Integrins are critical in thrombosis and hemostasis1. Antagonists of the platelet integrin αIIbβ3 are potent anti-thrombotic drugs, but also have the life-threatening adverse effect of bleeding2,3. It is thus desirable to develop new antagonists that do not cause bleeding. Integrins transmit signals bidirectionally4,5. Inside-out signaling activates integrins via a talin-dependent mechanism6,7. Integrin ligation mediates thrombus formation and outside-in signaling8,9, which requires Gα13 and greatly expands thrombi. Here we show that Gα13 and talin bind to mutually exclusive, but distinct sites within the integrin β3 cytoplasmic domain in opposing waves. The first talin binding wave mediates inside-out signaling and also “ligand-induced integrin activation”, but is not required for outside-in signaling. Integrin ligation induces transient talin dissociation and Gα13 binding to an ExE motif, which selectively mediates outside-in signaling and platelet spreading. The second talin binding wave is associated with clot retraction. An ExE motif-based inhibitor of Gα13-integrin interaction selectively abolishes outside-in signaling without affecting integrin ligation, and suppresses occlusive arterial thrombosis without affecting bleeding time. Thus, we have discovered a novel mechanism for the directional switch of integrin signaling and, based on this mechanism, we designed a potent new anti-thrombotic that does not cause bleeding.

Objective Reactive oxygen species (ROS) are known to regulate platelet activation; however, the mechanisms of ROS production during platelet activation remain unclear. Platelets express different isoforms of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) oxidases (NOXs). Here we investigated the role of NOX1 and NOX2 in ROS generation and platelet activation using NOX1 and NOX2 knockout mice. Approach and Results NOX1−/Y platelets showed selective defects in G protein-coupled receptor (GPCR)-mediated platelet activation induced by thrombin and thromboxane A2 analog U46619, but were not affected in platelet activation induced by collagen-related peptide (CRP), a glycoprotein VI (GPVI) agonist. In contrast, NOX2−/− platelets showed potent inhibition of CRP-induced platelet activation, and also showed partial inhibition of thrombin-induced platelet activation. Consistently, production of ROS was inhibited in NOX1−/Y platelets stimulated with thrombin, but not CRP, whereas NOX2−/− platelets showed reduced ROS generation induced by CRP or thrombin. Reduced ROS generation in NOX1/2 deficient platelets is associated with impaired activation of Syk and phospholipase Cγ2 (PLCγ2), but minimally affected mitogen-activated protein kinase pathways. Interestingly, laser-induced arterial thrombosis was impaired but the bleeding time was not affected in NOX2−/− mice. WT thrombocytopenic mice injected with NOX2−/− platelets also showed defective arterial thrombosis, suggesting an important role for platelet NOX2 in thrombosis in vivo but not hemostasis. Conclusions NOX1 and NOX2 play differential roles in different platelet activation pathways and in thrombosis. ROS generated by these enzymes promotes platelet activation via the Syk/PLCγ/calcium signaling pathway.

The Akt family of serine/threonine kinases includes Akt1, Akt2, and Akt3 isoforms. Prior studies have reported that Akt1 and Akt2, but not Akt3, are expressed in platelets. Here, we show that Akt3 is expressed in substantial amounts in platelets. Akt3 ؊/؊ mouse platelets selectively exhibit impaired platelet aggregation and secretion in response to low concentrations of thrombin receptor agonists and thromboxane A 2 (TXA 2 ), but not collagen or VWF. In contrast, platelets from Akt1 ؊/؊ or Akt2 ؊/؊ mice are defective in platelet activation induced by thrombin, TXA 2 , and VWF, but only Akt1 ؊/؊ platelets show significant defects in response to collagen, indicating differences among Akt isoforms. Akt3 ؊/؊ platelets exhibit a significant reduction in thrombin-induced phosphorylation of glycogen synthase kinase 3␤ (GSK-3␤) at Ser9, which is known to inhibit GSK-3␤ function. Thus, Akt3 is important in inhibiting GSK-3␤. Accordingly, treatment of Akt3 ؊/؊ platelets with a GSK-3␤ inhibitor rescued the defect of Akt3 ؊/؊ platelets in thrombin-induced aggregation, suggesting that negatively regulating GSK-3␤ may be a mechanism by which Akt3 promotes platelet activation. Importantly, Akt3 ؊/؊ mice showed retardation in FeCl 3 -induced carotid artery thrombosis in vivo. IntroductionPlatelets are critical for hemostasis, but under pathologic conditions, are also important in thrombosis. 1 Platelet activation is initiated at sites of vascular injury on exposure to soluble agonists such as thrombin, ADP, and thromboxane A 2 , and adhesion to subendothelial matrix proteins, such as von Willebrand factor and collagen. 2 These adhesive proteins and agonists stimulate an intracellular signal transduction cascade leading to transformation of the major platelet adhesion receptor, integrin ␣ IIb ␤ 3 from its resting to active state (inside-out signaling), which allows the integrin to bind fibrinogen, and therefore mediate platelet aggregation. 3,4 Activated platelets secrete proaggregating factors and adhesive glycoproteins from granules, which further cause stabilization and amplification of aggregation, leading to thrombus formation. Ligand binding to the activated integrin ␣ IIb ␤ 3 also transmits "outside-in" signals, which are critically important in stable platelet adhesion, spreading, and clot retraction. 3,5,6 Elucidation of the signaling pathways regulating platelet activation is essential for the identification of novel anti-thrombotic targets for the prevention of thrombosis, a major cause of heart attack and stroke. It is established that phosphoinositide 3-kinases (PI3K) play important roles in platelet activation. [7][8][9][10][11][12] Akt (also known as Protein Kinase B or PKB), the most well known effector of PI3K, is activated downstream of PI3K during platelet activation. 9,13,14 Akt is a family of serine/threonine kinases with 3 isoforms: Akt1, Akt2, and Akt3 (for reviews see Manning et al 15 and Bhaskar et al 16 ). Akt isoforms are 80% homologous in their protein sequences. However, knockout mouse models of Akt...

Key Points GPIb-IX signaling cooperates with PAR signaling to promote platelet response to low concentrations of thrombin, which are important in vivo. Thrombin induces a GPIb-IX–specific signaling pathway that requires the cytoplasmic domain of GPIbα, 14-3-3 protein, Rac1, and LIMK1.

It is currently unclear why agonist-stimulated platelets require shear force to efficiently externalize the procoagulant phospholipid phosphatidylserine (PS) and release PS-exposed microvesicles (MVs). We reveal that integrin outside-in signaling is an important mechanism for this requirement. PS exposure and MV release were inhibited in β platelets or by integrin antagonists. The impaired MV release and PS exposure in β platelets were rescued by expression of wild-type β but not a Gα binding-deficient β mutant (EEE to AAA), which blocks outside-in signaling but not ligand binding. Inhibition of Gα or Src also diminished agonist/shear-dependent PS exposure and MV release, further indicating a role for integrin outside-in signaling. PS exposure in activated platelets was induced by application of pulling force via an integrin ligand, which was abolished by inhibiting Gα-integrin interaction, suggesting that Gα-dependent transmission of mechanical signals by integrins induces PS exposure. Inhibition of Gα delayed coagulation in vitro. Furthermore, inhibition or platelet-specific knockout of Gα diminished laser-induced intravascular fibrin formation in arterioles in vivo. Thus, β integrins serve as a shear sensor activating the Gα-dependent outside-in signaling pathway to facilitate platelet procoagulant function. Pharmacological targeting of Gα-integrin interaction prevents occlusive thrombosis in vivo by inhibiting both coagulation and platelet thrombus formation.