PB0417 - ASK1 Regulates Thrombin, But Not Collagen Induced TxA2 Generation in Human Platelets

Background: Apoptosis signal-regulating kinase 1 (ASK1), a MAP3K, activates JNK and p38 MAP kinase pathways. Using Ask1-/- mouse model, we have shown that Ask1 regulates platelet reactivity and thrombosis through regulation of TxA2 generation. In human platelets ASK1 is robustly activated downstream of most physiological agonists but its consequence is not known. It was recently reported that selonsertib (GS4997), a selective ASK1 inhibitor, failed to inhibit GPVI induced TxA2 generation and aggregation in human platelets.

Aims: To evaluate the role of ASK1 in human platelet activation.

Methods: Human platelets suspensions were pretreated with GS4997 and platelet functions such as aggregation, PAC-1 binding, granule secretion, and TxA2 generation were evaluated. Western blot analysis was used to assess the state of phosphorylation of cPLA2, Erk1/2 and p38.

Results: We found that pretreatment of human platelet suspension with GS4997 (1µM) did not inhibit collagen (2.5µg/mL) mediated platelet aggregation and TxA2 generation which is in agreement with previous report. Interestingly, pretreatment of platelet rich plasma with GS4997 (as low as 10fM) significantly (P < 0.001) inhibited platelet aggregation induced by thrombin receptor activating peptides (PAR1-AP (10µM) and PAR4-AP (60µM). Furthermore, thrombin (0.05U) induced aggregation was also significantly inhibited (P < 0.001) in resuspended platelets by GS4997 (1µM). Additionally, GS4997 mediated inhibition of PAR1- and PAR4-mediated platelet aggregation was restored by the addition of U46619 (2.5µM) or ADP (5µM) suggesting that TxA2 generation and granule secretion downstream of PARs is regulated by ASK1. Furthermore, ASK1 inhibition significantly reduced (P < 0.001) TxA2 generation following stimulation of PAR1-AP or PAR4-AP. Interestingly, GS4997 abrogated p38 and cPLA2 phosphorylation and significantly reduced Erk2 phosphorylation. Erk2 inhibitor U0126 (3µM) inhibited thrombin-induced platelet aggregation and TxA2 generation suggesting that both ASK1/p38 and Erk2 regulate TxA2 generation.

Conclusion(s): Our findings suggest that in human platelets ASK1 regulates TxA2 generation via p38 and Erk2 downstream of thrombin receptors.