Abstract
Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) 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 PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.
Highlights
In addition to specific interactions involving their Fab domains, immunoglobulins G can interact with various membrane receptors by their Fc region
PI 3-Kinase and phospholipase C (PLC) Are Rapidly Activated upon Fc␥RIIA Cross-linking—Cross-linking of platelet Fc␥RIIA has been shown to induce a transient association of PI 3-kinase to the ITAM sequences present in the cytoplasmic tail of the receptor; its consequences on a possible in vivo activation of the enzyme were not emphasized [23]
There is some evidence that Syk interacts with PI 3-kinase [23], which is recruited to the membrane and activated, leading to the rapid accumulation of PtdIns[3,4,5]P3. Another downstream effector of Syk has been identified as phospholipase C-␥2 (PLC-␥2), which is converted into an active form upon tyrosine phosphorylation, this might involve additional protein-tyrosine kinases such as Bruton’s tyrosine kinase (Btk), as shown for the B cell receptor (49 –51)
Summary
Fc␥RIIA, low affinity Fc␥ receptor IIA; ITAM, immunoreceptor tyrosine-based activation motif; Fc⑀R, high affinity IgE receptor; SH2, Src homology-2; PI 3-kinase, phosphoinositide. Calcium mobilization and activation of protein kinase C via the two second messengers produced upon hydrolysis of PtdIns[4,5]P2 [13, 14]. Besides the fact that Fc␥RIIA might play a key role in the hemostatic and inflammatory function of platelets, these cells are interesting to consider in so far they contain a single class of Fc␥ receptor (Fc␥RIIA), in contrast to neutrophils or monocytes, for instance [1, 3]. Clustering of platelet Fc␥RIIA promotes phosphorylation of the two tyrosine residues of the ITAM motif, which is followed by the classical set of signaling events occurring under similar conditions, i.e. various tyrosine phosphorylations and calcium mobilization, through activation of PLC␥2 (3, 20 –22). Our present data unravel a causal relationship between PI 3-kinase and PLC-␥2, which might function in the signaling cascade evoked by other membrane receptors involved in the immune response
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