Apoptosis Signal-Regulating Kinase (ASK1) Regulates Thrombosis in Part By Regulating cPLA2 phosphorylation-Dependent TxA2 Generation

When vascular endothelium is injured, circulating platelets are activated by primary agonists. Activation causes platelets to change shape, aggregate, and release secondary agonists which reinforce initial platelet activation as well as help recruit additional platelets to the site of vascular injury. MAP kinases have been shown to be important regulators of platelet function and secondary agonist production. One important secondary agonist released by activated platelets is TxA2. TxA2 is generated by metabolism of Arachidonic acid (AA). AA is released from platelet membrane phospholipids via the activity of PLAs. In platelets cPLA2 activity has been shown to be regulated by MAP kinases, however, the mechanisms which regulate platelet MAP kinase activity are not well understood. Our laboratory has identified that ASK1 (a Ser/Thr kinase of the MAP3K family) is present in both human and murine platelets and is activated by physiological agonists. ASK1 is known to be activated by a number of cellular stress response pathways. When challenged by cellular stress, ASK1 auto phosphorylates Thr845 on its activation loop, which is required for its ability to phosphorylate its substrates. Here we show that ASK1 regulates platelet function in part by regulating agonist-induced TxA2 generation. To determine the role of Ask1 in hemostasis and thrombosis, we evaluated in vivo thrombosis using carotid artery injury induced by 10% FeCl3 or pulmonary thromboembolism induced by injecting mixture of collagen/epinephrine. We found that genetic ablation of Ask1 renders mice significant protection from thrombosis. To determine the mechanism by which Ask1 regulates platelet activation leading to thrombosis, we evaluated the MAP kinase cascade using Ask1 null platelets. We found that genetic ablation of Ask1 blocked agonist-induced activation of the MAP2Ks (MKK3 and MKK4) in murine platelets. Since MKK3 can activate p38 and MKK4 can activate both p38 and JNK, we assessed MAPKs activation in murine platelets. When stimulated by various agonists, activation of p38 was entirely lost in Ask1 null platelets while activation of ERK1/2 and JNK remained unaffected indicating that Ask1 solely regulates p38 activity in platelets. Activity of p38 has been linked to agonist-induced generation of TxA2, an important contributing factor to thrombosis. We therefore evaluated agonist-induced production of TxA2 by measuring TxB2 (a stable metabolite of TxA2). We saw a substantial reduction (~50% in thrombin- and ~70% in convulxin-induced) production of TxA2 in Ask1 null platelets suggesting a separate Ask1 independent mechanism for TxA2 generation. Since TxA2 is a metabolite of AA, whose production in platelets is caused by cPLA2 enzymatic activity and cPLA2 activity is regulated by phosphorylation of its Ser505 residue by p38, we evaluated phosphorylation of cPLA2 (p-Ser505). We found that agonist-induced phosphorylation of cPLA2 (Ser505) was completely lost in Ask1 null platelets. Although in Ask1 null platelets cPLA2 phosphorylation (Ser505) is completely abolished, substantial amount (~50%) of TxA2 was generated in response to thrombin suggesting that there exists an Ask1 independent mechanism of activation of cPLA2. To rule out the possibility that an alternative PLA2 is responsible for the residual TxA2 production found in Ask1 null platelets, we evaluated agonist-induced TxA2 production in the presence of pyrrophenone, a cPLA2 specific inhibitor. Pretreatment with pyrrophenone completely abolished agonist-induced TxA2 production in murine as well as human platelets, suggesting that cPLA2 is solely responsible for the majority of agonist induced AA/TxA2 in platelets. In addition to its phosphorylation, it is documented that cPLA2 activity is also dependent on intracellular Ca2+, which facilitates translocation of cPLA2 to AA containing membranes. It is therefore possible that the remainder of TxA2 formed is dependent on Ca2+-dependent activity of cPLA2. Taken together these in vivo and in vitro results strongly suggest that ASK1 plays a key role in regulating thrombosis, in part, by regulating the signaling mechanisms involved in agonist-induced production of TxA2. Disclosures No relevant conflicts of interest to declare.