High concentrations of adenosine-5P P-diphosphate ADP are able to induce partial aggregation without shape change of P2Y 1 receptor-deficient mouse platelets through activation of the P2Y 12 receptor. In the present work we studied the transduction pathways selectively involved in this phenomenon. Flow cytometric analyses using R-phycoerythrin-conjugated JON/A antibody (JON/A-PE), an antibody which recognizesAdrenaline induced no such activation but strongly potentiated the effect of ADP in a dose-dependent manner. Global phosphorylation of 32 P-labeled platelets showed that P2Y 12 -mediated aggregation was not accompanied by an increase in the phosphorylation of myosin light chain (P 20 ) or pleckstrin (P 47 ) and was not affected by the protein kinase C (PKC) inhibitor staurosporine. On the other hand, two unrelated phosphoinositide 3-kinase inhibitors, wortmannin and LY294002, inhibited this aggregation. Our results indicate that (i) the P2Y 12 receptor is able to trigger a P2Y 1 receptor-independent inside-out signal leading to K K IIb L L 3 integrin activation and platelet aggregation, (ii) ADP and adrenaline use different signaling pathways which synergize to activate the K K IIb L L 3 integrin, and (iii) the transduction pathway triggered by the P2Y 12 receptor is independent of PKC but dependent on phosphoinositide 3-kinase. ß
NTPDase1 is the major enzyme regulating nucleotide metabolism at the surface of VSMCs and thus contributes to the local regulation of vascular tone by nucleotides.
Phosphoinositide (PI) 3-kinases play an important role in regulating the adhesive function of a variety of cell types through affinity modulation of integrins. Two type I PI 3-kinase isoforms (p110 and p110␥) have been implicated in G i -dependent integrin ␣ IIb  3 regulation in platelets, however, the mechanisms by which they coordinate their signaling function remains unknown. By employing isoform-selective PI 3-kinase inhibitors and knock-out mouse models we have identified a unique mechanism of PI 3-kinase signaling co-operativity in platelets. We demonstrate that p110 is primarily responsible for G i -dependent phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ) production in ADP-stimulated platelets and is linked to the activation of Rap1b and AKT. In contrast, defective integrin ␣ IIb  3 activation in p110␥ ؊/؊ platelets was not associated with alterations in the levels of PI(3,4)P 2 or active Rap1b/AKT. Analysis of the effects of active site pharmacological inhibitors confirmed that p110␥ principally regulated integrin ␣ IIb  3 activation through a non-catalytic signaling mechanism. Inhibition of the kinase function of PI 3-kinases, combined with deletion of p110␥, led to a major reduction in integrin ␣ IIb  3 activation, resulting in a profound defect in platelet aggregation, hemostatic plug formation, and arterial thrombosis. These studies demonstrate a kinaseindependent signaling function for p110␥ in platelets. Moreover, they demonstrate that the combined catalytic and non-catalytic signaling function of p110 and p110␥ is critical for P2Y 12 /G i -dependent integrin ␣ IIb  3 regulation. These findings have potentially important implications for the rationale design of novel antiplatelet therapies targeting PI 3-kinase signaling pathways.The phosphoinositide (PI) 2 3-kinases are a well defined family of lipid kinases that participate in a broad range of signaling processes downstream of growth factor, antigen, hormone, and adhesion receptors (1, 2). They are classified into several distinct groups (types I-III), based on their primary structure, mode of regulation, and substrate specificity (3, 4). The most intensely studied members of the PI 3-kinase superfamily are the type I PI 3-kinases, due to their involvement in the regulation of fundamental cell processes, including proliferation, glucose metabolism, survival, and migration (1). PI 3-kinases principally transduce signals through the catalytic generation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P 3 ) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P 2 ); second messengers that facilitate the recruitment of pleckstrin homology domain-containing signaling proteins to the plasma membrane (5). The type I enzymes are divided into two subtypes, Ia and Ib; type Ia isoforms include p110␣, -, and -␦ and type Ib includes a single isoform p110␥. p110␣, -, and -␦ share common regulatory subunits (p85␣, p85, p55␣, p55␥, p50␣) and are classically regulated by tyrosine kinase-linked receptors, although G protein-coupled receptor-medi...
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