A critical role for Gly25 in the B chain of human thrombin

We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989-97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild-type prothrombin (FII-WT) and mutant prothrombin (designated FII-G25((330))S) cDNA in COS-7 cells showed a mild reduction (50%) in FII-G25((330))S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by TaYpan snake or Echis carinatus venoms. We show that the G25((330))S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9-fold reduction in catalytic efficiency of the mutant toward S-2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25((330)) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombin