and comparision with the derivatives of noncrosslinked fibrin confirmed the identity of these components. These data provide conclusive evidence that crosslinking of human fibrin involves formation of peptide bonds between two γ-chains to form γ-γ-dimer and between multiple α-chains to form high molecular weight polymers of α-chains.