INFLUENCE OF ARGININE-93, ARGININE-97, AND ARGININE-101 OF THROMBIN TO ITS FUNCTIONAL SPECIFICITY

Mutation of three Arg residues, 93, 97, and 101, to Ala in thrombin (t hrombin R93,97,101A) has previously been shown to eliminate most hepar in acceleration of thrombin inhibition by antithrombin and most of the ability of chondroitin sulfate (CS) on thrombomodulin (TM) to enhance affinity for TM and to eliminate the characteristic high-affinity int eraction with protein C observed with TM lacking CS. In this study we examined the relative impact of mutation of these Arg residues alone a nd in combination on the above reactions and, in addition, on the abil ity of rabbit TM to accelerate thrombin inhibition by antithrombin. Th e order of importance for heparin acceleration of inhibition by antith rombin was Arg 101, 93, and 97. In contrast, Arg 97 was the major resi due required for TM-dependent acceleration of reactivity with antithro mbin and for CS-dependent enhancement of TM affinity. Arg 101 and 93 w ere critical for TM-dependent, high-affinity protein C interaction at low Ca2+ concentrations, while Arg 97, which was critical for the othe r TM-dependent effects, played no detectable role in this metal depend ence. These results illustrate that these Arg residues in anion bindin g exosite 2 contribute very differently to the diverse reactions depen dent on that domain in thrombin.