Molecular mapping of thrombin‐receptor interactions

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Abstract
In addition to its procoagulant and anticoagulant roles in the blood coagulation cascade, thrombin works as a signaling molecule when it interacts with the G‐protein coupled receptors PAR1, PAR3, and PAR4. We have mapped the thrombin epitopes responsible for these interactions using enzymatic assays and Ala scanning mutagenesis. The epitopes overlap considerably, and are almost identical to those of fibrinogen and fibrin, but a few unanticipated differences are uncovered that help explain the higher (90‐fold) specificity of PAR1 relative to PAR3 and PAR4. The most critical residues for the interaction with the PARs are located around the active site where mutations affect recognition in the order PAR4 > PAR3 > PAR1. Other important residues for PAR binding cluster in a small area of exosite I where mutations affect recognition in the order PAR1 > PAR3 > PAR4. Owing to this hierarchy of effects, the mutation W215A selectively compromises PAR4 cleavage, whereas the mutation R67A abrogates the higher specificity of PAR1 relative to PAR3 and PAR4. 3D models of thrombin complexed with PAR1, PAR3, and PAR4 are constructed and account for the perturbations documented by the mutagenesis studies. Proteins 2001;45:107–116.