Abstract 014: Interferon-induced Transmembrane 3 (IFITM3) on Megakaryocytes and Platelets Regulates Fibrinogen Endocytosis and Thrombosis During Inflammation

Abstract

Introduction: IFITM3, an interferon (IFN) responsive gene, restricts pathogen replication through vesicular trafficking mechanisms. IFITM3 in megakaryocytes (MKs) and platelets has not been examined. We hypothesized that in MKs and platelets, IFITM3 regulates the endocytosis of pro-coagulant proteins, aggregation, and thrombosis. Methods: the regulation of IFITM3 gene expression was determined in MKs and platelets under inflammatory stimuli. Fibrinogen (Fgn) endocytosis, a clathrin-mediated event requiring αIIbβ3, and transferrin endocytosis, a clathrin-mediated, αIIbβ3-independent event, were examined in stimulated MKs and platelets from wild type (WT) and Ifitm -/- mice (KO). Integrin αIIbβ3 activation, platelet aggregation, and thrombosis was determined in WT and Ifitm -/- mice upon IFNα-stimulation. Co-immunoprecipitation identified interaction partners for IFITM3. To establish human relevance, IFITM3 expression, Fgn content, and aggregation was measured in platelets from septic patients, where systemic IFNs are increased. Results: IFNs significantly induced IFITM3 expression in MKs and platelets (p<0.001). Upregulation of IFITM3 by IFNs increased Fgn and transferrin endocytosis in MKs (2-fold vs. NT, p<0.05). Co-IP demonstrated a specific IFITM3 interaction between clathrin and αIIb. Upon IFN stimulation, IFITM3, clathrin, and αIIb shifted into lipid rafts. Increased Fgn endocytosis by IFNs enhanced platelet aggregation and accelerated death (~2-fold increased) due to platelet-dependent thrombosis (p<0.05 for all comparisons). In KO mice, enhanced Fgn endocytosis, platelet aggregation, and thrombosis to IFNs was completely prevented, demonstrating the necessity of IFITMs for these functional responses. Platelets from septic patients mirrored findings in mice with a ~20-fold increase in IFITM3 protein expression, with shifts into lipid rafts, compared to healthy controls (p<0.0001). Increased platelet IFITM3 expression was associated with greater platelet Fgn content and significant increases in platelet aggregation (20% increase, p<0.05). Conclusions: Our data identify IFITM3 as a previously unknown regulator of MK and platelet endocytosis and thrombosis under inflammatory stimuli in humans and mice.