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 β 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.