Ask1 regulates murine platelet granule secretion, thromboxane A2 generation, and thrombus formation.

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 αIIbß3 activation 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 TxA2 generation. 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 cPLA2 in 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 Ask1 also 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.

Deposition of fibrinogen on the surface of in vitro thrombi prevents platelet adhesion.

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion.

Integrin αIIbß3: a novel effector of Gα13.

Under physiological conditions, circulating platelets are discoid in shape. On these platelets, the fibrinogen receptor (integrin αIIbß3) is in a low-affinity state, unable to bind soluble fibrinogen (Fg). Activation by agonists such as ADP and thrombin leads to a change in the conformation of the integrin αIIbß3 through a process known as inside-out signaling. This enables the integrin to bind soluble Fg, which initiates a cascade of events referred to as outside-in signaling. Outside-in signaling control processes such as platelet spreading and clot retraction by regulating small G-proteins such as RhoA, Rac and cdc42.