title: Cleavage of the thrombin receptor: identification of potential activators and inactivators.
abstract: The kinetic parameters were determined for the hydrolysis of a peptide based on the activation site of the thrombin receptor (residues 38-60) by thrombin and 12 other proteases. The kcat and Km values for the cleavage of this peptide (TR39-40) by thrombin were 107 s-1 and 1.3 microM; the kcat/Km of TR39-40 is among the highest observed for thrombin. A model is presented that reconciles the parameters for cleavage of the peptide with the concentration dependence of cellular responses to thrombin. Cleavage of TR39-40 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR39-40 efficiently (kcat/Km > 10(6) M-1.s-1). Whereas trypsin cleaved TR39-40 at the thrombin activation site (Arg41-Ser42), chymotrypsin hydrolysed the peptide after Phe43. This chymotryptic cleavage would result in inactivation of the receptor. The efficient cleavage of TR39-40 by chymotrypsin (kcat/Km approximately 10(6) M-1.s-1) was predominantly due to a low Km value (2.8 microM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR39-40 at the Arg41-Ser43 bond, but exhibited kcat/Km values that were at least 10(3)-fold lower than that observed with thrombin. Both tissue and urokinase plasminogen activators as well as granzyme B and neutrophil elastase were unable to cleave TR39-60 at appreciable rates. However, neutrophil cathepsin G hydrolysed the receptor peptide after Phe55. Like the chymotryptic cleavage, this cleavage would lead to inactivation of the receptor, but the cathepsin G reaction was markedly less efficient; the kcat/K(m) value was almost four orders of magnitude lower than that for thrombin. In addition to the above cleavage sites, a secondary site for thrombin and other arginine-specific proteases was identified at Arg46, but the cleavage at this site only occurred at very low rates and is unlikely to be significant in vivo.
authors.affiliation: Department of Haematology, University of Cambridge, U.K.
authors.forename: M A
authors.initials: MA
authors.lastname: Parry
authors.forename: T
authors.initials: T
authors.lastname: Myles
authors.forename: J
authors.initials: J
authors.lastname: Tschopp
authors.forename: S R
authors.initials: SR
authors.lastname: Stone
mesh_terms: D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008969:Molecular Sequence Data; D018179:Receptors, Thrombin / Q000819:agonists / Q000037:antagonists & inhibitors / Q000378:metabolism*
chemical_list: D018179:Receptors, Thrombin; D010450:Endopeptidases