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

SUMMARY1. Pancreatic phospholipase A2 (phosphatide acyl-hydrolase, EC 3.1.1.4) and phospholipase C (phospharidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Bacillus cereus appeared not to be lyric for human erythrocytes, either before or after treatment of the cells with trypsin, pronase or neuraminidase. No significant breakdown of phospholipids could be observed.2. Both phospholipases were found to evoke hemolysis in the presence of sublytic concentrations of sodium deoxycholate, whereas sublytic concentrations of Triton X-100 were effective only in combination with phospholipase C.3. Treatment of human red cell ghosts with either phospholipase A2 or phospholipase C resulted in a complete hydrolysis of lecithin, phosphatidylethanolamine and phosphatidylserine, whereas sphingnmyelin was not attacked. Similar results were obtained with liposomes derived from human erythrocytes, indicating that the degree of hydrolysis depends only upon the cl~emical nature of the phospholipids involved.4. In the native human erythrocyte membrane the fatty acid-ester linkage at C2 and the phosphoryl-glycerol linkage at C3 of the phosphoglyceride molecules apparently are not accessible to phospholipase A and C attack. Removal of the sialic acid residues from the membrane surface does not promote the action of these lipolytic enzymes. Changes in membrane architecture occurring during membrane isolation or as induced by nonlyric concentrations of detergents lead to exposure ofmembrane phosphoglycerides to phospholipases A and C.Previous reports from this laboratory dealt with the action of crude phosphc~ lipases on intact erythrocytes. Whereas Crotalus adamanteus phospholipase A (phosphatide acyl-hydrolase, EC 3.1.1.4) has been shown not to be lyric I , the action of Clostridium welchii phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) exhibited a complete hemolysis of intact erythrocytes from human and various animal species 2. Snake venom phospholipase A has been shown to be lyric in combination with a Biochim. Biophys. Acta, 241 (1971) [925][926][927][928][929]

SUMMARY1. Phospholipase C (phosphatidylcholine cholinephosphohydrolase, E.C. 3.1.4.3) from Bacillus cereus evoked hemolysis of intact human erythrocytes in hypotonic sucrose solutions at sucrose concentrations below 120 mM, whereas pancreatic phospholipase A2 (phosphatide acyl-hydrolase, E.C. 3.1.1.4) became lytic below 100 mM sucrose. Treatment of intact cells with proteolytic enzymes prior to the incubations with phospholipases A: and C did not alter the lytic behavior of these phospholipases.2. Resealed ghosts made in saline in the presence of 2 mM CaC12 retained 16-20% of their hemoglobin and were sensitive to lysis by phospholipase C, but not by phospholipase A2; resealed ghosts made in saline in the presence of 1 mM EDTA retained only 1-2% of their hemoglobin and were lysed by both phospholipases A2 and C. Resealed ghosts prepared in the presence of Ca 2+ after increasing durations of lysis prior to the resealing procedure, showed a decrease in lysis due to phospholipase C, but no lysis due to phospholipase A2.3. Resealed ghosts made in sucrose in the presence of 2 mM CaC12 retained 24-30% of their hemoglobin and showed increasing lysis by phospholipase C with increase in the lysing tonicity prior to resealing. Phospholipase A2 showed a minimum in the lysis of resealed ghosts, which were hemolysed at 20 mM sucrose prior to resealing. Treatment with subtilopeptidase A of resealed ghosts followed by treatment with phospholipase A2 or C showed no increased lysis as compared with non-proteolysed cells.

Oxidized low-density lipoprotein (oxLDL) and associated oxidized phosphocholine-headgroup phospholipids (oxPCs) activate blood platelets through ligation of the scavenger receptor CD36. Previously, we found that oxLDL stimulated phosphorylation of phospholipase Cγ2 (PLCγ2). However, the functional relevance of PLCγ2 phosphorylation in oxLDL-mediated platelet hyperactivity remained elusive. Here, we set out to explore the functional importance of PLCγ2 in oxLDL-mediated platelet activation using human and genetically modified murine platelets. The CD36-specific oxidized phospholipid (oxPC) triggered the generation of reactive oxygen species (ROS) in platelets under static and arterial flow conditions. The ROS generation in response to oxPC was sustained for up to 3 h but ablated in CD36- and PLCγ2-deficient platelets. The functional importance of ROS generation in response to atherogenic lipid stress was examined through measurement of P-selectin expression. OxPC induced P-selectin expression, but required up to 60 min incubation, consistent with the timeline for ROS generation. P-selectin expression was not observed in CD36- and PLCγ2-deficient mice. The ability of oxPC and oxLDL to stimulate P-selectin expression was prevented by incubation of platelets with the ROS scavenger N-acetyl-cysteine (NAC) and the NOX-2 inhibitor gp91ds-tat, but not with the NOX-1 inhibitor ML171. In summary, we provide evidence that prolonged exposure to oxLDL-associated oxidized phospholipids induces platelet activation via NOX-2-mediated ROS production in a CD36- and PLCγ2-dependent manner.

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