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)

Intensive diabetes control has been associated with increased mortality in type 2 diabetes (T2DM); this has been suggested to be due to increased hypoglycemia. We measured hypoglycemia-induced changes in endothelial parameters, oxidative stress markers and inflammation at baseline and after a 24-hour period in type 2 diabetic (T2DM) subjects versus age-matched controls. Case-control study: 10 T2DM and 8 control subjects. Blood glucose was reduced from 5 (90 mg/dl) to hypoglycemic levels of 2.8 mmol/L (50 mg/dl) for 1 hour by incremental hyperinsulinemic clamps using baseline and 24 hour samples. Measures of endothelial parameters, oxidative stress and inflammation at baseline and at 24-hours post hypoglycemia were performed: proteomic (Somalogic) analysis for inflammatory markers complemented by C-reactive protein (hsCRP) measurement, and proteomic markers and urinary isoprostanes for oxidative measures, together with endothelial function. Between baseline and 24 -hours after hypoglycemia, 15 of 140 inflammatory proteins differed in T2DM whilst only 1 of 140 differed in controls; all returned to baseline at 24-hours. However, elevated hsCRP levels were seen at 24-hours in T2DM (2.4 mg/L (1.2-5.4) vs. 3.9 mg/L (1.8-6.1), Baseline vs 24-hours, P < 0.05). In patients with T2DM, between baseline and 24-hour after hypoglycemia, only one of 15 oxidative stress proteins differed and this was not seen in controls. An increase (P = 0.016) from baseline (73.4 ng/mL) to 24 hours after hypoglycemia (91.7 ng/mL) was seen for urinary isoprostanes. Hypoglycemia resulted in inflammatory and oxidative stress markers being elevated in T2DM subjects but not controls 24-hours after the event.While type 2 diabetes (T2DM) is associated with an increased risk of cardiovascular disease 1 , strict glycemic control does not result in obvious cardiovascular benefit in people with T2DM 2-4 . A link between strict glycemic control, hypoglycemia and increased cardiovascular morbidity and mortality has been observed in clinical studies 5,6 . Although the underlying mechanism remains unclear, increased inflammatory cytokines and a leukocytosis are reported after hypoglycemia 7,8 , suggesting a link between hypoglycemia and inflammation.It is well recognized that oxidative stress leads to damage of proteins and deoxyribonucleic acid (DNA) 9 and contributes to the diabetic complications of retinopathy, nephropathy, neuropathy and cardiovascular disorders 10-13 , and is directly linked to vascular inflammation, precipitating both endothelial cell dysfunction and vascular damage 14 . Oxidative stress results from excessive generation of free radicals and/or deficient defense mechanisms 15 , and leads to a disturbance of the physiological redox state 16 . The membrane associated 1