Rapid and transient thrombin stimulation of phosphatidylinositol 4,5-bisphosphate synthesis but not of phosphatidylinositol 3,4-bisphosphate independent of phospholipase C activation in platelets

When platelets are stimulated by thrombin they immediately undergo inositol lipid hydrolysis via phospholipase C activation. However, subsequently an increased production of phosphatidylinositol 4,5-bisphosphate is observed. Phospholipases C were inhibited by lowering the cytoplasmic free calcium concentration by preincubation with Quin-2-tetra(acetoxymethyl) ester. Aggregation and secretion were also totally suppressed. Under these conditions we observed an increased labeling of phosphatidylinositol 4,5-bisphosphate, indicating a stimulation of inositol lipid kinases, independent of lipid hydrolysis by phospholipase C. Conversely the production of phosphatidylinositol 3,4-bisphosphate was totally abolished. These results suggest a different regulation of the kinases/phosphatases responsible for the production of phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate.     

Protein kinase C-mediated formation of phosphatidylinositol 3,4-bisphosphate in human platelets

The effect of phorbol 12,13-dibutyrate on the formation of phosphatidylinositol 3,4-bisphosphate in washed human platelets was studied. Platelets labelled with [32P]Pi were stimulated with phorbol 12,13-dibutyrate or thrombin in the presence or absence of staurosporine. Lipids were extracted, and deacylated, and the glycerophosphoinositol derivatives were analyzed by high performance liquid chromatography. Phorbol 12,13-dibutyrate increased formation of phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4-bisphosphate in a dose- and time-dependent manner. Thrombin also increased formation of phosphatidylinositol 3,4-bisphosphate. Staurosporine completely inhibited phorbol 12,13-dibutyrate or thrombin-stimulated production of phosphatidylinositol 3,4-bisphosphate. These data indicate that production of phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 4-monophosphate is mediated by protein kinase C. It is widely recognized that production of phosphatidylinositol 3,4-bisphosphate is caused by the tyrosine kinase-mediated activation of phosphatidylinositol 3-kinase. However, in platelets, production of phosphatidylinositol 3,4-bisphosphate might be related to stimulation of phosphatidylinositol 4-kinase, which is activated by protein kinase C.     

Inositol 1,3,4,5-tetrakisphosphate and not phosphatidylinositol 3,4-bisphosphate is the probable precursor of inositol 1,3,4-trisphosphate in agonist-stimulated parotid gland.

When [3H]inositol-prelabelled rat parotid-gland slices were stimulated with carbachol, noradrenaline or Substance P, the major inositol trisphosphate produced with prolonged exposure to agonists was, in each case, inositol 1,3,4-trisphosphate. Much lower amounts of radioactivity were present in the inositol 1,4,5-trisphosphate fraction separated by anion-exchange h.p.l.c. Analysis of the inositol trisphosphate head group of phosphatidylinositol bisphosphate in [32P]Pi-labelled parotid glands showed the presence of phosphatidylinositol 4,5-bisphosphate, but no detectable phosphatidylinositol 3,4-bisphosphate. Carbachol-stimulated [3H]inositol-labelled parotid glands contained an inositol polyphosphate with the chromatographic properties and electrophoretic mobility of an inositol tetrakisphosphate, the probable structure of which was determined to be inositol 1,3,4,5-tetrakisphosphate. Since an enzyme in erythrocyte membranes is capable of degrading this tetrakisphosphate to inositol 1,3,4-trisphosphate, it is suggested to be the precursor of inositol 1,3,4-trisphosphate in parotid glands.     

Direct activation of the epithelial Na(+) channel by phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate produced by phosphoinositide 3-OH kinase

The phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) is accepted to be a direct modulator of ion channel activity. The products of phosphoinositide 3-OH kinase (PI3K), PtdIns(3,4)P(2) and phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), in contrast, are not. We report here activation of the epithelial Na(+) channel (ENaC) reconstituted in Chinese hamster ovary cells by PI3K. Insulin-like growth factor-I also activated reconstituted ENaC and increased Na(+) reabsorption across renal A6 epithelial cell monolayers via PI3K. Neither IGF-I nor PI3K affected the levels of ENaC in the plasma membrane. The effects of PI3K and IGF-I on ENaC activity paralleled changes in the plasma membrane levels of the PI3K product phospholipids, PtdIns(3,4)P(2)/PtdIns(3,4,5)P(3), as measured by evanescent field fluorescence microscopy. Both PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) activated ENaC in excised patches. Activation of ENaC by PI3K and its phospholipid products corresponded to changes in channel open probability. We conclude that PI3K directly modulates ENaC activity via PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3). This represents a novel transduction pathway whereby growth factors, such as IGF-I, rapidly modulate target proteins independent of signaling elicited by kinases downstream of PI3K.     

Calpain I activation is not correlated with aggregation in human platelets.

