Summary
Three 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.
Keywords
Proteases - protease-activated receptors - thrombin - cathepsin G
