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
P. Renesto et al., 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, Blood, 89(6), 1997, pp. 1944-1953
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.