Evaluation of proteinase-activated receptor-1 (PAR(1)) agonists and antagonists using a cultured cell receptor desensitization assay: Activation of PAR(2) by PAR(1)-targeted ligands

We developed a calcium signaling-based assay, using cultured human embryoni c kidney cells (HEK), that evaluates simultaneously, the activation/desensi tization or blockade of the proteinase-activated receptors, PAR(1) and PAR( 2). Using this assay, we analyzed the actions of a number of previously des cribed putative PAR(1)-targeted peptide agonists and antagonists. We found that most of the previously described PAR(1)-targeted agents can also activ ate/desensitize PAR(2), and most of these peptides can also activate a calc ium signaling pathway in a target cell that possesses PAR(2) along with PAR (1). Furthermore, we used this assay to develop a PAR(1) receptor-activatin g probe [Ala-parafluoroPhe-Arg-Cha-Cit-Tyr-NH2 (Cit-NH2)], which displays a high degree of specificity for PAR(1) over PAR(2), and we used the assay t o quantitate the ability of trypsin to disarm the activation of PAR(1) by t hrombin. The abilities of the PAR(1)-targeted agents to desensitize or bloc k PAR(1) in the HEK cell assay were compared with their activities in a hum an platelet aggregation assay. Our data illustrate the usefulness of the HE K cell assay for evaluating the PAR(1)/PAR(2) selectivity of PAR-activating agonists. The PAR(1)-selective agonist that we developed using the assay s hould prove useful for studying the effects of selectively activating PAR(1 ) in vivo.