PAR1 Antagonists Development and Clinical Utility

Thrombin is a potent activator of platelets and other cells. The mechanism by which thrombin, a protease, regulates cellular behaviors like a hormone was revealed by expression cloning of a G protein-coupled receptor now known as protease-activated receptor-1 (PAR1). Thrombin activates PAR1 by cleaving its N-terminal ectodomain to reveal a new N-terminus that then serves as a tethered peptide ligand, binding to the heptahelical domain to trigger G protein activation. A synthetic hexapeptide mimicking this new N-terminus activates PAR1 without receptor cleavage. Point mutations that prevent receptor cleavage render PAR1 unactivatable by thrombin without altering activation by exogenous tethered ligand peptide. Replacement of the thrombin cleavage site with sites for other proteases allows PAR1 to signal in response to those other proteases. Removal of sequence N-terminal to the cleavage site and creation of the new protonated amino group at N-terminal of the tethered ligand sequence is necessary for its function, explaining how the tethered ligand is silent in the intact receptor and activated by receptor cleavage. Thus, PARs are, in essence, peptide receptors that carry their own ligands, which can be unmasked by receptor cleavage. Mammalian cells utilize four PARs to respond to coagulation proteases and other proteases with trypsin-like specificity. Three, PAR1, PAR3 and PAR4 can mediate responses to thrombin, with PAR3 and PAR4 mediating platelet activation by thrombin in mice and PAR1 and PAR4 platelet activation in humans. Inhibition of thrombin signaling via PARs in platelets decreases thrombus formation in animal models of platelet-dependent thrombosis. These and other studies led to development of the PAR1 antagonist vorapaxar (Zontivity), a first-in-class antiplatelet agent approved for secondary prevention of atherothrombotic events in selected patients with prior myocardial infarction and peripheral arterial disease. What was and was not learned from animal and human studies and remaining questions related to clinical utility of PAR inhibition will be discussed. The structure-function studies outlined above support the tethered ligand model, but a structure that reveals how the tethered ligand binds and how binding leads to transmembrane signaling and G protein activation is needed. Vorapaxar provided a tool for stabilizing PAR1 protein during solubilization, purification and crystallization. In collaboration with Brian Kobilka and colleagues, we solved a crystal structure of off-state PAR1 in complex with its antagonist vorapaxar, which explains pharmacological properties of vorapaxar and efforts to solve a complementary on-state structure are ongoing.