Activation of protein C by the thrombin-thrombomodulin complex: cooperative roles of Arg-35 of thrombin and Arg-67 of protein C.

The binding of Ca(2+) to the 70-80 loop of protein C inhibits protein C activation by thrombin in the absence of thrombomodulin (TM), but the metal ion is required for activation in the presence of TM. Structural data suggests that the 70-80 loop is located between two antiparallel beta strands comprised of residues 64-69 and 81-91 on the protease domain of protein C. To test the hypothesis that a salt-bridge/hydrogen bond interaction between Arg-67 of the former strand and Asp-82 of the latter strand modulates the unique Ca(2+)-binding properties of protein C, we engineered a disulfide bond between the two strands by substituting both Arg-67 and Asp-82 with Cys residues. The activation of this mutant was enhanced 40- to 50-fold independent of TM and Ca(2+). Furthermore, the Arg-67 to Ala mutant of protein C was activated in the absence of TM by the Arg-35 to Glu mutant of thrombin with the same efficiency as wild-type protein C by wild-type thrombin-TM complex. These results suggest that TM functions by alleviating the Ca(2+)-dependent inhibitory interactions of Arg-67 of protein C and Arg-35 of thrombin.