Cleavage of the thrombin receptor: identification of potential activators and inactivators

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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 (TR38–60) by thrombin were 107 s-1 and 1.3 µM; the kcat/Km of TR38–60 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 TR38–60 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR38–60 efficiently (kcat/Km > 106 M-1·s-1). Whereas trypsin cleaved TR38–60 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 TR38–60 by chymotrypsin (kcat/Km ≈ 106 M-1·s-1) was predominantly due to a low Km value (2.8 µM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR38–60 at the Arg41-Ser42 bond, but exhibited kcat/Km values that were at least 103-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 TR38–60 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/Km 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.