Apoptosis Signal-Regulating Kinase 1 Regulates Platelet Function through Integrin aIIbb3 Outside-In Signaling

Abstract 328

Atherosclerosis tends to develop in areas of the circulation where the blood flow is either low or turbulent, such as regions where the arteries bifurcate. Studies have shown that shear stress stimulates endothelial cells and modulates platelet function. However, the intracellular signaling events that coordinate these effects are not completely understood. The mitogen-activated protein kinase (MAPK) cascades are crucial in regulating cellular stress responses. It is known that a number of diseases, including cardiovascular diseases, are intimately related to stress related mechanisms mediated by MAPK cascades. Apoptosis signal-regulating kinase 1 (ASK1) is a member of the MAP kinase-kinase-kinase family which responses to diverse array of stresses. Here, we show for the first time that ASK1, a 155 kDa protein, is present in human and murine platelets and its role as a novel regulator of platelet function. In human platelets, when stimulated with thrombin, ASK1 is rapidly activated within 30 sec, as indicated by phosphorylation of Thr 845 on its activation loop. Interestingly, we found that ASK1 is rapidly inactivated after 3 min by dephosphorylation of phospho-Thr 845. Concomitantly, ASK1 is phosphorylated on Ser 967, a known inhibitory phosphorylation site. The activation of ASK1 coincided well with its downstream effector, p38 Map kinase. These results suggested that ASK1 may play a role in platelet function. We therefore tested the hemostatic functions in congenic Ask-1 knockout mice and compared it to C57/BL6 wild type (Wt) mice. We found a significant delay in tail bleeding time (P=0.2×10⁻⁹) in Ask1 null mice. While all Wt mice stopped bleeding with an average bleeding time of 100 sec, the Ask1 null mice had an average bleeding time of 576 sec, with 5 out of 9 mice that did not stop bleeding. A 10% FeCl₃-induced carotid artery injury, a well-established in vivo thrombosis model, 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. This finding was further supported by the histological examination of the lung tissue of mice surviving after 3 min of injection, which showed very few thrombi in Ask1 null mice compared to Wt mice. We next asked if the platelet function is affected in Ask1 null mice. We found that low dose of PAR-4 peptide-induced aggregation is significantly less in Ask1 null mice compared to Wt mice. When analyzed for the ability of Ask1 null platelets to retract clots, we found that while clot retraction was completed within 2h in Wt platelets, Ask1 null platelets failed to retract clots even after 18h. When assessed for phosphorylation of the integrin b₃ subunit as an indicator of outside-in signaling, we found that Wt platelet exposure to immobilized fibrinogen showed a robust phosphorylation of b₃ subunit. Interestingly, Ask1 null platelets failed to show any induction of b₃ phosphorylation. These in vitro and in vivo results strongly suggest that ASK1 plays a significant role in the regulation of platelet function and hemostasis.