Influence of Arginines 93, 97, and 101 of thrombin to its functional specificity.
Mutation of three Arg residues, 93, 97, and 101, to Ala in thrombin (thrombin R93,97,101A) has previously been shown to eliminate most heparin 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 interaction with protein C observed with TM lacking CS. In this study we examined the relative impact of mutation of these Arg residues alone and in combination on the above reactions and, in addition, on the ability of rabbit TM to accelerate thrombin inhibition by antithrombin. The order of importance for heparin acceleration of inhibition by antithrombin was Arg 101, 93, and 97. In contrast, Arg 97 was the major residue required for TM-dependent acceleration of reactivity with antithrombin and for CS-dependent enhancement of TM affinity. Arg 101 and 93 were critical for TM-dependent, high-affinity protein C interaction at low Ca2+ concentrations, while Arg 97, which was critical for the other TM-dependent effects, played no detectable role in this metal dependence. These results illustrate that these Arg residues in anion binding exosite 2 contribute very differently to the diverse reactions dependent on that domain in thrombin.
He
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Cardiovascular Biology Research Program, Oklahoma Medical Research Foundation, University of Oklahoma Health Science Center, Oklahoma City, Oklahoma 73104, USA.
Ye
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Esmon
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CT
Rezaie
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AR
D000818:Animals; D000990:Antithrombin III / Q000378:metabolism; D001120:Arginine / Q000235:genetics / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002384:Catalysis; D002809:Chondroitin Sulfates / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008297:Male; D016297:Mutagenesis, Site-Directed; D011486:Protein C / Q000378:metabolism; D011817:Rabbits; D013917:Thrombin / Q000235:genetics / Q000502:physiology*
D011486:Protein C; D000990:Antithrombin III; D006493:Heparin; D002809:Chondroitin Sulfates; D001120:Arginine; D013917:Thrombin; D002118:Calcium