The MAP3K Apoptosis Signal-Regulating Kinase 1 (ASK1) Functions As a Calcium-Dependent Regulator of Platelet Activation Downstream of the G(α)q-PLC Signaling Axis

Abstract

Upon vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins and activate to form a hemostatic platelet plug. Dysregulation of the molecular mechanisms dictating platelet plug formation is responsible for numerous thrombotic disorders. While it is known that MAPK signaling proteins such as p38, JNK, and ERK play a central role in platelet activation, little is known about the upstream MAP3K proteins regulating their activation following receptor stimulation. Our laboratory has identified the presence and activation of Apoptosis Signal-regulating Kinase 1 (ASK1), a member of the MAP3K family in human and mouse platelets. Ablation of Ask1 from mice results in a strong anti-thrombotic phenotype, and Ask1-/- platelets display significant functional defects such as integrin αIIbβ3 activation, granule secretion, and thromboxane A2 (TxA2) generation. To elucidate the signaling pathway leading to ASK1 activation, washed human platelets (4 x 108 platelets/mL) were evaluated for ASK1 phosphorylation at residue T845 (activated ASK1) by western blot analysis. Blocking TxA2 generation with aspirin (1 mM) severely diminished ASK1 activation induced by PAR1 peptide SFLLRN (100 μM), convulxin (10 ng/mL), and ADP (20 μM). Interestingly, PAR4 peptide AYPGKF (100 μM)-induced ASK1 activation was unaffected by aspirin treatment, suggesting AYPGKF-induced ASK1 activation to occur independently of TxA2 generation. The TxA2 mimetic, U46619, also induced strong T845 ASK1 phosphorylation. This data suggests ASK1 activation in human platelets to occur downstream of the TxA2 receptor TP-α and thrombin receptor PAR4. In nucleated cells, ASK1 is primarily activated by reactive oxygen species (ROS) following oxidative stress. The mechanism of ASK1 activation in platelets has never been studied. Treatment of washed human platelets (4 x 108 platelets/mL) with the ROS donor hydrogen peroxide (1 μM) failed to induce ASK1 activation. Additionally, scavenging of intracellular ROS by the ROS-scavenging compound MnTMPyP (200 μM) had no effect on agonist-induced ASK1 activation suggesting that ASK1 is activated by a ROS-independent mechanism. Interestingly, inducing intracellular calcium rise with the SERCA inhibitor, thapsigargin (1 µM), resulted in strong activation of ASK1 in human platelets. Blocking calcium rise using the calcium chelator BAPTA (50 μM) blocked both thapsigargin and agonist-induced ASK1 activation. This apparent dependency on calcium for ASK1 activation was also observed in the megakaryoblastic cell line MEG-01. Inhibition of IP3-mediated calcium release using the PLC inhibitor U73122 (10 μM) was sufficient to block ASK1 activation induced by U46619 (5 μM) and AYPGKF (100 μM), providing further evidence of a calcium-dependent mechanism for ASK1 activation in human platelets. The identification of ASK1 in platelets and the observed defects in Ask1-/- murine platelets as well as the impaired thrombosis highlights the central role of ASK1 in mediating platelet activation which, until this point, has largely been overlooked. In addition, the observation that ASK1 activation in platelets is exclusively calcium-dependent makes platelets to be a unique model for studying calcium-dependent ASK1 signaling that is independent of ROS. Collectively, we strongly believe that ASK1 is a novel target for anti-thrombotic drug therapy. DisclosuresNo relevant conflicts of interest to declare.