Forskolin increases cAMP in a receptor-independent manner, thereby providing evidence that oxLDL did not affect the conversation of PGI2 with the IP receptor or target adenylyl cyclases. cAMP signaling were blocked by pharmacological inhibition of CD36, mimicked by CD36-specific oxidized phospholipids and ablated in CD36?/? murine platelets. The injection of oxLDL into wild-type mice strongly promoted FeCl3-induced carotid thrombosis and diminished PKA signaling. In contrast, platelet sensitivity to a PDE-resistant cAMP analog remained normal. Genetic deletion of CD36 guarded dyslipidemic animals from PGI2 hyposensitivity and restored PKA signaling. These data suggest that CD36 can translate atherogenic lipid stress into platelet hyperactivity through modulation of inhibitory cAMP signaling. Introduction Myocardial infarction (MI) is usually characterized by platelet-driven atherothrombotic events that lead to acute occlusion of a coronary vessel. Excessive platelet activation is usually controlled by endothelial derived nitric oxide (NO) and prostacyclin (PGI2),1 but action of these protective agents is overcome in MI by mechanisms that are yet to be elucidated. A key risk factor for MI is usually dyslipidemia, which is usually strongly associated with a pro-thrombotic phenotype linked to atherothrombosis and platelet hyperactivity.2,3 The blood of high-risk individuals with dyslipidemia is characterized by increased plasma lipid peroxides, with low density lipoproteins (LDL) serving as a highly abundant carriers for these oxidatively-modified lipids.4C6 Oxidized LDL (oxLDL) are circulating pathological ligands that can enhance thrombosis through their ability to promote platelet hyperactivity. experimentation demonstrates that these altered lipoproteins can cause direct activation of platelets and also potentiate platelet activation induced by physiological agonists such as thrombin, ADP and epinephrine.7C10 However, the potential pathophysiological importance of these observations for thrombosis in vivo remain unclear. The scavenger receptor CD36 has emerged as a potential conduit for transducing plasma lipid stress into platelet hyperactivity and thrombosis, through the recognition of lipoprotein associated molecular patterns (LAMPs). CD36, alone or potentially in combination with Toll-Like Receptor (TLR)2 and TLR6 drive a complex series of intracellular signaling events that are associated with platelet activation.11C15 Upon ligation of CD36, Src family kinases constitutively associated with the receptor, drive the activation of Syk, Vav-1, PLC2, ERK5 and JNK that are associated with platelet activation.13,16C18 More recently, data have emerged to suggest that 4-Aminobutyric acid the signaling events promote the generation of reactive oxygen species (ROS).14,16,17 ROS in turn activate ERK to drive thrombosis directly by platelet hyperactivity and caspase-dependent procoagulant activity.18,19 Moreover, we found that ROS diminish sensitivity to the nitric oxide (NO)-stimulated cGMP-PKG inhibitory signaling cascade to reduce the threshold for platelet activation.17 These data suggest that the 4-Aminobutyric acid translation of atherogenic lipid 4-Aminobutyric acid stress by platelet CD36 is functionally linked to both stimulation of activatory signaling pathways and to an as of yet Rabbit Polyclonal to LIMK1 ill-defined modulation of inhibitory pathways. PGI2 is the most potent endogenous regulator of platelet function with both genetic and pharmacological modulation of the pathway linked to accelerated thrombosis control), without stimulating aggregation directly (Physique 1A). In contrast, PGI2-mediated inhibition was unaffected by nLDL (5.81.2%). Comparable data were obtained when platelets were stimulated with collagen (basal). When platelets were treated with nLDL (50 g/mL), the ability of the prostanoid to elevate cAMP was unaffected (1885203 fmol cAMP/1108 platelets), while oxLDL (50 g/mL) prevented PGI2-induced accumulation of cAMP (48123 fmol cAMP/1108 platelets; CD36. (A) Washed human platelets (5108/mL) incubated with apyrase, indomethacin and EGTA were incubated with FA6-152 or IgG (1 g/mL) for 20 minutes (min). Platelets were then incubated alone or with control native LDL (nLDL) or oxLDL (50 mg/mL) for 2 min and subsequently stimulated by PGI2 (50nM) for 1 min. Treated platelets were lysed in Laemmli buffer, separated by SDS-PAGE and immunoblotted with anti-phosphoVASPser157 or anti- tubulin. (Top) Representative blot of three impartial experiments. (Bottom) Densitometry of pVASPser157 fold-change above basal meanstandard error of mean (SEM) (n=3 *associated with dyslipidemia.32 Western diet significantly raised cholesterol levels (6.20.2-fold increase over basal; is usually prevent by inhibition of phosphodiesterase 3A (PDE3A). Intravital microscopy was performed as described in the thrombosis under flow, normal chow WT blood formed small thrombi on immobilized collagen in a time dependent manner, which was abolished by PGI2 (20 nM) (Physique 5Di). High-fat fed WT blood showed an accelerated thrombotic response with increased surface area (113.6% compared to 16.24.3% at 2 mins). In addition, dyslipidemia caused significant hyposensitivity to PGI2, with the prostanoid causing 31.710.7% inhibition compared 61.65.6% with normal chow (is prevented by inhibition of phosphodiesterase 3A To examine the role of oxLDL in thrombosis we used intravital microscopy following ferric chlorideC.
