Cathepsin G Activates Protease-activated Receptor-4 in Human Platelets

Abstract

Of the four known protease-activated receptors (PARs), PAR1 and PAR4 are expressed by human platelets and mediate thrombin signaling. Whether these receptors are redundant, interact, or play at least partially distinct roles is unknown. It is possible that PAR1 and/or PAR4 might confer responsiveness to proteases other than thrombin. The neutrophil granule protease, cathepsin G, is known to cause platelet secretion and aggregation. We now report that this action of cathepsin G is mediated by PAR4. Cathepsin G triggered calcium mobilization in PAR4-transfected fibroblasts, PAR4-expressing Xenopus oocytes, and washed human platelets. An antibody raised against the PAR4 thrombin cleavage site blocked platelet activation by cathepsin G but not other agonists. Desensitization with a PAR4 activating peptide had a similar effect. By contrast, inhibition of PAR1 function had no effect on platelet responses to cathepsin G. When neutrophils were present, the neutrophil agonist fMet-Leu-Phe triggered calcium signaling in Fura-2-loaded platelets. Strikingly, this neutrophildependent platelet activation was blocked by the PAR4 antibody. These data show that PAR4 mediates platelet responses to cathepsin G and support the hypothesis that cathepsin G might mediate neutrophil-platelet interactions at sites of vascular injury or inflammation.Proteases regulate cellular behaviors in part via a family of G protein-coupled protease-activated receptors (PARs) 1 for which the thrombin receptor, PAR1, is the prototype. PAR1 is activated by an unusual mechanism in which thrombin cleaves the receptor amino-terminal exodomain to expose a new amino terminus that then serves as a tethered ligand (1, 2). The synthetic peptide SFLLRN, which mimics the first six amino acids of this newly unmasked amino terminus, can activate PAR1 independent of receptor cleavage and thrombin. The cognate P1Ј-P6Ј peptides of other PARs have been useful as agonists for probing the role of these receptors in various cellular responses (3-5).Four distinct PARs are now known. PAR1, PAR3, and the recently characterized PAR4 can be activated by thrombin (1, 5-7). PAR2 is activated by trypsin and trypsin-like enzymes (4). The importance of platelet activation in myocardial infarction and other thrombotic diseases has prompted considerable focus on identifying the PARs that mediate platelet activation by thrombin. Available data suggest that thrombin signaling in human platelets is mediated largely by hPAR1 and hPAR4 (8). PAR1 is necessary for platelet activation at low concentrations of thrombin, and PAR4 can mediate signaling at higher concentrations. The presence of two receptors raises the question of whether PAR1 and PAR4 are redundant, interact, or serve at least partially distinct functions in human platelets. In particular, it is possible that PAR4 might confer responsiveness to proteases other than thrombin.Platelet PARs may be exposed to a variety of proteases at sites of inflammation or coagulation, and leukocytes are one potential source. For example, neutr...

Chymase Participates in Chronic Dermatitis by Inducing Eosinophil Infiltration

“…This finding naturally indicates that the DNFB-and chymase-induced eosinophil accumulation in mice is, at least in part, due to the chemotactic activity of chymase. The direct action of chymase in stimulating eosinophil migration might be mediated via putative receptor(s), such as protease-activated receptor(s) (PAR) (Cocks and Moffatt, 2000) like cathepsin G, an enzyme closely related to chymase, which acts on the platelet by interacting with PAR-4 (Sambrano et al, 2000). An alternative explanation for chymase-induced eosinophil migration is that chymase promotes production of chemokines such as eotaxin.…”

N-Linked Glycosylation Regulates Human Proteinase-activated Receptor-1 Cell Surface Expression and Disarming via Neutrophil Proteinases and Thermolysin

“…The mass spectrometry study by Renesto et al (13) Because cathepsin G has been reported to have both activating and disarming actions on PARs (12,13,30,31), we tested the role of N-terminal glycosylation of hPAR 1 in regulating this proteinase. We have demonstrated that cathepsin G could clearly disarm N62Q/N75Q and WT hPAR 1 to the same efficiency.…”

Protease activated receptors: theme and variations

“…For example, cathepsin G causes an increase in cytosolic Ca 2+ in human platelets, but has no apparent agonist e ect on human endothelial cells or ®broblasts, despite the fact that PAR1 is present on all three. A recent report provides an explanation for these observations by showing that cathepsin G is an agonist for PAR4 (Sambrano et al, 2000). Human platelets express PAR4, endothelial cells and ®broblasts do not.…”

“…Trypsin (Kahn et al, 1998b). Cathepsin G (Sambrano et al, 2000). g PAR1 antagonists: (Bernatowicz et al, 1996;Hoekstra et al, 1998;McComsey et al, 1999;Andrade-Gordon et al, 1999;Kawabata et al, 1999) sion, a meticulous, tissue by tissue approach may be required to better understand the role of PARs in development and homeostasis.…”