Abstract 23: EphB2 Mediated Contact-Dependent and Independent Signaling in the Regulation of Thrombosis and Hemostasis

Abstract

Introduction: Eph/ephrin signalling plays important roles in the development of central nervous system and vasculature. The presence of EphA4, EphB1 and ephrinB1 in platelets was reported previously where forced clustering of EphA4 or ephrinB1 resulted in cytoskeletal rearrangements, fibrinogen binding and granule secretion. Further studies have emphasized the role of EphA4 in regulation of integrin αIIbβ3 mediated outside-in signalling. We have recently reported the presence of EphB2 in platelets and established its involvement in regulation of platelet function. Hypothesis and Methods: We assessed the hypothesis that Eph/ephrin signalling is mainly mediated through the cytoplasmic domains [including a kinase domain, sterile alpha motif and PDZ motif] of Eph kinases using a mouse model in which the intracellular region of EphB2 (EphB2LacZ) was replaced with β-galactosidase. Use of this mouse model enabled us to explore the importance of EphB2 cytoplasmic signalling in regulating platelet function without interfering with its ligand binding. Results and Conclusions: The absence of EphB2 cytoplasmic tail resulted in reduced agonist-induced platelet activation, fibrinogen binding, granule secretion and thrombus formation. Integrin αIIbβ3 mediated outside-in signalling was also reduced and associated with a diminished level of myosin binding to integrin β3 tail in EphB2LacZ mouse platelets. Moreover, a reduced level of calcium mobilisation and phosphorylation of protein kinase B and phospholipase Cγ2 in EphB2LacZ platelets suggest a potential role in the regulation of phosphoinositide-3 kinase signalling. Reduced levels of fibrinogen binding, granule secretion and platelet spreading observed on individual EphB2LacZ platelets suggest a role for EphB2 signalling without the need for cell-cell contact. Conversely, diminished platelet aggregation, clot retraction and thrombus formation in EphB2LacZ platelets point towards the importance of EphB2 mediated intracellular signalling in a contact-dependent manner when platelet-platelet contacts occur. In conclusion, this study implicates the EphB2 cytoplasmic region in mediating contact-dependent and independent signalling in platelets.