Calpain-catalysed hydrolysis of platelet substrates such as cytoskeletal and calmodulin-binding proteins, and of protein kinase C, is assumed to contribute to platelet aggregation. We have measured calpain I activation by immunoblotting, and [Ca2+]i (cytoplasmic Ca2+ concn.) by fura-2 fluorescence, in parallel with measurement of aggregation, in stirred human platelets treated at different [Ca2+]ext (extend Ca2+ concns.) with A23187, leupeptin, phorbol ester and thrombin. Hydrolysis of actin-binding protein, and [3H]5-hydroxytryptamine release, were also measured in some cases. A rise in [Ca2+]i, platelet aggregation and calpain activation often occurred together. With some combinations of agonists and [Ca2+]ext, however, this correlation was clearly not maintained. It was shown: (a) that activation of calpain and its hydrolysis of platelet substrates were not strictly necessary conditions for platelet secretion and aggregation; (b) conversely, that calpain activation could occur without aggregation.     

Protein kinase C phosphorylation of syntaxin 4 in thrombin-activated human platelets

We postulated that the syntaxins, because of their key role in SNARE complex formation and exocytosis, could be important targets for signaling by intracellular kinases involved in secretion. We found that syntaxin 4 was phosphorylated in human platelets treated with a physiologic agent that induces secretion (thrombin) but not when they were treated with an agent that prevents secretion (prostacyclin). Syntaxin 4 phosphorylation was blocked by inhibitors of activated protein kinase C (PKC), and, in parallel assays, PKC inhibitors also blocked secretion from thrombin-activated platelets. In platelets, cellular activation by thrombin or phorbol 12-myristate 13-acetate decreased the binding of syntaxin 4 with SNAP-23, another platelet t-SNARE. Phosphatase inhibitors increased syntaxin 4 phosphorylation and further decreased syntaxin 4-SNAP-23 binding induced by cell activation. Conversely, a PKC inhibitor blocked syntaxin 4 phosphorylation and returned binding of syntaxin 4-SNAP-23 to that seen in nonstimulated platelets. In vitro, PKC directly phosphorylated platelet syntaxin 4 and recombinant syntaxin 4. PKC phosphorylation in vitro inhibited (71 +/- 8%) the binding of syntaxin 4 to SNAP-23. These results provide evidence that extracellular activation can be coupled through intracellular PKC signaling so as to modulate SNARE protein interactions involved in platelet exocytosis.     

Thrombin stimulates the production of phosphatidylinositol 3,4-bisphosphate in human erythroleukemia cells.

The human erythroleukemic cell line, HEL, which has numerous platelet markers, shows enhanced inositol phosphate production in response to thrombin. We investigated the production of phosphoinositides in HEL cells and showed that thrombin stimulates the turnover of several phosphoinositides including the synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). Phosphatidylinositol 3-monophosphate is also produced in HEL cells and its synthesis is not stimulated by thrombin. Pretreatment of HEL cells with the stable prostacyclin analog iloprost inhibits the thrombin-induced increase in the production of PtdIns(3,4)P2. 3-Phosphorylated phosphoinositides have been implicated in signal transduction and regulation of cell proliferation in other cells and may be involved in signal transduction in HEL cells.     

Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils

We recently showed that phosphatidylinositol trisphosphate (PIP3) was present in a unique lipid fraction generated in neutrophils during activation. Here, we demonstrate that the band containing this fraction isolated from thin layer chromatography consists primarily of PIP3 and that only small amounts of radiolabeled PIP3 exist prior to activation. In addition, high performance liquid chromatography of deacylated phospholipids from stimulated cells reveals an increase in a fraction eluting ahead of glycerophosphoinositol 4,5-P2. After removal of the glycerol we found that it coeluted with inositol 1,3,4-P3 when resubjected to high performance liquid chromatography. Thus, we have detected a second, novel form of phosphatidylinositol bisphosphate in activated neutrophils, PI-(3,4)P2. The elevation of PIP3 through the formyl peptide receptor is blocked by pretreatment with pertussis toxin, implicating mediation of the increase in PIP3 by a guanosine triphosphate-binding (G) protein. The rise in PIP3 is not secondary to calcium elevation. Buffering the rise in intracellular calcium did not diminish the increase in PIP3. The elevation of PIP3 appears to occur during activation with physiological agonists, its level varying with the degree of activation. Leukotriene B4, which elicits many of the same responses as stimulation of the formyl peptide receptor but with minimal oxidant production, stimulates a much attenuated rise in PIP3. Isoproterenol, which inhibits oxidant production also reduces the rise in PIP3. Hence formation of PI(3,4)P2 and PIP3 (presumed to be PI(3,4,5)P3) correlates closely with the early events of neutrophil activation.     

