Protein Palmitoylation Participates in PAR1-Mediated Platelet Activation.

Platelet accumulation at sites of vascular injury is the primary event in arterial thrombosis. We have performed a screen of 16,320 small molecules to identify compounds that inhibit platelet activation mediated by the PAR1 specific agonist, SFLLRN. JF081204{5C} (9-methylene-4-pentyl-2,3,4,9-tetrahydro-1H-cyclopenta(b)quinoline) was identified to be a potent antiplatelet and antithrombotic compound. We have previously shown that JF0812405{5C} inhibited thrombosis in a murine model by up to 80% with an IC₅₀ of 2 mg/kg. Assays of structure activity relationships revealed that length of the alkyl chain of the compound is a critical determinant of its activity. Initial target-directed studies suggested that this compound inhibited protein palmitoylation. We therefore sought to clarify the effects of this compound on platelet protein palmitoylation and determine the role of palmitoylation in signaling induced by PAR1. We first analyzed the effects of JF081204{5C} on palmitoylation in resting platelets. Resting platelets were incubated with a cell-permeable peptide (MyrGCK(NBD)) that renders cells fluorescent upon palmitoylation. By confocal microscopy, the peptide was observed within platelets primarily localized around the periphery. Incubation of resting platelets with JF081204{5C} inhibited palmitoylation-induced fluorescence of the MGC peptide. The inhibitory effect of JF081204{5C} on palmitoylation was confirmed by ³H-palmitate labeling experiments. Resting platelets were incubated with ³H-palmitate in the presence or absence of JF081204{5C} and proteins were extracted by acetone precipitation. ³H-palmitate incorporation was decreased in platelets incubated with JF081204{5C} as evidenced by scintillation counting. We next analyzed the effects of JF081204{5C} on PAR1-mediated activation. JF081204{5C} inhibited SFLLRN-induced P-selectin expression, ¹⁴C-serotonin release, aggregation, and α_IIbβ₃ activation with an IC₅₀ of 5 μM. In contrast, JF081204{5C} failed to inhibit platelet activation induced by ADP, PMA, A23187, or collagen. However, JF081204{5C} inhibited epinephrine-induced platelet aggregation with an IC₅₀ of 5 μM, indicating that JF081204{5C} acts on an intracellular target. Stimulation with SFLLRN resulted in increased ³H-palmitate-incorporation into platelet proteins as evidenced by scintillation counting and autoradiography. In contrast, this increase was not observed following stimulation with U46619 or epinephrine. Autoradiography of the labeled proteins separated by SDS-PAGE demonstrated that JF081204{5C} inhibited SFLLRN-induced ³H-palmitate incorporation into select bands by up to 50%, while not affecting ³H-palmitate incorporation into other bands. We next sought to assess the role of palmitoylation in signaling through PAR1 using alternative inhibitors. 2-Bromopalmitate, a palmitic acid analog, abolished ³H-palmitate-incorporation into platelet protein and inhibited SFLLRN-induced platelet activation with an IC₅₀ of 30 μM. Acyl-protein thioesterase 1 (APT1) is an enzyme that specifically depalmitoylates proteins. Infusion of APT1 into permeabilized platelets inhibited SFLLRN-induced P-selectin expression in a dose-dependent manner with an IC₅₀ of 350 nM. The observations that SFLLRN is a more potent inducer of protein palmitoylation than other platelet agonists and that inhibitors of protein palmitoylation block SFLLRN-mediated platelet activation demonstrate that protein palmitoylation plays a prominent role in signaling downstream of PAR1.