Activation of Apoptosis Signal‐regulating Kinase 1 (ASK1) Occurs Downstream of PAR4 and TP‐α Receptors in Human Platelets

Upon 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. Dysregulation of the molecular mechanisms dictating platelet plug formation are responsible for numerous thrombotic disorders. We have identified the presence and activation of a MAP‐Kinase Kinase Kinase in human and mouse platelets known as Apoptosis Signal‐regulating Kinase 1 (ASK1). Ablation of Ask1 from mice results in a strong anti‐thrombotic phenotype, as evidenced by a significantly increased time of vessel occlusion (P = 0.0003) following carotid artery injury with 10% FeCl 3 , as well as protection from pulmonary thromboembolism induced by intravenous injection with collagen and epinephrine. Western blot analysis has indicated ASK1 to be rapidly activated through phosphorylation of T838 by all physiological agonists (Thrombin, ADP, Thromboxane A2, Collagen) in human platelets, however the precise signaling pathways leading to ASK1 activation in platelets remain to be elucidated. To study this, washed human platelets (4 × 10 8 Platelets/mL) were pre‐treated with apyrase (1 U/mL) or aspirin (1 mM) to evaluate specific dependencies on secreted ADP and thromboxane A2, respectively, for ASK1 activation by the stated physiological agonists. The ratio of activated ASK1 to total ASK1 signal obtained by western blot was assessed over a series of time points, and statistical significance between average ratios obtained for treated and non‐treated samples was determined using a two‐tailed, paired t‐test (Significance P < 0.05). Thrombin (0.1 U/mL)‐induced ASK1 activation was mildly attenuated by apyrase treatment but strongly attenuated by aspirin treatment, suggesting a primary dependency on thromboxane A2 relative to secreted ADP for thrombin‐induced ASK1 activation. Independent stimulation of the PAR1 and PAR4 thrombin receptors using synthetic PAR1 peptide SFLLRN (100 μM) and PAR4 peptide AYPGKF (100 μM) revealed PAR1‐induced ASK1 activation to be mildly attenuated by apyrase treatment but strongly attenuated by aspirin treatment, whereas PAR4‐induced ASK1 activation was unaffected by apyrase or aspirin treatment. This suggested that thrombin, at 0.1 U/mL, was acting primarily through PAR1 while PAR4‐induced ASK1 activation was occurring through a distinct signaling axis and likely requires higher thrombin concentrations for activation. As an agonist alone, ADP (20 μM) could only weakly activate ASK1 and was completely abolished by aspirin treatment. Conversely, the thromboxane A2 mimetic U46619 (5 μM) could strongly induce ASK1 activation and was unaffected by apyrase treatment. This data suggested that secondary signaling through secreted ADP was serving to reinforce ASK1 activation by means of thromboxane A2 generation. Activation of ASK1 by collagen (2 μg/mL) was mildly attenuated by apyrase treatment but strongly attenuated by aspirin treatment, again highlighting a primary dependency on thromboxane A2 generation relative to secreted ADP for ASK1 activation. Collectively, our data indicates ASK1 activation to occur downstream of the PAR4 and TP‐α receptors in human platelets. Support or Funding Information Research reported in this presentation was supported by the National Heart, Lung, and Blood Institute of the National Institutes of Health under Award Number 1R01HL113118.