Thrombin interaction with a recombinant N-terminal extracellular domain of the thrombin receptor in an acellular system

Abstract

The cDNA of the human endothelial cell thrombin receptor has been cloned and a chimeric fusion protein consisting of glutathione-S-transferase (GST) and the portion 25-97 corresponding to the N-terminal first extracellular domain of the thrombin receptor (TRE) has been expressed in Escherichia coli. Introduction of a factor Xa cleavage site in the fusion protein allowed purification of TRE after removal from the GST carrier protein. Purified GST-TRE or TRE have been tested in solution for their ability to-interact with thrombin. α-Thrombin cleaved the fusion protein at position Arg-41-Ser-42 of TRE in a time- and concentration-dependent manner and GST-TRE competed with the tripeptidic substrate S-2238 for hydrolysis by thrombin (K(i) = 0.5 μM). γ-Thrombin that lacks the anion-binding exosite was 100-fold less potent than α-thrombin at cleaving GST-TRE. TRE competed with polymerizing fibrin monomers for binding to thrombin (K(i) = 7.5 μM). The cleavage of GST-TRE by α-thrombin was inhibited by several α-thrombin exosite ligands such as the C-terminal peptide of hirudin, thrombomodulin and fibrin(ogen) fragment E. In contrast, platelet glycocalicin did not inhibit GST-TRE cleavage. In conclusion, the use of purified soluble GST-TRE allowed us to derive an affinity constant for thrombin interaction with the N-terminal domain of the receptor and to confirm the location of the cleavage site at Arg-41-Ser-42 of the receptor. The importance of the thrombin anion-binding exosite for thrombin receptor recognition is highlighted by the low reactivity of γ-thrombin for GST-TRE and by competition experiments, which in addition indicate that binding sites for fibrin(ogen), thrombomodulin and GST-TRE are overlapping. In contrast, binding of thrombin to GST-TRE and glycocalicin are not mutually exclusive, indicating that glycocalicin and TRE interact with discrete subsites within the large groove that constitutes the anion-binding exosite.