title: Effect of the hirudin carboxy-terminal peptide 54-65 on the interaction of thrombin with platelets.
abstract: The carboxy-terminal region of hirudin (residues 54-65) has previously been shown to inhibit thrombin clotting activity without binding to the catalytic site of the enzyme. In the present study, the effect of hirudin 54-65 on thrombin interaction with specified platelet proteins has been investigated. Hirudin 54-65 was found to inhibit thrombin-induced platelet aggregation and secretion in a dose-dependent manner. Substitution of either Phe56, Glu57, Ile59, Pro60 or Leu64 showed that these residues were critical for inhibition of thrombin-induced platelet activation whereas sulfation of Tyr63 increased the inhibitory potency of the peptide. Hydrolysis of glycoprotein V, a platelet membrane substrate for thrombin, was only partially inhibited by hirudin 54-65. Although hirudin 54-65 did not decrease the amount of thrombin bound to platelets during cross-linking experiments, it was found to inhibit the specific binding of thrombin to platelet glycoprotein Ib. Since the carboxy-terminal region of hirudin has previously been reported to bind near the trypsin-catalyzed beta cleavage site, we have analyzed the consequences of alpha to beta-thrombin conversion on both thrombin-hirudin 54-65 interaction and thrombin activity toward platelets. The beta cleavage induced a decrease in the affinity of thrombin for both glycoprotein Ib and hirudin 54-65. Altogether, our results indicate that thrombin recognition sites for hirudin 54-65 and platelet membrane glycoprotein Ib share common structures located near the beta cleavage site at Arg 73 on the thrombin B chain.
authors.affiliation: Laboratoire de Recherche sur l'Hémostase et la Thrombose, Faculté Xavier Bichat, Paris, France.
authors.forename: M
authors.initials: M
authors.lastname: Jandrot-Perrus
authors.forename: M G
authors.initials: MG
authors.lastname: Huisse
authors.forename: J L
authors.initials: JL
authors.lastname: Krstenansky
authors.forename: A
authors.initials: A
authors.lastname: Bezeaud
authors.forename: M C
authors.initials: MC
authors.lastname: Guillin
mesh_terms: D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000187:drug effects* / Q000378:metabolism; D002918:Chymotrypsin; D006629:Hirudins / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000494:pharmacology*; D015539:Platelet Activation / Q000502:physiology; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology*; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D013917:Thrombin / Q000378:metabolism*
chemical_list: D006629:Hirudins; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D010980:Platelet Membrane Glycoproteins; D002918:Chymotrypsin; D013917:Thrombin