Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aα Arg-16 → His substitution

Abstract

Fibrinogen, Purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performace liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an Aα Arg-16 → His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His¹⁶]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His¹⁶]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant k_cat/K_m for hydrolysis of the abnormal Aα chain by thrombin was calculated from Lineweaver-Burk plots to be 16–30-fold less than that for the normal Aα chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P 1 site can be accommodated within the enzyme hydrophobic active-site pocket.