Key Points• oxLDL binds platelet CD36 to stimulate tyrosine kinaseand PKC-dependent activation of NOX2 and generation of ROS.• oxLDL-and hyperlipidemiainduced ROS mediate platelet desensitization to inhibitory cGMP signaling to facilitate platelet activation and thrombus formation.Oxidized low-density lipoprotein (oxLDL) promotes unregulated platelet activation in dyslipidemic disorders. Although oxLDL stimulates activatory signaling, it is unclear how these events drive accelerated thrombosis. Here, we describe a mechanism for oxLDLmediated platelet hyperactivity that requires generation of reactive oxygen species (ROS). Under arterial flow, oxLDL triggered sustained generation of platelet intracellular ROS, which was blocked by CD36 inhibitors, mimicked by CD36-specific oxidized phospholipids, and ablated in CD36 2/2 murine platelets. oxLDL-induced ROS generation was blocked by the reduced NAD phosphate oxidase 2 (NOX2) inhibitor, gp91ds-tat, and absent in NOX2 2/2 mice. The synthesis of ROS by oxLDL/CD36 required Src-family kinases and protein kinase C (PKC)-dependent phosphorylation and activation of NOX2. In functional assays, oxLDL abolished guanosine 39,59-cyclic monophosphate (cGMP)-mediated signaling and inhibited platelet aggregation and arrest under flow. This was prevented by either pharmacologic inhibition of NOX2 in human platelets or genetic ablation of NOX2 in murine platelets. Platelets from hyperlipidemic mice were also found to have a diminished sensitivity to cGMP when tested ex vivo, a phenotype that was corrected by infusion of gp91ds-tat into the mice. This study demonstrates that oxLDL and hyperlipidemia stimulate the generation of NOX2-derived ROS through a CD36-PKC pathway and may promote platelet hyperactivity through modulation of cGMP signaling. (Blood. 2015;125(17):2693-2703

Key Points• Protein kinase A (PKA) phosphorylates RhoA on serine 188 to inhibit RhoA membrane translocation and RhoA kinase (ROCK) signaling.• Inhibition of RhoA/ROCK2 promotes myosin light chain (MLC) phosphatase activity, which prevents the phosphorylation of MLC and platelet shape change.Cyclic adenosine monophosphate (cAMP)-dependent signaling modulates platelet shape change through unknown mechanisms. We examined the effects of cAMP signaling on platelet contractile machinery. Prostaglandin E 1 (PGE 1 )-mediated inhibition of thrombinstimulated shape change was accompanied by diminished phosphorylation of myosin light chain (MLC). Since thrombin stimulates phospho-MLC through RhoA/Rhoassociated, coiled-coil containing protein kinase (ROCK)-dependent inhibition of MLC phosphatase (MLCP), we examined the effects of cAMP on this pathway. Thrombin stimulated the membrane localization of RhoA and the formation of a signaling complex of RhoA/ROCK2/myosin phosphatase-targeting subunit 1 (MYPT1). This resulted in ROCK-mediated phosphorylation of MYPT1 on threonine 853 (thr 853 ), the disassociation of the catalytic subunit protein phosphatase 1d (PP1d) from MYPT1 and inhibition of basal MLCP activity. Treatment of platelets with PGE 1 prevented thrombin-induced phospho-MYPT1-thr 853 in a protein kinase A (PKA)-dependent manner. Examination of the molecular mechanisms revealed that PGE 1 induced the phosphorylation of RhoA on serine 188 through a pathway requiring cAMP and PKA. This event inhibited the membrane relocalization of RhoA, prevented the association of RhoA with ROCK2 and MYPT1, attenuated the dissociation of PP1d from MYPT1, and thereby restored basal MLCP activity leading to a decrease in phospho-MLC. These data reveal a new mechanism by which the cAMP-PKA signaling pathway regulates platelet function. (Blood. 2013;122(20):3533-3545)

Findings from this study demonstrate that ablation of LAIR-1 in megakaryocytes leads to increased Src family kinase activity and downstream signaling in response to collagen that is transmitted to platelets, rendering them hyper-reactive specifically to agonists that signal through Syk tyrosine kinases, but not to G-protein-coupled receptors.