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.