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 (TxA) generation, as well as P2Y 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 TxA 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 TxA formation in murine platelets, with both GS4997 and p38 inhibitors reducing TxA 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 PLA phosphorylation, TxA formation and platelet aggregation, demonstrating that this pathway is redundant in human platelets. Together, these results demonstrate that ASK1 contributes to TxA formation in murine, but not human platelets and highlight the importance of confirming findings from genetic murine models in humans.