Platelets express a single class of Fc␥ receptor (Fc␥RIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. Fc␥RIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-␥2 (PLC-␥2), and calcium signaling. Here, we show that in response to Fc␥RIIA cross-linking, phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLC␥2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin ␣ IIb /␤ 3 engagement. In contrast, the stable association of PLC␥2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLC␥2 was able to preferentially interact in vitro with PtdIns(3,4,5)P 3 . Finally, exogenous PtdIns(3,4,5)P 3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P 3 play a key role in the activation and adequate location of PLC␥2 induced by Fc␥RIIA cross-linking.

Abstract. Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29 % of the p85o~ regulatory subunit of phosphoinositide 3-kinase (Ptdlns 3-kinase) and is accompanied by a significant increase in Ptdlns 3-kinase activity in this subcellular fraction. Actually, Ptdlns(3,4)P2 accumulation and Ptdlns 3-kinase, pp60 ~-s'~, and p125 eA~ translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of Ptdlns(3,4)P2 and the relocalization of Ptdlns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85o~ was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85a. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85et with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with Ptdlns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85tx with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125 rAK. In addition, we show that Ptdlns 3-kinase is significantly activated by the p125 rAK proline-rich sequence binding to the src homology 3 domain of p85a subunit. This interaction may represent a new mechanism for Ptdlns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of o~ro/~3 integrin and platelet aggregation evoked by thrombin.T HE accumulation of phosphatidylinositol 3,4-bisphosphate (Ptdlns(3,4)P2) m and Ptdlns(3,4,5)P3 in thrombin-stimulated human platelets is now well established (16,24,38,41) and seems to be regulated differently (38). However, the exact mechanism of activation of phosphoinositide 3-kinase (Ptdlns 3-kinase) in these ceils is still obscure.

Platelets homozygous for the Pl(A1) allele appear to be less sensitive to inhibitory action of low-dose aspirin. This differential sensitivity to aspirin may have potential clinical implications whereby specific antiplatelet therapy may be best tailored according to the patient's Pl(A) genotype.

From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.

Admission plasma glucose, even after adjustment on HbA(1c), is a prognostic factor associated with mortality after acute myocardial infarction. Acute rather than the chronic pre-existing glycometabolic state accounts for the prognosis after acute myocardial infarction.

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