Role of ASK1 in Platelet Activation and Immune-Induced Thrombocytopenia and Thrombosis
Apoptosis signal-regulating kinase 1 (ASK1) is a MAPK kinase kinase (MAPKKK) that regulates activation of the c-Jun N-terminal kinase (JNK)- and p38-stress response pathways leading to apoptosis in nucleated cells. In platelets, ASK1 potentiates agonist-induced platelet activation and thrombosis. However, the mechanism(s) regulating ASK1 activation, ASK1’s function in regulating platelet activation, and ASK1’s role in regulating immune-induced thrombocytopenia and thrombosis (ITT) remain unknown. Here we investigate these mechanisms to clarify ASK1’s role in platelets and ITT. We show that platelet agonists induce ASK1 activation by increasing intracellular Ca2+ concentration. This increased Ca2+ state corresponds with ASK1 dissociating from calcium and integrin-binding protein 1 (CIB1), and then associating with tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), both of which are well characterized negative and positive regulators of ASK1 in neuronal cells respectively. We further found that loss of Ask1 attenuated integrin activation and a- granule secretion. Closer inspection showed that activation of p38 was blocked in Ask1 knockout platelets, attenuating cPLA2-Ser505 phosphorylation and thromboxane A2 (TxA2) generation. Accounting for TxA2 generation removed any genotype differences in integrin activation and α-granule secretion. Additionally, we found that GS-4997 (a small-molecule ASK1 inhibitor) was as effective as aspirin for inhibiting platelet aggregation. Dual inhibition with aspirin+GS-4997 showed little to no effect over monotherapy. To study ASK1’s role in regulating ITT we used an anti-CD9-induced mouse model of immune complex (IC)-induced pathologies in vitro and in vivo as it mimics the action of pathologic ICs. We found that ASK1 regulated anti-CD9- induced platelet aggregation. Anti-CD9-induced cPLA2 phosphorylation and TxA2 generation were also delayed in Ask1 knockout platelets, contributing to diminished α- and δ-granule secretion. Knockout of Ask1 protected mice from anti-CD9-induced thrombocytopenia, shock, and pulmonary thromboembolism. Whole blood microfluidic testing revealed that platelet adhesion and thrombosis was enhanced by Ask1 dependent TxA2 generation. When tested using a mouse model of heparin-induced thrombocytopenia, inhibition of Ask1 protected mice from shock. Together our results suggest that (1) ASK1 is negatively regulated in a Ca2+ dependent manner by CIB1. (2) that ASK1 regulates platelet functions by regulating TxA2 generation. And (3) that ASK1 may be a potential target for the treatment of IC-induced shock and other immune-mediated thrombotic disorders.
Patel, Pravin, "Role of ASK1 in Platelet Activation and Immune-Induced Thrombocytopenia and Thrombosis" (2020). ETD Collection for Thomas Jefferson University. AAI27666839.