Evidence that Thrombin Releases Fibrinopeptide B From non-Polymerizing Fibrin

Abstract

When thrombin acts upon fibrinogen (Fg), substantial amounts of fibrinopeptide A (FPA) are released prior to significant release of fibrinopeptide B(FPB). It has been claimed that polymerization of des-AA fibrin is a prerequisite for the release of FPB from des-AA fibrin, by exposing structures (conformational changes) required for the FPB part of the B beta chain to be recognized by thrombin (Blombäck, Hogg and Blombäck, 1976, 1978). To test this theory, either Fg or des-AA fibrin(made by exposure of Fg to insol. reptilase in urea) were exposed to thrombin (l or 100 NTH units) in a non-polymerizing system (2.5 M urea, pH 7.4,I=0.15, temp.+20°C), measuring FPA release(FPA-RIA +N-terminal analysis) and FPB release (N-terminal analysis). At 1 NIH unit of thrombin, complete release of FPA from Fg in urea occurred, whilst no FPB release from Fg or des-AA in urea were noted, irrespective of the incubation time,(Potency of thrombin during incubation time was unchanged). Incubation with 100 NIH units of thrombin caused complete release of both FPA and FPB from 30 mg of Fg or des-AA fibrin in urea within 5 min, and chromatography of the resulting des-AABB fibrin, (80 × 2 cm, Sepharose 4B, 2.5 M urea) demonstrated a homogenous elution profile, indicating absent polymerization. It seems likely that urea in some way interferes with the affinity of thrombin for FPB, which might be compensated for by the use of high thrombin concentrations. It is concluded that polymerization is not a prerequisite for the release of FPB by thrombin.