Evaluation of Two Structurally Distinct Novel Inhibitors of Apoptosis Signal-Regulating Kinase 1 (MAP3K5), As Potent Anti-Platelet Agents

Platelets play a critical role in both hemostasis and thrombosis. Anti-platelet drugs currently available apart from aspirin are directed against platelet agonist receptors or fibrinogen receptor integrin a_IIbb₃. These antagonists, although having potent anti-thrombotic activities, cause severe bleeding due to their effect on hemostasis. It is therefore of utmost important to develop new drugs that will protect from thrombosis with minimal effect on hemostasis. Apoptosis signal-regulating kinase (ASK1) is a redox sensitive serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its role in platelets is not known. We found that ASK1 is rapidly activated downstream of all platelet agonists. Ablation of Ask1 gene results in impaired platelet functions such as granule secretion, thromboxane A₂ generation, as well as fibrinogen receptor activation, which translates into attenuated platelet aggregation compared to WT littermates. We also found that thrombin failed to activate p38 in Ask1 null platelets, showing that Ask1 is indispensable for p38 activation by thrombin. FeCl₃-induced carotid artery injury model of thrombosis showed a significantly increased (P=0.0003) time of occlusion and unstable thrombus formation in Ask1 null mice. These results indicated that ASK1 plays a central role in regulating platelet function, making it a potential target for combating thrombosis. We therefore synthesized two novel and highly specific ASK1 inhibitors based on the published reports, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) and GS-4997. We found that GS-4997 (500nM) and IPTB (5mM) inhibit agonist-induced ASK1 activation in human platelets. They do not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1/2. We also found that IPTB and GS-4997 dose-dependently inhibited activation of p38, a downstream effector kinase, induced by a variety of platelet agonists. Furthermore, these compounds dose-dependently inhibited ADP, collagen, convulxin and PAR4 activating peptide AYPGKF-induced platelet aggregation as well as platelet spreading on immobilized fibrinogen. In-vivo carotid artery thrombosis assay revealed that WT mice injected intraperitoneally with either IPTB (100mg/kg) of GS-4997 (100mg/kg), showed a significantly increased time of occlusion (P=0.028 and P=0.005 respectively) and thrombus formed were unstable as compared to control WT mice treated with saline alone. Furthermore, injection of either IPTB or GS-4997 protected mice against collagen/epinephrine-induced pulmonary thromboembolism. Out of 14 saline-treated mice only two survived whereas, 10 out of 11 mice treated with GS-4997 (100mg/kg) survived (P=0.0002). In case of IPTB 1 out of 12 control mice survived as compared to 9 out of 12 treated mice (P=0.0028). Interestingly, tail-bleeding studies revealed that WT mice treated with either IPTB (1mg/kg) of GS-4997 (1mg/kg), did not affect the average bleeding time (100s) seen in the WT mice treated with saline alone, suggesting that both inhibitors had no effect on in-vivo hemostasis. Moreover, pretreatment of the whole blood with these inhibitors significantly reduced thrombus formation under arterial flow (800s^-1) without affecting platelet adhesion to collagen as assessed using a microfluidic device. Our results strongly suggest that both IPTB and GS-4997 protect the mice from thrombosis without affecting hemostasis. Further development of these inhibitors as a potential therapeutic agent to combat thrombotic disorders is highly warranted.