Redundant role of ASK1-mediated p38MAPK activation in human platelet function

Apoptosis signal-regulating kinase 1 (ASK1) is a member of mitogen-activated protein kinase kinase kinase (MAP3K) family, which recently has been implicated in the regulation of p38 MAPK/PLA2/thromboxane (TxA₂) generation, as well as P2Y₁₂ signalling in murine platelets. ASK1 has therefore been proposed as a potential target for anti-thrombotic therapy. At present it is unknown whether ASK1 also contributes to TxA₂ formation and platelet function in human. In this study we therefore examined the role of ASK1 using the ASK1 inhibitor selonsertib (GS-4997). We established that ASK1 is responsible for p38 phosphorylation and TxA₂ formation in murine platelets, with both GS4997 and p38 inhibitors reducing TxA₂ formation. Similar to murine platelets, activation of human platelets resulted in the rapid and transient phosphorylation of ASK1 and the MAP2Ks MMK3/4/6. In contrast, phosphorylation of p38 and its substrate; MAPKAP-kinase2 (MAPKAPK2) was much more sustained. In keeping with these findings, inhibition of ASK1 blocked early, but not later p38/MAPKAPK2 phosphorylation. The latter was dependent on non-canonical autophosphorylation as it was blocked by the p38 inhibitor; SB203580 and the SYK inhibitor; R406. Furthermore, ASK1 and p38 inhibitors had no effect on PLA₂ phosphorylation, TxA₂ formation and platelet aggregation, demonstrating that this pathway is redundant in human platelets. Together, these results demonstrate that ASK1 contributes to TxA₂ formation in murine, but not human platelets and highlight the importance of confirming findings from genetic murine models in humans.