ASK1 Activation in Platelets By Oxidized LDL Is Independent of CD36

Oxidized low-density lipoprotein (oxLDL) is a major contributor to platelet hyper-activation in hyperlipidemic conditions. OxLDL induces a prothrombotic phenotype by sensitizing the platelets for activation. Several receptors have been identified for oxLDL on platelets, but majority of the studies were focused on CD36. OxLDL binding to CD36 results in activation of multiple signaling molecules including Src family kinases (SFKs), Vav 1&3, JNK2, ERK5, and reactive oxygen species (ROS). The mechanism(s) by which oxLDL sensitizes platelets for activation is poorly understood. We have recently identified Apoptosis Signal Regulating Kinase 1 (ASK1), a MAP kinase kinase kinase, in platelets. Here, we show that in human platelets ASK1 is activated by oxLDL as determined by its activation-specific phosphorylation on T⁸⁴⁵. ASK1 activation was evident with oxLDL concentrations as low as 10μg/ml, with maximal activation observed with 100μg/ml. In contrast, native LDL (nLDL, 10-200μg/ml) failed to stimulate ASK1 activation. The activation of ASK1 was rapid peaking at 1 minute and returning to base line by 5 minutes. To determine if ROS is responsible for the activation of ASK1, we pretreated platelets with MnTMPyP, a ROS scavenger. MnTMPyP failed to inhibit the activation of ASK1 induced by oxLDL. To determine the mechanism by which oxLDL induces ASK1 activation, we pretreated platelets with 10μM each of pharmacological inhibitors, PP2 for SFKs, Y27632 for Rho kinase, bisindolylmaleimide for PKC, LY294002 for PI3 Kinase, U73122 for PLC, and 25μM of BAPTA-AM to chelate calcium. We found that platelets pretreated with U73122 and BAPTA-AM abolished oxLDL induced ASK1 activation, whereas others had no effect suggesting that ASK1 is activated in a calcium-dependent manner. To investigate the role of CD36 in oxLDL induced ASK1 activation, we used FA6-152, a function-blocking antibody for human CD36, and murine CD36nullplatelets. Interestingly, either blocking or lack of CD36 had no effect on oxLDL-induced ASK1/p38 activation suggesting that CD36 is not involved in oxLDL induced ASK1 activation. To determine if ASK1 play a role in oxLDL-induced sensitization of platelets for activation, we treated platelets with GS-4997, a specific ASK1 inhibitor, before stimulating them with ADP and oxLDL. ADP (10µM) or oxLDL (100µg/ml), showed no aggregation, but combination of oxLDL and ADP showed robust aggregation of vehicle-treated platelets. However, pretreatment of platelets with GS-4997 significantly inhibited oxLDL-induced potentiation of ADP-induced platelet aggregation suggesting that ASK1 plays a role in platelet activation under hyperlipidemic conditions.