Thrombin is a serine protease that plays an essential role in blood coagulation and also induces various responses in endothelial cells. The actions of thrombin on the conversion of fibrinogen to fibrin are inhibited by peptides based on the amino acid sequence of hirudin, a natural anticoagulant from leeches. We show in these studies that the peptides Hir45-64 and sulfated Hir53-64 block the effects of thrombin on endothelial cells. These peptides inhibited, in a concentration-dependent manner, the synthesis of prostaglandin I2 and platelet-activating factor, and the acquisition of an adhesive surface for leukocytes that occur in response to thrombin. These actions of the peptides occurred even though the catalytic site of thrombin was not blocked.