Agonist-Induced Thromboxane A2 Generation Requires Signaling Through Apoptosis Signal-Regulating Kinase 1 (ASK1): Genetic Ablation of Ask1 Severely Affects In Vivo Thrombosis
Authors: Naik Meghna Ulhas , Turaga Ramya , Ichijo Hidinori , Naik Ulhas P 
Published: October 2019

Abstract 187

Oxidative stress is a major contributing factor for cardiovascular diseases. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family, which responds to a diverse array of cellular stresses including oxidative stress. Although its downstream signaling effectors, the JNK and p38 MAP kinases, have been shown to be present and activated in platelets, the presence of ASK1 is not known. Here we show that ASK1 is highly expressed in human platelets. During activation of platelets by a variety of physiological agonists, ASK1 is rapidly activated by phosphorylation of threonine (T845) in its activation loop. In order to determine the physiological significance of activation of ASK1 we used Ask1 knockout mouse. Genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2×10−9). While all WT mice stopped bleeding with an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s, with 5 out of 9 mice that did not stop bleeding for up to 10 min. A carotid artery injury induced by 10% FeCl3 showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. Furthermore, we found that loss of Ask1 renders significant protection to the mice from pulmonary thromboembolism induced by intravenous injection of a mixture of collagen and epinephrine as determined by increased survival and a lack of exclusion of Evans blue dye from the lungs. To determine the cause of such a severe defect in thrombosis, we evaluated ex vivo platelet functions. We found that low doses of agonist-induced platelet aggregation were significantly reduced in Ask1 null mice compared to WT mice. Defective aggregation was found to be due in part to impaired activation of fibrinogen receptor as indicated by reduced fibrinogen binding. When the ability of Ask1 platelets to release platelet granular contents was analyzed, both a- and d-granule secretion were significantly inhibited as assessed by P-selectin exposure and 14C-serotonin release, respectively. Interestingly, aspirin treatment had no effect on the amount of 14C-serotonin release from Ask1 null platelets, but reduced the amount in WT platelets, bringing it to the same level as that of Ask1 null platelets, suggesting that agonist-induced thromboxane generation was impaired in the Ask1 null platelets. This was further confirmed by the complete lack of thrombin-induced TxA2 production in Ask1 null platelets as compared to WT. Since cPLA2 is a key enzyme in the pathway of TxA2 generation, we analyzed the effect of the absence of Ask1 on cPLA2 activation. We found a complete absence of cPLA2 activation-induced by thrombin in Ask1 null platelets while a robust activation of cPLA2 occurred in WT. Additionally, agonist-induced activation of the p38 MAP kinase, known to be involved in cPLA2 activation, was absent in Ask1 null platelets. Furthermore, the TxA2 mimetic U46619-induced platelet aggregation was also greatly reduced in Ask1 null platelets, suggesting that signaling through the TxA2 receptor is also impaired in Ask1 null platelets. These in vitro and in vivo results strongly suggest that ASK1 plays a major role in the regulation of hemostasis and thrombosis by regulating a number of signaling events that are critical for proper platelet function.

Disclosures:

No relevant conflicts of interest to declare.