Recombinant fibrinogen studies reveal that thrombin specificity dictates order of fibrinopeptide release

Abstract

During cleavage of fibrinogen by thrombin, fibrin-opeptide A (FpA) release precedes fibrinopeptide B (FpB) release. To examine the basis for this ordered release, we synthesized A'β fibrinogen, replacing FpB with a fibrinopeptide A-like peptide, FpA' (G14V). Analyses of fibrinopeptide release from A'β fibrinogen showed that FpA release and FpA' release were similar; the release of either peptide followed simple first-order kinetics. Specificity constants for FpA and FpA' were similar, demonstrating that these peptides are equally competitive substrates for thrombin. In the presence of Gly-Pro-Arg-Pro, an inhibitor of fibrin polymerization, the rate of FpB release from normal fibrinogen was reduced 3-fold, consistent with previous data; in contrast, the rate of FpA' release from A'β fibrinogen was unaffected. Thus, with A'β fibrinogen, fibrinopeptide release from the β chain is similar to fibrinopeptide release from the α chain. We conclude that the ordered release of fibrinopeptides is dictated by the specificity of thrombin for its substrates. We analyzed polymerization, following changes in turbidity, and found that polymerization of A'β fibrinogen was similar to that of normal fibrinogen. We analyzed clot structure by scanning electron microscopy and found that clots from A'β fibrinogen were similar to clots from normal fibrinogen. We conclude that premature release of the fibrinopeptide from the N terminus of the β chain does not affect polymerization of fibrinogen.