SPECIFIC-INHIBITION OF THROMBIN-INDUCED CELL ACTIVATION BY THE NEUTROPHIL PROTEINASES ELASTASE, CATHEPSIN-G, AND PROTEINASE-3 - EVIDENCE FOR DISTINCT CLEAVAGE SITES WITHIN THE AMINOTERMINAL DOMAIN OF THE THROMBIN RECEPTOR

The aim of this study was to investigate the inhibitory effects of hum an leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR 3) on the activation of endothelial cells (ECs) and platelets by throm bin and to elucidate the underlying mechanisms. Although preincubation of ECs with HLE or Cat G prevented cytosolic calcium mobilization and prostacyclin synthesis induced by thrombin, these cell responses were not affected when triggered by TRAP42-55, a synthetic peptide corresp onding to the sequence of the tethered ligand (Ser(42)-Phe(55)) unmask ed by thrombin on cleavage of its receptor, Using IlaR-A, a monoclonal antibody directed against the sequence encompassing this cleavage sit e, flow cytometry analysis showed that the surface expression of this epitope was abolished after incubation of ECs with HLE or Cat G. Furth er experiments conducted with platelets indicated that not only HLE an d Cat G but also PR3 inhibited cell activation induced by thrombin, al though they were again ineffective when TRAP42-55 was the agonist, Sim ilar to that for ECs, the epitope for IlaR-A disappeared on treatment of platelets with either proteinase, These results suggested that the neutrophil enzymes proteolyzed the thrombin receptor dowstream of the thrombin cleavage site (Arg(41)-Ser(42)) but left intact the TRAP42-55 binding site (Gins3 Ser(93)) within the extracellular aminoterminal d omain. The capacity of these proteinases to cleave five overlapping sy nthetic peptides mapping the portion of the receptor from Asn(35) to p ro(85) was then investigated. Mass spectrometry studies showed several distinct cleavage sites, ie, two for HLE (Val(72)-Ser(73) and lle(74) -Asn(75)), three for Cat G (Arg(41)-Ser(42) phe(55)-Trp(56) and Tyr(69 )-Arg(70)), and one for PR3 (Val(72)-Ser(73)). We conclude that this s ingular susceptibility of the thrombin receptor to proteolysis account s for the ability of neutrophil proteinases to inhibit cell responses to thrombin. (C) 1997 by The American Society of Hematology.