Abstract 269: Stimulation of Toll-like Receptor 2 on Human Platelets Increases Platelet Adhesion in an AKT-Dependent Manner
Cells of the innate immune system utilize Toll-like receptors (TLRs) to recognize pathogen-associated molecular patterns and to initiate a defense response. In addition to their primary roles in thrombosis and hemostasis, platelets contribute to the immune response by releasing proinflammatory and antimicrobial mediators upon activation. Studies suggest that live bacteria and select bacterial components directly induce platelet aggregation, and a recent major study in humans indicates that acute infections are associated with a transient increase in the risk of vascular thrombotic events. Nevertheless, the precise mechanism by which platelets respond to bacteria has not been well studied. Previous findings in our laboratory indicate that human platelets express functional TLR2, as incubation with the TLR2 specific ligand, Pam3CSK4, induces platelet aggregation. In the present study, we further investigated the effects of TLR stimulation on platelet function. Stimulation of washed platelets with Pam3CSK4 significantly induced production of reactive oxygen species (ROS), as measured with flow cytometry by changes in 2′,7′-dichlorofluorescin-fluorescence. Inhibition of TLR2 with monoclonal antibody (mAb) significantly reduced platelet-mediated ROS generation. Additionally, in a model of recirculating platelet flow, Pam3CSK4 robustly increased adhesion of platelets to fibrinogen- and collagen-coated slides, whereas preincubation with anti-TLR2 mAb reduced adhesion. The contribution of relevant intracellular signaling pathways to these downstream responses was also examined. Pam3CSK4 dose-dependently induced phosphorylation of AKT, and this effect was inhibited by pretreating platelets with anti-TLR2 mAb or LY294002, an inhibitor of phosphoinositide 3-kinase. In addition to inhibiting Pam3CSK4-induced platelet aggregation, LY294002 also significantly reduced Pam3CSK4-induced platelet adhesion and ROS production. In summary, this is the first report of TLR stimulation on human platelets leading to increased free radical production and platelet adhesiveness. These findings suggest that innate immunity is relevant to the thrombo-inflammatory response and it is mediated by the expression of TLRs on platelets.