Stability of the disulfide bonds of fibrinogen and identification of specific subsets of surface-oriented histidine residue highly susceptible to alkylation.

Abstract

The alkylating agents, iodoacetate and iodoacetamide, were spontaneously incorporated into fibrinogen at pH above 5.5 and 37°C. These alkylating agents were covalently bound by fibrinogen, and the association was not dependent upon the presence of factor XIII or plasminogen. Approximately 18 mol of either alkylating agent were bound per mol of fibrinogen. By amino acid analysis, no S‐carboxymethylcysteine was detected; but the histidine derivatives, 3‐carboxymethylhistidine and 1,3‐dicarboxymethylhistidine, were identified as the only modified residues, and these were present at an approximate 2: 1 ratio. The alkylation reaction was not altered by systematic cleavage of fibrinogen by plasmin, and the alkylated residues in the D · E complex were again restricted to the 3‐carboxymethylhistidine and 1,3‐dicarboxymethylhistidine derivatives in an approximate 2: 1 ratio. Approximately 72% of the incorporated alkylating agent associated with the D region of fibrinogen and 28% with the E region. Alkylation of fibrinogen prolonged its thrombin time, and alkylated fibrinogen inhibited the coagulation of unmodified fibrinogen. This effect was associated with interference with the polymerization of fibrin monomers. The results indicate that the disulfide bonds of native fibrinogen are stable, and plasmin‐mediated catabolism of fibrinogen does not induce disulfide interchange. Specific subsets of surface‐oriented histidine residues located within the relatively plasmin‐resistant D · E core of fibrinogen are highly susceptible to alkylation, and some of these specific histidine residues may play an important role in the intermolecular alignment and polymerization of fibrin monomers.