ASK1, a Novel Regulator of Thrombosis, Is Activated Downstream of G-Protein Coupledreceptors in Human Platelets

In the event of vascular injury, platelets rapidly adhere to sub-endothelial matrix proteins such as collagen and Von Willebrand factor and activate to form a stable hemostatic platelet plug. Defects in the molecular mechanisms dictating platelet plug formation are responsible for numerous thrombotic disorders. Elucidating the signaling pathways and molecular mechanisms of platelet activation is paramount to the development of safer and more effective anti-thrombotic drugs. While it is known that MAP-Kinase signaling participates in platelet activation, it is unknown how MAP-Kinase signaling specifically mediates platelet activation. Our laboratory has identified the presence and activation of a MAP-Kinase Kinase Kinase known as Apoptosis Signal Regulating Kinase 1 (ASK1). We have demonstrated using an ASK1 knockout mouse model that ablation of ASK1 leads to a significantly increased (p = .0003) time of vessel occlusion associated with unstable thrombus formation following a carotid artery injury induced by 10% FeCl₃. Furthermore, ASK1 knockout mice display protection from pulmonary thromboembolism induced by an intravenous injection of collagen and epinephrine. In order to determine the kinetics of ASK1 activation by physiological agonists, washed human platelets (4 x 10⁸ platelets/mL) were treated with 0.1 U/mL of thrombin for 30”, 1’, 3’, 5’, and 8’. Robust activation of ASK1 by thrombin occurred as early as 30 seconds up until 5 min, after which ASK1 activation decreased sharply. Platelets treated with 100 µM of PAR1 (SFLLRN) or PAR4 (AYPGKF) peptides resulted in strong ASK1 activation, suggesting that both the PAR1 and PAR4 receptors lead to ASK1 activation. Inhibition of Src family kinases by PP2 or PI3K by wortmannin or Rho kinase by Y-27632 had no effect on thrombin-induced ASK1 activation. However, inhibition of PLC-β2, a mediator of platelet activation downstream of the PAR1/4 receptors, strongly inhibited ASK1 activation by thrombin. We next determined whether TxA2 generation was responsible for ASK1 activation by thrombin. Washed platelets were pre-treated with 1 mM aspirin to block TxA2 generation, followed by treatment with 0.1 U/mL of thrombin. It was found that blocking TxA2 generation eliminated ASK1 activation by thrombin at 30” and 1’, but not at a later time point, suggesting there may be an additional pathway contributing to ASK1 activation. The observation that TxA2 generation contributes to ASK1 activation by thrombin seemed to correlate with the finding that treatment of platelets with 1 µM of the TxA2 mimetic U46619, which activates the TP-α receptor, could also activate ASK1. We also determined whether ADP released from dense granules, which would activate the P2Y₁ and P2Y₁₂ receptors, leads to ASK1 activation. To test this, washed platelets were pre-treated with 1 U/mL of apyrase to hydrolyze secreted ADP. It was found that apyrase treatment completely eliminates ASK1 activation by thrombin, suggesting a strong dependency of thrombin-induced ASK1 activation on ADP release from dense granules. To further investigate this possibility, washed platelets were pre-treated with 50 µM of the P2Y₁ antagonist MRS2179 or P2Y₁₂ antagonist 2-MeSAMP, followed by treatment with 0.1 U/mL of thrombin. Antagonism of the P2Y₁₂ receptor and not P2Y₁ receptor severely diminished ASK1 activation by thrombin. This indicates that ASK1 activation by thrombin is also dependent on ADP released from dense granules and subsequent activation of the P2Y₁₂ receptor. Surprisingly, collagen, a strong activator of platelets, was unable to activate ASK1 in washed platelets at a concentration of 2 µg/mL. Similarly, 2 µM epinephrine treatment also had no effect. However, when washed platelets were treated with 2 µg/mL collagen and 2 µM epinephrine together, a strong ASK1 activation was observed (p=.0012). This suggests the existence of a novel mechanism for ASK1 activation by simultaneous stimulation of the collagen receptors GPVI/α₂β₁ and epinephrine receptor α2_A. The finding that ASK1 activation occurs downstream of TP-α, P2Y₁₂, and possibly α2_A receptors highlights the importance of ASK1 in regulation of these G-Protein Coupled Receptors in platelet activation. In conclusion, our data indicates ASK1 to be a key mediator in platelet activation and represents a novel target for anti-thrombotic drug therapy.