Abstract
Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-Kgamma. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-Kgamma, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC50 = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC50 = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-Kgamma. betaPMA or SFLLRN promotes the conversion of platelet integrin alphaIIb/beta3 into a fibrinogen-binding form required for platelet aggregation. Activation of alphaIIb/beta3 in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC50 of 1 nM in each case. Wortmannin inhibits neither activation of alphaIIb/beta3 by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of "outside-in" signaling by alphaIIb/beta3 is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-Kgamma, contributes to activation of alphaIIb/beta3 when the thrombin receptor or PKC is stimulated.
Highlights
Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-K␥
1 The abbreviations used are: PtdIns, phosphatidylinositol, PtdIns(3,4,5)P3 and PtdIns(3,4)P2; PI 3-K, phosphoinositide 3-kinase; p85, non-enzymatic subunit of heterodimeric PI 3-K; PI 3-K␥, non-p85-containing PI 3-K that is activated by G␥; G␥, ␥ subunits of heterotrimeric GTP-binding proteins; 3-PPI, 3-phosphorylated phosphoinositides; PtdOH, phosphatidic acid; Phospholipase C (PLC), phospholipase C; PKC, protein kinase C; PMA, phorbol myristate acetate; and PtdIns(3,4)P2 as a function of the stimulation of two phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-K␥, both of which are dependent upon GTP-binding proteins (1, 2)
Stimulated 3-PPI Accumulation in Platelets—SFLLRN, a thrombin receptor-directed agonist peptide, and PMA, a potent agonist for PKC, each stimulated the accumulation of 3-PPI in a dose- and time-dependent manner (Fig. 1, A-C), whereas ␣PMA, a phorbol ester that is ineffective in activating PKC, was without effect in this regard
Summary
Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-K␥. In this study we examined the relative susceptibility to inhibition by wortmannin of PMA- and thrombin receptor-stimulated 3-PPI production as well as the effects of wortmannin on p85/PI 3-K and PI 3-K␥ activities in platelets.
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