Neutrophil proteases can inactivate human PAR3 and abolish the co-receptorfunction of PAR3 on murine platelets

Three members of the protease-activated receptor family, PAR1, PAR3 and PAR 4, are activated when thrombin Cleaves the receptor N-terminus, exposing a tethered ligand. Proteases other than thrombin can also cleave PAR family m embers and, depending upon whether this exposes or removes the tethered lig and, either activate or disable the receptor. For example, on human platele ts PAR1 is disabled by cathepsin G, although aggregation still occurs becau se cathepsin G can activate PARA The present studies examine the interactio n of cathepsin G and a second neutrophil protease, elastase, with PAR3 usin g two model systems: COS-7 cells transfected with human PAR3 and mouse plat e lets, which express PAR3 and PAR4, but not PAR1. In contrast to human pla telets, 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 expressi ng human PAR3, both cathepsin G and elastase prevented activation of phosph olipase C by thrombin. Notably, this inhibition occurred without loss of th e binding sites for two monoclonal antibodies that flank the tethered ligan d on human PAR3; We therefore conclude that 1) exposure to cathepsin G disa bles signaling through human PAR3, and prevents murine PAR3 from serving it s normal role, which is to facilitate PAR4 cleavage at low thrombin concent rations, 2) elastase disables human, but not murine, PAR3, 3) in contrast t o 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 el astase involves a mechanism other than amputation of the tethered ligand do main. These results extend the range of possible interactions between PAR f amily members and proteases, and provide further support for species-specif ic differences in the interaction of these receptors with proteases other t han thrombin.