Identification Of a Novel Small Molecule Inhibitor Of ASK1 As a Potent Anti-Platelet Agent

Apoptosis signal-regulating kinase (ASK1) is a serine/threonine kinase, belonging to the MAP kinase-kinase-kinase family, which is activated in response to stress. However, its presence and role in platelets are not known. We found that ASK1 is expressed in platelets and is rapidly activated during platelet stimulation by various agonists in a dose-dependent manner. In addition, we found that TRAF2/6, known endogenous activators of ASK1, are expressed in platelets and associate with ASK1 upon platelet activation with agonists. Furthermore, genetic ablation of Ask1 significantly delayed tail-bleeding time (P=0.2x10^-9). While WT mice showed an average bleeding time of 100 s, the Ask1 null mice had an average bleeding time of 576 s. 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 a mixture of collagen and epinephrine as determined by increased survival and lack of large occlusive thrombi in the lung. We also found that ADP- and AYPGKF (PAR4 receptor peptide) -induced platelet aggregation was diminished in Ask1 null mice compared to WT mice. Furthermore, PAR4 peptide-induced alpha- and dense-granular secretion was also reduced in Ask1 null platelets compared to WT. Interestingly, we also found that Ask1 null platelets bind less FITC-fibrinogen compared to the WT upon activation by PAR4 peptide. Furthermore, thrombin failed to activate MKK6 and p38 in Ask1 knockout platelets, showing that Ask1 is indispensable for p38 activation by thrombin. These results indicated that ASK1 regulates platelet function by augmenting platelet secretion as well as fibrinogen receptor activation, making it an important target for combating thrombosis. We therefore synthesized a novel and highly specific ASK1 inhibitor, N-(6-(1H-imidazol-1-yl)imidazo[1,2-a]pyridin-2-yl)-4-(tert-butyl)benzamide (IPTB) based on the published report. IPTB has been found to be a very potent inhibitor that inhibits ASK1 activity at nM concentrations. IPTB is also highly specific to ASK1 and does not affect activities of related protein kinases such as ASK2, MEKK1, TAK1, and ERK1. We found that in human platelets, IPTB dose-dependently inhibits p38 activation induced by a variety of platelet agonists. Furthermore, IPTB dose-dependently inhibited ADP and PAR4 peptide-induced platelet aggregation. Interestingly, IPTB also dose-dependently inhibited platelet spreading on immobilized fibrinogen. Our results strongly suggest that the dose of IPTB could be adjusted so that it attenuates thrombosis without affecting hemostasis. This development would make IPTB a novel potential therapeutic agent to be used to combat thrombotic disorders.