Differential Expression of Micro RNAs Accompanies Differential Reactivity Via Platelet FcγRIIa in Humans and Transgenic Mice.

Abstract

AbstractAbstract 2165One of the major clinical challenges in heparin-induced thrombocytopenia (HIT) is that antibodies to the heparin/PF4 complex are necessary but not sufficient to cause thrombocytopenia and thrombosis. While contributions of macromolecular antigen structure, antibody recognition and procoagulant activation of leukocytes and endothelial cells are increasingly appreciated, the sine qua non of HIT is platelet activation via FcγRIIa. It has been recognized for over 20 years that there is substantial inter-individual variation in human platelet reactivity to IgG immune complex agonists (such as in HIT) acting via FcγRIIa. In the course of our Platelet RNA And eXpression-1 (PRAX1) study on the genomics of platelet reactivity, we observed differential reactivity among 154 healthy donors of platelets when platelet-rich plasma aggregated in response to the agonist anti-CD9, a well-established model of FcγRIIa-mediated activation relevant to HIT. Importantly, even after accounting for the known sources of variation in reactivity - level of platelet surface FcγRIIa expression and for the H/R131 ligand-binding polymorphism - most of the variation in platelet reactivity remained unexplained. In parallel, we established human FcγRIIa transgenic mouse lines of the C57Bl6 strain and the 129SvJ strain. Unexpectedly, we identified differential reactivity to FcγRIIa-mediated platelet activation between these different mouse strains. The availability of models of variation in FcγRIIa-mediated platelet activation in both humans and mice allowed us a unique opportunity to test the hypothesis that differential expression (DE) of platelet RNA species accounts for differential reactivity. Total RNA was isolated from leukocyte-depleted platelets from each human donor and from each transgenic mouse strain. Using the species-appropriate Nanostring digital quantification assay, microRNAs (miRs) expressed in platelets were identified. 4 DE miRs in humans and 7 in mice were identified with a statistically significant difference in normalized expression, after Benjamini-Hochberg correction for multiple testing. DE was validated by qRT-PCR. Of note, several DE miRs were shared by human and mouse platelets. Work is ongoing as to the identity and functions of the mRNAs targeted by these DE miRs. Disclosures:No relevant conflicts of interest to declare.