Ectopic Olfactory Receptor Expression in Platelets as Novel Antithrombotic Targets

Introduction: Platelets are enriched in orphan G-protein-Coupled Receptors (GPCRs) that could potentially regulate platelet reactivity. A remote study in patients with hereditary anosmia reported dysfunctional platelet responses suggesting the presence of platelet olfactory receptor (ORs) that could regulate thrombosis. Hypothesis: Platelet ORs serve as an alternative pharmacological strategy to generate platelet cyclic adenosine monophosphate (cAMP) to prevent thrombosis. Methods: Ribonucleic Acid sequencing (mRNAseq) of human megakaryocytes (MKs) was conducted. Various ORs were validated in platelets by RT-qPCR and immunoblotting (IB). Reporter cell lines with stable integration of cloned ORs identified OR ligands and later validated in adult platelets. Fluorophores for OR second messengers (Fura2 for Ca 2+ and MQAE for Cl - ) and an ELISA for cAMP were used for adult platelets. Platelet reactivity in response to OR agonists was evaluated by FACS and aggregometry, and thrombosis by biomechanical platelet activation was evaluated using ex vivo microfluidics and flow cones to simulate disturbed flow (D-flow). OR agonist injection into mice and CRISPR-Cas9 OR knockdown was conducted, with platelet reactivity and thrombosis tested as proof-of-concept. Results: Only 15 of >400 ORs in the genome were identified in MKs, and only 3 in all adult platelets (highest expression was OR2L13). A terpene family was identified as OR2L13 agonists, generating cAMP in the platelet, and structural modifications were made with IC 50 values in the low-high μM range for inhibiting agonist-induced platelet activation, ex vivo human blood thrombosis by microfluidics, and in vivo platelet activation in mice with prolonged tail bleeding. Deletion of OR2L13 (olfr168 -/- ) in mice augmented platelet reactivity with loss of OR agonist-mediated prolonged tail bleeding in olfr168 -/- . OR2L13 in human platelets is stored and trafficked by alpha granules following surface agonist stimulation and biomechanical activation. Conclusions: Like neurons, platelets have a fully functional OR signal transduction cascade which operates as a negative regulator of platelet reactivity and thrombosis in response to both chemical as well as biomechanical platelet activation.