Objective-ADP-induced P2y 12 signaling is crucial for formation and stabilization of an arterial thrombus. We demonstrated recently in platelets from healthy subjects that insulin interferes with Ca 2ϩ increases induced by ADP-P2y 1 contact through blockade of the G-protein G i , and thereby with P2y 12 -mediated suppression of cAMP. Methods and Results-Here we show in patients with type 2 diabetes mellitus (DM2) that platelets have lost responsiveness to insulin leading to increased adhesion, aggregation, and procoagulant activity on contact with collagen. Using Ser 473 phosphorylation of protein kinase B as output for insulin signaling, a 2-fold increase is found in insulin-stimulated normal platelets, but in DM platelets there is no significant response. In addition, DM2 platelets show increased P2y 12 -mediated suppression of cAMP and decreased P2y 12 inhibition by the receptor antagonist AR-C69931MX. Key Words: P2y 12 receptor Ⅲ Ca 2ϩ regulation Ⅲ clopidogrel Ⅲ protein kinase B/Akt Ⅲ IRS-1 P latelet activation leads to release of components that initiate formation of a thrombus and start inflammatory responses that contribute to atherosclerosis. 1 Signaling through the P2y 12 receptor is crucial for formation and stabilization of a thrombus. 2,3 Inhibition of the P2y 12 receptor reduces collagen-induced adhesion, aggregation and thrombin generation. 3,4 Subjects with a P2y 12 deficiency have a bleeding tendency 3,5 and individuals with an increased P2y 12 receptor copy number have platelets with an increased responsiveness to agonists, and these subjects experience peripheral arterial thrombosis. 6 The CAPRIE trial shows that long-term administration of the P2y 12 antagonist clopidogrel is more effective than aspirin in reducing the combined risk of ischemic stroke, myocardial infarction, or vascular death in subjects with a prothrombotic condition such as diabetes mellitus type 2 (DM2). 7 These findings illustrate the crucial role of P2y 12 signaling in platelet activation in vitro and in vivo. Conclusion-TheThe importance of P2y 12 signaling is explained by its capacity to initiate 2 pathways that directly interfere with platelet activating or inhibiting mechanisms. First, there is the activation of the G-protein subunit G i ␣, which inhibits adenylyl cyclase and thereby formation of the platelet inhibitor cAMP. 8 This property is particularly evident after treatment with prostacyclin, 9 and also in the absence of cAMP elevating agents, P2y 12 signaling controls cAMP production through adenylyl cyclase. 10,11 cAMP inhibits platelets through cAMPdependent protein kinase (protein kinase A [PKA]), 12 which inhibits almost all platelet functions through blockade of multiple steps in platelet activation cascades including receptor activation, signaling through the mitogen-activated protein kinases pathway, formation of thromboxane A 2 (TxA 2 ), and the activation of key enzymes such as phospholipase C ␤ and protein kinase C (PKC). 13 Second, there is the release of the G i ␤␥ dimer leading to the activation ...

Patients with diabetes mellitus have a 2-4-fold increased risk for coronary artery disease. They suffer from both microvascular (nephropathy and retinopathy) and macrovascular (peripheral artery disease) complications (1). Apart from increased concentrations of certain coagulation factors (2), patients with diabetes mellitus type I and II have platelets that show increased adhesion, aggregation, thromboxane production, and P-selectin expression (3). The hyperactivity might be caused by the absence of insulin inhibition, since intensive insulin treatment in diabetic patients reduced platelet aggregation (4).The insulin receptor is a heterotetrameric transmembrane glycoprotein composed of two extracellular ␣ subunits (135 kDa each) and two transmembrane ␤ subunits (95 kDa each) that function as allosteric enzymes in which the ␣ subunit inhibits the tyrosine kinase activity of the ␤ subunit. Insulin binding to the ␣ subunit relieves the inhibition of the kinase activity in the ␤ subunit leading to autophosphorylation of the ␤ subunits and a conformational change that further increases the kinase activity. The insulin receptor tyrosine kinase phosphorylates proteins such as Shc and the insulin receptor substrates IRS-1 (165-185 kDa) and . IRS-1 and IRS-2 have a highly conserved amino terminus, which contains a pleckstrin homology domain, a phosphotyrosine binding domain, and a carboxyl terminus with several tyrosine phosphorylation sites. IRS-1 and IRS-2 are complementary and act as "docking sites" to several Src homology 2 domains containing proteins, such as the regulatory subunits of phosphatidylinositol 3-kinase (PI3K) 1 (5). GTP-binding proteins (G-proteins) can also act as signal transducers for the insulin receptor. G-proteins are guanine nucleotide-binding regulatory proteins that function as molecular switches between a GTP-bound "on state" and a GDPbound "off state." These proteins amplify, transmit, and integrate signals. The major G-proteins involved in platelet aggregation and secretion are G q ␣, which mediates increases in cytosolic Ca 2ϩ concentration, [Ca 2ϩ ] i , and G i ␣, which inhibits adenylyl cyclase thereby suppressing cAMP that is an inhibitor of platelets (6). Receptors that couple to G-proteins are generally seven-transmembrane proteins, but there are important exceptions. The insulin-like growth factor II receptor has a single transmembrane domain and couples directly to G i2 in a manner similar to that of conventional G-protein-coupled receptors (7). Studies have been reported suggesting that the insulin receptor binds G i ␣ 2 (8, 9).The insulin receptor is present on muscle, liver, and adipose tissue but also on endothelial cells, lymphocytes, erythrocytes, and platelets. A human platelet contains ϳ570 insulin receptors (10). Insulin binding induces phosphorylation of the ␤ subunits (11,12), demonstrating that the receptor is func-

In insulin-responsive tissues, insulin is a potent activator of protein kinase B (PKB)-mediated glucose uptake through the facilitative glucose transporter GLUT4. In platelets, glucose uptake is mediated through GLUT3, which is present in plasma (15%) and intracellular alpha-granule (85%) membranes. Here we report the PKB-mediated glucose uptake by platelets by agents that do (thrombin) or do not (insulin) induce alpha-granule translocation to the plasma membrane. Both thrombin and insulin activate PKB and induce glucose uptake albeit with different kinetics. Inhibition of PKB by the pharmacological inhibitor ML-9 decreases thrombin-induced alpha-granule release and thrombin- and insulin-induced glucose uptake. At low glucose (0.1 mm), both agents stimulate glucose uptake by lowering the Km for glucose (thrombin and insulin) and increasing Vmax (thrombin). At high glucose (5 mm), stimulation of glucose uptake by insulin disappears, and insulin becomes an inhibitor of thrombin-induced glucose uptake via mechanisms independent of PKB. We conclude that in platelets glucose transport through GLUT3 is regulated by changes in surface expression and affinity modulation, which are both under control of PKB.