PB0810 - Oxidized-LDL Induces two Distinct Platelet Phenotypes: Aggregatory Platelets Through Gq-Dependent ASK1 Activation and Procoagulant Platelets Through CD36 Dependent SFK Activation

Background: It is well evidenced that oxidized LDL (oxLDL) sensitizes platelets for activation, but the mechanism(s) by which it primes platelets is poorly understood. Apoptosis Signal Regulating Kinase 1 (ASK1) is robustly activated by all platelet agonists, but its role in oxLDL-induced platelet sensitization is not known.

Aims: To investigate the role of ASK1 in oxLDL-induced platelet sensitization.

Methods: Human and mouse platelet functional studies and signaling pathway evaluations were performed by Lumi-aggregometer and western blotting respectively. Intracellular Ca2+ level was determined by Fluo-4AM using flow cytometer. PSer exposure was quantified using Annexin-V binding.

Results: Hyperlipidemic mouse platelets showed significantly elevated Ask1/p38 activation compared to control. In human platelets, ASK1 is rapidly activated by oxLDL, but not by native LDL. Inhibition of CD36 failed to inhibit ASK1 activation. Additionally, genetic deletion of CD36 had no effect on oxLDL induced Ask1/p38 activation suggesting a CD36 independent pathway. Furthermore, pharmacological inhibition signaling molecules downstream of CD36 such as SFKs and ROS, failed to inhibit oxLDL induced ASK1 activation, pointing to an alternative signaling pathway. Inhibition of PLC, Gq, or chelation of intracellular Ca2+ abolished ASK1 activation by oxLDL, suggesting a GPCR-dependent pathway. Pharmacological inhibition of known platelet GPCRs failed to inhibit ASK1/p38 activation suggesting involvement of an unidentified GPCR. Interestingly, oxLDL synergistically enhanced platelet aggregation induced by subthreshold concentration of Gi/Gz- but not Gq-coupled receptors. OxLDL/epinephrine combination induced robust pulmonary thromboembolism in mice. Furthermore, oxLDL/ADP induced platelet hyper-aggregation was abolished by inhibition of P2Y12 but not P2Y1. Moreover, inhibition of ASK1 abrogated the oxLDL/ADP induced platelet hyper-activation but not PS-exposure, which was attenuated by inhibition of SFKs.

Conclusion(s): OxLDL signals through two independent platelet receptors, CD36 and a Gq-coupled receptor. CD36 signaling is involved in ROS generation and PSer exposure, whereas Gq signaling induces ASK1/p38 activation to prime platelets to be activated by Gi/Gz pathway.