CalDAG-GEFI deficiency protects mice from FcγRIIa-mediated thrombotic thrombocytopenia induced by CD40L and β2GPI immune complexes

IntroductionPlatelet activation via the Fc receptor IIa (FcRIIa) is implicated in the pathogenesis of immune complex (IC)-mediated thrombocytopenia and thrombosis (ITT). We previously showed that ICs composed of antigen and antibodies targeting CD40 ligand (CD40L) or 2 Glycoprotein I (2GPI) induce ITT in mice transgenic for human FcRIIa (hFcR) but not wild-type controls (which lack FcRIIa). Here we evaluated the contribution of the guanine nucleotide exchange factor, CalDAG-GEFI, and P2Y12, key regulators of Rap1 signaling in platelets, to ITT induced by these clinically relevant ICs. MethodsPre-formed anti-CD40L or anti-2GPI ICs were injected into hFcR/Caldaggef1(+/+) or hFcR/Caldaggef1(-/-) mice, with or without clopidogrel pretreatment. Animals were observed for symptoms of shock for 30min, during which time core body temperature was monitored. Platelet counts were obtained before and 30min after IC injection. Lungs were assessed for thrombosis by histology or near-infrared imaging. ResultsBoth CD40L and 2GPI ICs rapidly induced severe thrombocytopenia, shock and a reduction in body temperature in hFcR/Caldaggef1(+/+) mice. hFcR/Caldaggef1(-/-) mice were protected from CD40L and 2GPI IC-induced thrombocytopenia and shock, whereas P2Y12 inhibition had only a modest effect on IC-induced ITT. Consistent with these findings, IC-induced integrin activation in vitro and the accumulation of activated platelets in the lungs of IC-challenged mice was strongly dependent on CalDAG-GEFI. ConclusionsOur studies demonstrate that CalDAG-GEFI plays a critical role in platelet activation, thrombocytopenia and thrombosis induced by clinically relevant ICs in mice. Thus, CalDAG-GEFI may be a promising target for the intervention of IC-associated, FcRIIa-mediated thrombotic conditions.