Abstract 4146605: A Non-odorant Olfactory Receptor Ligand Depolymerizes the Platelet Actin Cytoskeleton to Prevent Thrombosis

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Background: Despite antiplatelet therapy use, some patients have residual thrombotic risks due to High residual platelet reactivity (HRPR). We identified ectopic expression of Olfactory Receptor (OR) 2L13, a G protein-coupled receptor, in human platelets that suppresses platelet reactivity when activated. Our goal was to identify agonists in a high throughput screen for OR2L13 as an alternative strategy to suppress HRPR. Methods: Using an OR2L13 reporter cell line, 8K bioactive compounds were screened. Lead compounds were validated, and orthogonally tested for antiplatelet effects by Light Transmission Aggregometry (LTA) and Flow cytometry. Using murine models, the impact of OR2L13 agonists was determined by evaluating thrombosis and hemostasis. Lastly, the impact of a lead OR2L13 agonist on HRPR in patients with coronary artery disease (CAD) and peripheral artery disease (PAD) was determined. Biased and unbiased platelet phospho-proteomic profiling examined the OR2L13 signaling pathway. Results: 169 molecular hits were identified exceeding the 25% threshold, of which 6 ligands (CCF0054500, CCF0054432, CCF0053070, CCF0052249, CCF0051970 and CCF0058399) were persistently specific for OR2L13. Only CCF0054500 suppressed platelet aggregation and α-granule exocytosis through P2Y 12, PAR1, TP and GPVI receptors suggesting a downstream common mediator. In a murine cremaster arterial injury model, platelet accumulation at the injury site was reduced by 88.9% by CCF0054500 than vehicle (P<0.0003) without altering fibrin generation. CCF0054500 had no impact on hemostasis in mice (tail bleeding assay). CCF0054500 inhibited HRPR through P2Y 12, PAR1, TP and GPVI in patients with CAD and PAD, even when taking other anti-platelet agents. Phospho-proteomics revealed CCF0054500 phosphorylates heat shock protein 27 (HSP27) which depolymerizes platelet actin cytoskeleton that we further validated by clot retraction assay (CCF0054500 70.6 area vs. 5.2 area vehicle. P<0.0001), and this protective effect is reversed by an HSP27 inhibitor, area 3.56, P<0.0001) Conclusion: We identified the first non-olfactory ligand therapeutic that is bioavailable to engage a platelet OR and inhibit platelet activation and thrombosis via HSP27-mediated cytoskeleton rearrangement. Furthermore, CCF0054500 suppresses platelet reactivity irrespective of the receptor-stimulated and circumvents the issue of HRPR in patients with established vascular disease.

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