Abstract 514: Inhibiting The P2Y12 Receptor In Megakaryocytes And Platelets Suppresses Interferon-associated Inflammatory Responses

Abstract

Megakaryocytes (MKs) and platelets are recognized as mediators of inflammation and immunity. Antiplatelet therapies are widely used to prevent cardiovascular events; however, their role in attenuating platelet-mediated inflammation is uncertain. We investigated whether antiplatelet drugs, aspirin (ASA) and P2Y 12 inhibitors, alter the MK and platelet transcriptomes. We hypothesized that antiplatelet therapy reduces MK- and platelet-mediated inflammation. RNA-seq was performed in CD34 + derived MKs treated with ASA (100 μM), P2Y 12 inhibitor (AZD1283, 5 μM) or PBS for 24 hours. We validated our MK model using RNA-seq data from platelets of healthy volunteers randomized in a crossover design to low-dose ASA (n = 70, 64% female, 43.6 ± 9 years) or P2Y 12 inhibitor (ticagrelor, n = 57, 61% female, 43.9 ± 10 years) for 4 weeks each. The clinical relevance of our findings was assessed using RNA-seq data from platelets of SLE patients (n = 54, 100% female, 40.6 ± 12 years) and matched controls (n = 18, 100% female, 42.1 ± 15 years). Finally, we validated our findings in vitro using RT-qPCR and western blot. We identified 74 ASA- and 2,499 P2Y 12 inhibitor MK-responsive genes. In contrast to ASA, P2Y 12 inhibitor-mediated gene changes between MK and platelet transcriptomes were positively correlated (R=0.37, p<0.001). P2Y 12 inhibition downregulated IFNα response pathway in both MKs (NES = -2.3, p = 0.002) and platelets (NES = -2.24, p = 3.22 х 10 -8 ). SLE is characterized by activation of the IFN system. Consistently, the IFNα pathway was upregulated in platelets from SLE patients versus controls (NES = 3.38, p = 1.25 х 10 -9 ). Among 72 upregulated IFNα genes in SLE patients, 33 were inhibited with P2Y 12 inhibition in MKs or platelets, including IRF7 and IFITM3. Finally, we validated the P2Y 12 inhibitory effect on IFN-associated responses. While IFNα (1000 U/mL) significantly upregulated both IRF7 and IFITM3, pre-treatment with P2Y 12 receptor inhibition attenuated this response on both, the transcriptomic and proteomic levels. Our data indicate that targeting the P2Y 12 receptor attenuates MK- and platelet-mediated IFN signaling pathways. P2Y 12 -mediated suppression of platelet-induced IFNα signaling may be beneficial in inflammatory and autoimmune diseases including SLE.