Cleavage of the thrombin receptor: identification of potential activators and inactivators Available to Purchase

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 k_cat and K_m values for the cleavage of this peptide (TR^38–60) by thrombin were 107 s^-1 and 1.3 µM; the k_cat/K_m of TR^38–60 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 TR^38–60 was not specific for thrombin. The pancreatic proteases trypsin and chymotrypsin hydrolysed TR^38–60 efficiently (k_cat/K_m > 10⁶ M^-1·s^-1). Whereas trypsin cleaved TR^38–60 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 TR^38–60 by chymotrypsin (k_cat/K_m ≈ 10⁶ M^-1·s^-1) was predominantly due to a low K_m value (2.8 µM). The proteases factor Xa, plasmin, plasma kallikrein, activated protein C and granzyme A also hydrolysed TR^38–60 at the Arg⁴¹-Ser⁴² bond, but exhibited k_cat/K_m 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 TR^38–60 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 k_cat/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.