The role of the Phe105 and Ile106 residues of thrombin-activated protein C for the inhibition of platelet aggregation. A major thrombotic complication in patients treated with hirudin is attributed to its inhibitory activity towards factor XIII. Therefore, a new generation of thrombin-specific anticoagulants is being developed, for example, hirulogs. The anticoagulant activity of hirulogs resides in a synthetic peptide derived from the amino-terminus of thrombin-activated protein C (residues 1 to 38), whereas the specificity resides in the three amino acids at position 34. It was recently demonstrated that the peptide derivative P34 [Hirulog(R)], unlike protein C, inhibits platelet aggregation. An interesting question is whether amino acids other than arginine (like Ile34) might be equally effective in the thrombin-specific activity of protein C. Hence, the effects of the Thr34, Phe35, Lys37 and Phe105, Ile106, Thr107 and Glu109 residues on the inhibition of platelet aggregation by thrombin-activated protein C were investigated. Ile34 and Arg34 appear to have comparable effects, while Ala34 results in only 7.4% of the platelet aggregation inhibition of Arg34. In contrast, Ile106, Thr107 and Glu109 are equally important to the thrombin-specific activity of protein C, while Phe105 has a limited role in platelet aggregation inhibition. This study shows that the thrombin-specific site(s) resides not only within the 34 amino acids of hirulog(R), but also within the 108 amino acids of protein C. Moreover, Ile106, Thr107 and Glu109 are crucial to the platelet aggregation-inhibitory activity of thrombin-activated protein C. Thus, the three residues of hirulog(R) and the five residues of protein C may be candidates for the design of an improved thrombin inhibitor.