The anucleate platelet is not amenable to traditional forward genetic analysis, but is a good candidate for a chemical genetic screen. In collaboration with the Broad Institute Probe Development Center, we have performed a high throughput screen using a modified luciferin-luciferase detection system to identify inhibitors of Par1-mediated dense granule secretion. For primary screening, over 300,000 compounds were assayed in duplicate using freshly outdated platelet-rich plasma supplied by several blood banks across the United States. Computational analyses of the primary data demonstrated that approximately 0.2% of compounds showed ≥50% inhibition relative to maximally inhibitory concentrations of the known antiplatelet agent, cilostazol. Secondary screening using 8-point dose response curves and counterscreening to exclude luciferase inhibitors identified 137 small molecules that inhibited Par1-mediated ATP/ADP release without significant inhibition of luciferase. Twenty eight compounds were selected for further testing based on their IC50s in confirmatory assays (<10 μM), lack of activity in unrelated bioassays, and chemical structure. Known platelet inhibitors were excluded. Of the 28 compounds, 16 compounds potently inhibited SFLLRN-induced α-granule release from washed platelets, as monitored by P-selectin expression. IC50s for these compounds ranged from <0.3 to 1 μM. None of the compounds that failed to inhibit α-granule release demonstrated significant inhibition of SFLLRN-induced 14C-serotonin release at 10 μM. Of the 16 confirmed inhibitory compounds, 12 demonstrated >50% stimulation of cAMP levels compared to platelets exposed to PGE1 and were not further analyzed. One of the 4 remaining compounds lost activity upon resynthesis. The three compounds that demonstrated activity upon resynthesis included a diaminophenyl, an acylhydrazone, and a cyanopyridone. A series of chemical analogs for each of the three series was synthesized and/or obtained from commercial vendors. Structure activity relationships were determined by testing these compounds in the luciferin-luciferase assay. The most potent analog of each series was then further analyzed. The representative compounds (10 μM) failed to block platelet granule release induced by the Par4 activating peptide AYPGFK, PMA, calcium ionophore, or collagen, suggesting that the compounds act at or near Par1. However, only one of the three compounds, termed DAP, significantly inhibited thrombin-induced platelet activation. Determination of DAP activity in cultured cells expressing Par1 confirmed DAP-mediated inhibition of Par1 activity. Analysis of dose-response curves at multiple inhibitor concentrations were consistent with non-competitive inhibition of Par1-mediated granule secretion. In functional studies, DAP inhibited platelet aggregation and secretion, but not platelet shape change. In contrast, the orthosteric Par1 inhibitor Sch79797 blocked aggregation, secretion, and shape change. Despite its inability to inhibit activation through human Par4, DAP blocked activation through mouse Par4, demonstrating complete inhibition of AYPGFK-mediated aggregation of mouse platelets at 5 μM. Platelets obtained from mice 45 minutes after infusion of 10 mg/kg DAP failed to aggregate in response to AYPGFK. The effect of DAP on thrombus formation was evaluated using intravital microscopy. These studies showed that infusion of 10 mg/kg DAP potently inhibited platelet accumulation following laser-induced vascular injury of cremaster arterioles in mice. These studies demonstrate the feasibility of using blood-banked platelets in a large-scale chemical genetic screen. Compounds that block platelet function via elevation of cAMP are common, indicating a need to include cAMP assays early in the screening funnel. The novel antithrombotic DAP identified in this screen appears to block Par1 via an allosteric mechanism. The ability of DAP to inhibit mouse Par4, but not human Par4, indicates that DAP acts outside the ligand binding pocket of Par1. This compound will provide a useful probe to study the effect of small molecule modulation of Par1 on receptor conformation and downstream signaling.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.