Neutrophil Proteases Can Inactivate Human PAR3 and Abolish the Co-receptor Function of PAR3 on Murine Platelets

Abstract

SummaryThree members of the protease-activated receptor family, PAR1, PAR3 and PAR4, are activated when thrombin cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family members and, depending upon whether this exposes or removes the tethered ligand, either activate or disable the receptor. For example, on human platelets PAR1 is disabled by cathepsin G, although aggregation still occurs because cathepsin G can activate PAR4. The present studies examine the interaction of cathepsin G and a second neutrophil protease, elastase, with PAR3 using two model systems: COS-7 cells transfected with human PAR3 and mouse platelets, which express PAR3 and PAR4, but not PAR1. In contrast to human platelets, cathepsin G did not aggregate murine platelets, and prevented their activation only at low thrombin concentrations. Elastase had no effect on thrombin responses in mouse platelets, but when added to COS cells expressing human PAR3, both cathepsin G and elastase prevented activation of phospholipase C by thrombin. Notably, this inhibition occurred without loss of the binding sites for two monoclonal antibodies that flank the tethered ligand on human PAR3. We therefore conclude that 1) exposure to cathepsin G disables signaling through human PAR3, and prevents murine PAR3 from serving its normal role, which is to facilitate PAR4 cleavage at low thrombin concentrations, 2) elastase disables human, but not murine, PAR3, 3) in contrast to human PAR4, mouse PAR4 will not support platelet aggregation in response to cathepsin G, and 4) the inactivation of human PAR3 by cathepsin G and elastase involves a mechanism other than amputation of the tethered ligand domain. These results extend the range of possible interactions between PAR family members and proteases, and provide further support for species-specific differences in the interaction of these receptors with proteases other than thrombin.

Novel mechanisms for activated protein C cytoprotective activities involving noncanonical activation of protease-activated receptor 3

“…Cleavage of endothelial PAR3 was confirmed using antibody Mab19b with its epitope previously mapped to PAR3 residues 39 to 51. 31 As anticipated, Mab19b did not recognize the P3R L peptide C-terminal of the APC cleavage site, but recognition of the suggested epitope encompassing P3K L peptide was ,5% of the parental P3 peptide, suggesting that recognition of PAR3 by Mab19b was cleavage sensitive and that additional residues N-terminal of Thr39 likely contribute to its epitope ( Figure 4B). Confirming the H103 antibody results, reactivity of endothelial cells with Mab19b significantly diminished after thrombin or APC treatment ( Figure 4D).…”

Neutrophil Serine Proteinases Activate Human Nonepithelial Cells to Produce Inflammatory Cytokines Through Protease-Activated Receptor 2

“…With regard to the effect of neutrophil serine proteinases on the PAR family, it is reported that PAR-1 on human platelets and endothelial cells is inactivated by HLE, Cat G, and PR3 by cleavage downstream of the tethered ligand (41) and that HLE and Cat G inactivate human PAR-3 (42). It is also predicted that HLE, Cat G, and PR3 are able to inactivate human PAR-2 by cleavage downstream of the tethered ligand using the recombinant receptors (43).…”

Noncanonical PAR3 activation by factor Xa identifies a novel pathway for Tie2 activation and stabilization of vascular integrity

“…Antibodies blocking PAR3 proteolytic activation (combination of H103 and Mab19b 19,29 ) prevented FXa-induced Tie2 phosphorylation at Ser1119 ( Figure 6B) and at Y992 (supplemental Figure 3). Similarly, EPCR blocking antibodies inhibited FXa-induced phosphorylation of Tie2 consistent with the observation that PAR3 activation at Arg41 by FXa required EPCR ( Figure 1C).…”