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Bidirectional signaling through platelet integrin αIIbβ3is essential in hemostasis and thrombosis. In quiescent platelets, αIIbβ3is in a low-affinity ligand binding state. However, on platelet activation by agonists through inside-out signaling, a rapid switch in the conformation of the integrin results in a high affinity ligand binding state capable of binding soluble fibrinogen. Ligand binding to the αIIbβ3induces a signaling termed outside-in signaling that regulate platelet spreading and clot retraction. These events are often interchangeably used to represent outside-in signaling pathway. Using pharmacological inhibitors of known signaling molecules that have been implicated to regulate outside-in signaling, we assessed human platelet spreading and clot retraction. We found that inhibition of phosphoinositide-3-kinase, phospholipase C, protein kinase C, and focal adhesion kinase strongly attenuated both platelet spreading and clot retraction suggesting that they are essential for both clot retraction and platelet spreading, whereas inhibition of Rac1, rho-associated, coiled-coil containing protein kinase, p38, and MEK did not affect platelet spreading but significantly delayed clot retraction suggesting that these signaling molecules do not participate in platelet spreading. Interestingly, Src family kinases are required for platelet spreading and FAK activation but suppress clot retraction because their inhibition causes faster clot retraction. Thus, it becomes evident that platelet spreading, and clot retraction are differently regulated through αIIbβ3outside-in signaling and should not be used interchangeably as readout for αIIbβ3outside-in signaling assessment.

Cardiovascular diseases are currently among the leading causes of death in mankind. Although significant progress has been made in prevention and treatment, the currently available pharmacological interventions have a significant rate of severe bleeding side effects due to their effect on hemostasis. Recently, we have shown that apoptosis signal-regulating kinase 1 (ASK1) plays an important role in platelet activation and thrombus formation. Here, we show that two chemically distinct ASK1 inhibitors, dose-dependently inhibited thrombin-induced phosphorylation of ASK1 and its downstream effectors MKK3/4/6, p38 MAPK, and cPLA2 in human platelets, as detected by western blotting using phospho-specific antibodies. ASK1 inhibitors also dose-dependently inhibited platelet a granule release and α IIbβ 3activation which are the readouts for inside-out signaling events as analyzed by flow cytometry. Moreover, pretreatment of human platelets with ASK1 inhibitors completely impaired clot retraction and platelet spreading on immobilized fibrinogen two events regulated by outside-in signaling. To investigate the in-vivoeffect of Ask1 inhibitor on thrombus formation, we intraperitoneally injected C57BL/6 mice with Ask1 inhibitor (1mg/kg), and the blood was withdrawn at 1-, 4-, and 24-hour time points and analyzed for in vitrothrombus formation on collagen matrix under arterial flow. Ask1 inhibitor significantly decreased thrombus growth ( P<0.01) which was sustained for at least 24 hours. Platelet adhesion to collagen was not affected as determined by surface coverage.In a 10% FeCl 3-induced carotid artery injury model of thrombosis, we found that Ask1 inhibitor (100μg/kg)showed a significantly extended time of vessel occlusion ( P<0.01) .Moreover, Ask1 inhibitor (100μg/kg) treated C57BL/6 mice were protected from collagen/epinephrine-induced pulmonary thromboembolism ( P<0.001). Furthermore, in a well-established ischemic stroke model of transient middle cerebral artery occlusion, C57BL/6 mice treated with Ask1 inhibitor (1mg/kg) showed increased survival and improved neurological deficits than vehicle treated mice. The extent of the brain damage was assessed by staining the brain sections with 2% 2,3,5-triphenyltetrazoliun chloride, which showed significant reduction in infarct size and edema ( P<0.0001). Interestingly, mice treated with Ask1 inhibitor (1mg/kg), a 10-fold more than the amount needed to inhibit thrombosis, had no effect on hemostasis as observed by both the tail bleeding assay and laser-induced cremaster injury model. Whereas clopidogrel (5-30mg/kg) showed significantly increased ( P<0.0001) tail bleeding time. Next, we performed cerebral bleeding assay in which brain injury was induced by inserting a 26-gauge needle through the skull in the cerebrum 2mm laterally to the bregma and 4mm deep into the cerebrum and rotated 360 degrees. After 20 minutes the extent of bleeding in the brain was assessed by imaging. Treatment of C57BL/6 mice with clopidogrel (30mg/kg) showed significantly enhanced bleeding ( P<0.05) compared to vehicle treated mice. However, Ask1 inhibitor treated mice had same extent of bleeding as that of vehicle treated mice. Additionally, a liver bleeding assay was performed by excising a calibrated piece (~4-5mg) of inferior edge of the right lobe of the liver of the mouse under anesthesia. After 20 minutes the bleeding was assessed by counting red blood cells in the peritoneal lavage. As observed in brain bleeding assay, treatment of mice with clopidogrel (30mg/kg), showed significantly increased liver bleeding ( P<0.05) compared to vehicle treated mice whereas Ask1 inhibitor treated mice had same extent of liver bleeding as that of vehicle treated mice. Taken together, our results provide evidence that ASK1 inhibitors are novel therapeutic agents to combat thrombotic disorders without any side effects of bleeding, which is inherent to current anti-thrombotic drugs.

Mitogen-activated protein kinases (MAPKs) are expressed in platelets and are activated downstream of physiological agonists. Pharmacological and genetic evidence indicate that MAPKs play a significant role in hemostasis and thrombosis, but it is not well understood how MAPKs are activated upon platelet stimulation. Here, we show that apoptosis signal-regulating kinase 1 (ASK1), a member of the MAP3K family, is expressed in both human and murine platelets. ASK1 is rapidly and robustly activated upon platelet stimulation by physiological agonists. Disruption of Ask1(Ask1−/−) resulted in a marked functional defect in platelets. Ask1−/−platelets showed an impaired agonist-induced integrin αIIbβ3activation and platelet aggregation. Although there was no difference in Ca2+rise, platelet granule secretion and thromboxane A2(TxA2) generation were significantly attenuated in Ask1−/−platelets. The defective granule secretion observed in Ask1−/−platelets was a consequence of impaired TxA2generation. Biochemical studies showed that platelet agonists failed to activate p38 MAPK in Ask1−/−platelets. On the contrary, activation of c-Jun N-terminal kinases and extracellular signal-regulated kinase 1/2 MAPKs was augmented in Ask1−/−platelets. The defect in p38 MAPK results in failed phosphorylation of cPLA2in Ask1−/−platelets and impaired platelet aggregate formation under flow. The absence of Ask1 renders mice defective in hemostasis as assessed by prolonged tail-bleeding times. Deletion of Ask1also reduces thrombosis as assessed by delayed vessel occlusion of carotid artery after FeCl3-induced injury and protects against collagen/epinephrine-induced pulmonary thromboembolism. These results suggest that the platelet Ask1 plays an important role in regulation of hemostasis and thrombosis.