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 human leukocyte elastase (HLE), cathepsin G (Cat G), and proteinase 3 (PR3) on the activation of endothelial cells (ECs) and platelets by thrombin 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 corresponding to the sequence of the tethered ligand (Ser42-Phe55) unmasked by thrombin on cleavage of its receptor. Using IIaR-A, a monoclonal antibody directed against the sequence encompassing this cleavage site, flow cytometry analysis showed that the surface expression of this epitope was abolished after incubation of ECs with HLE or Cat G. Further experiments conducted with platelets indicated that not only HLE and Cat G but also PR3 inhibited cell activation induced by thrombin, although they were again ineffective when TRAP42-55 was the agonist. Similar to that for ECs, the epitope for IIaR-A disappeared on treatment of platelets with either proteinase. These results suggested that the neutrophil enzymes proteolyzed the thrombin receptor downstream of the thrombin cleavage site (Arg41-Ser42) but left intact the TRAP42-55 binding site (Gln83-Ser93) within the extracellular aminoterminal domain. The capacity of these proteinases to cleave five overlapping synthetic peptides mapping the portion of the receptor from Asn35 to Pro85 was then investigated. Mass spectrometry studies showed several distinct cleavage sites, i.e., two for HLE (Val(72)-Ser73 and Ile74-Asn75), three for Cat G (Arg41-Ser42, Phe55-Trp56 and Tyr69-Arg70), and one for PR3 (Val(72)-Ser73). We conclude that this singular susceptibility of the thrombin receptor to proteolysis accounts for the ability of neutrophil proteinases to inhibit cell responses to thrombin.
Renesto
P
P
Unité de Pharmacologie Cellulaire, Institut Pasteur, Paris, France.
Si-Tahar
M
M
Moniatte
M
M
Balloy
V
V
Van Dorsselaer
A
A
Pidard
D
D
Chignard
M
M
D000595:Amino Acid Sequence / Q000187:drug effects; D001665:Binding Sites / Q000187:drug effects; D001792:Blood Platelets / Q000378:metabolism; D002403:Cathepsins / Q000494:pharmacology*; D002460:Cell Line; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005110:Extracellular Space / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D019272:Leukocyte Elastase / Q000378:metabolism*; D008969:Molecular Sequence Data; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology*; D010446:Peptide Fragments / Q000494:pharmacology; D015539:Platelet Activation / Q000187:drug effects; D018179:Receptors, Thrombin / Q000096:biosynthesis / Q000097:blood / Q000737:chemistry / Q000378:metabolism*; D012697:Serine Endopeptidases / Q000378:metabolism*; D013917:Thrombin / Q000037:antagonists & inhibitors* / Q000378:metabolism / Q000494:pharmacology*; D014471:Umbilical Veins
D010446:Peptide Fragments; D018179:Receptors, Thrombin; C072032:thrombin receptor peptide (42-55); D002403:Cathepsins; D012697:Serine Endopeptidases; D019272:Leukocyte Elastase; D013917:Thrombin; D053146:Myeloblastin