Abstract 53: Immunoreceptor Tyrosine Activation Motif Signaling in Platelets Is Critical for the Maintenance of Vascular Integrity During Inflammation

Abstract

Introduction: Blood platelets, long recognized for their importance in primary hemostasis, have recently been identified as critical regulators of vascular integrity during inflammation and cancer. Interestingly, the contribution of platelets to the maintenance of vascular integrity in inflammation seems to be independent of their ability to form a clot. Goal: To identify signaling molecules critical for the contribution of platelets to vascular integrity in inflammation. Methods: Transgenic mice with platelet-specific expression of the interleukin (IL)4 receptor were rendered thrombocytopenic by infusion of antibodies to IL4R. Platelets from various knockout mice or inhibitor-treated wild-type (WT) platelets were transfused into thrombocytopenic (TP) mice and tested for their ability to support vascular integrity in a reverse passive Arthus reaction (rpA) model. Tissue hemoglobin (Hb) was used to quantify red blood cells in the inflamed tissue, a marker of disrupted vascular integrity. Results: Impaired vascular integrity at the site of inflammation was markedly higher in TP mice compared to controls (Hb: 1448±176 vs. 100±8 μg/cm2). Transfusion of WT platelets into TP mice strongly reduced tissue Hb (150±46 μg/ cm2). Transfused platelets from mice lacking the thrombin receptor PAR4 and/or functional P2Y12 (ADP receptor) also prevented disruption of vascular integrity (Hb: 210±66, 136±16 μg and 360±120 μg/cm2), suggesting that signaling by G protein-coupled receptors is not important for this response. Furthermore, infusion of heparin into WT mice did not lead to increased tissue Hb. Immunoreceptor tyrosine activation motif (ITAM) signaling, however, was critical for platelet-dependent maintenance of vascular integrity, as tissue Hb was markedly increased in TP mice transfused with platelets lacking the signal trandsucing adapter protein SLP-76 (1212±160 μg/cm2) or platelets lacking functional ITAM signaling receptors GPVI (852±196 μg/cm2), CLEC-2 (1062±188 μg/cm2), or both (1498±128 μg/cm2). Data are mean±SEM, n=4-5 mice per group. Conclusions: We have established a novel mouse model for the adoptive transfer of platelets into TP mice. Using this model we can show that the ITAM but not GPCR signaling in platelets is critical for the maintenance of vascular integrity during inflammation. Our studies highlight a potential clinical complication of future anti-platelet agents targeted towards ITAM signaling receptors such as GPVI.