Glycosaminoglycan contributions to both protein C activation and thrombin inhibition involve a common arginine-rich site in thrombin that includes residues arginine 93, 97, and 101.

Proteoglycans play pivotal roles in the regulation of thrombin. Thrombomodulin (TM) binds thrombin through protein-protein contacts and a chondroitin sulfate moiety. The complex activates the anticoagulant zymogen, protein C. Thrombin and a thrombin mutant with Arg93, Arg97, and Arg101 changed to Ala bind soluble TM lacking the chondroitin sulfate with comparable affinities, but the mutant binds TM containing chondroitin sulfate 45-fold weaker than thrombin. A simple hyperbolic relationship describes the Ca2+ dependence of protein C activation with the thrombin mutant-TM complex whether or not the TM contains chondroitin sulfate. A similar Ca2+ dependence is observed with wild type thrombin only when the TM contains chondroitin sulfate. Thus, charge neutralization of Arg93, Arg97, and Arg101 mimics the functional effects of the chondroitin sulfate. The mutant and wild type thrombin are inhibited at comparable rates by antithrombin +/- the pentasaccharide capable of inducing the "active" antithrombin conformation, but heparin acceleration of antithrombin inhibition of the mutant is reduced by more than 95%. Binding studies revealed that the mutant has a > or = 20-fold decrease in heparin affinity. We conclude that heparin and chondroitin sulfate interact with one or more of these Arg residues. These basic residues appear to play a critical role in the regulation of thrombin activity.