Platelet-derived growth factor-mediated Ca2+ entry is blocked by antibodies to phosphatidylinositol 4,5-bisphosphate but does not involve heparin-sensitive inositol 1,4,5-trisphosphate receptors.

Elevation of intracellular Ca2+ by platelet-derived growth factor (PDGF) and other growth factors involves both release of Ca2+ from intracellular Ca2+ stores and Ca2+ entry from the extracellular medium. Release from intracellular stores is believed to be mediated by inositol 1,4,5-trisphosphate (IP3) and the heparin-sensitive IP3 receptor. We studied the mechanism by which entry of extracellular Ca2+ is induced by PDGF. Intracellular free Ca2+ (Ca2+i) was measured in single cultured rat vascular smooth muscle cells using fura 2 microspectrofluorometry. In nominally Ca2(+)-free medium, PDGF (recombinant BB, 10 ng/ml) raised intracellular Ca2+ transiently (less than 5 min); addition of 2 mM Ca2+ to the bathing medium after 5 min caused a second, prolonged increase in intracellular Ca2+. Repeated changes in extracellular Ca2+ from 0 to 2 mM over 90 min caused rapid, parallel changes in Ca2+i of approximately 200 nM. This change in Ca2+i in response to changes in extracellular Ca2+ was virtually undetectable in control or thrombin-treated cells. The intracellular response to changes in medium Ca2+ after PDGF was completely blocked by 10 mM CoCl2, but not by 10(-7) M nicardipine. Microinjection of monoclonal antibodies to phosphatidylinositol 4,5-bisphosphate (PIP2) (kt 10, 2 mg/ml) totally abolished both mobilization of intracellular Ca2+ stores and entry of extracellular Ca2+. Consistent with this finding, maintenance of Ca2+ entry required ongoing receptor occupancy, since displacement of PDGF from its receptor with suramin (1 mM) eradicated extracellular Ca2+ entry in less than 5 min. To determine whether extracellular Ca2+ entry involves the heparin-sensitive IP3 receptor, cells were microinjected with heparin (4 mg/ml) prior to addition of PDGF. Heparin, but not chondroitin sulfate, prevented mobilization of intracellular Ca2+ stores but did not affect extracellular Ca2+ entry. We PDGF requires ongoing receptor occupancy and involves PIP2 or PIP2 metabolism. However, the signal which mediates PDGF-induced Ca2+ entry does not require the heparin-sensitive IP3 receptor.     

Inositol 1,2-cyclic 4,5-trisphosphate is not a product of muscarinic receptor-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis in rat parotid glands.

We have employed a neutral-pH extraction technique to look for inositol 1,2-cyclic phosphate derivatives in [3H]inositol-labelled parotid gland slices stimulated with carbachol. The incubations were terminated by adding cold chloroform/methanol (1:2, v/v), the samples were dried under vacuum and inositol phosphates were extracted from the dried residues by phenol/chloroform/water partitioning. Water-soluble inositol metabolites were separated by h.p.l.c. at pH 3.7. 32P-labelled inositol phosphate standards (inositol 1-phosphate, inositol 1,2-cyclic phosphate, inositol 1,4,5-trisphosphate and inositol 1,2-cyclic 4,5-trisphosphate) were quantitively recovered through both extraction and chromatography steps. Treatment of inositol cyclic phosphate standards with 5% (w/v) HClO4 for 10 min prior to chromatography resulted in formation of the expected non-cyclic compounds. [3H]Inositol 1-phosphate and [3H]inositol 1,4,5-trisphosphate were both present in parotid gland slices and both increased during stimulation with 1 mM-carbachol. There was no evidence for significant quantities of [3H]inositol 1,2-cyclic phosphate or [3H]inositol 1,2-cyclic 4,5-trisphosphate in control or carbachol-stimulated glands. Parotid gland homogenates rapidly converted inositol 1,4,5-trisphosphate to inositol bisphosphate and inositol tetrakisphosphate, but metabolism of the inositol cyclic trisphosphate was much slower. The results suggest that inositol 1,4,5-trisphosphate, but not inositol 1,2-cyclic 4,5-trisphosphate, is the water-soluble product of muscarinic receptor-stimulated phospholipase C in rat parotid glands.     

Diglyceride/monoglyceride lipases pathway is not essential for arachidonate release in thrombin-activated human platelets

Human platelets prelabeled with arachidonate exhibited a rapid and transient rise in arachidonoyl monoglyceride in addition to arachidonoyl diglyceride following thrombin stimulation. Substantial release of arachidonate and its metabolites also occurred at the early phase. Preincubation of labeled platelets with RHC 80267, a potent inhibitor of diglyceride lipase, prior to thrombin stimulation abolished the transient rise in monoglyceride but not the increase in diglyceride and the release of arachidonate and its metabolites. These results suggest that diglyceride does metabolize to monoglyceride and release arachidonate in intact platelets. However, the diglyceride/monoglyceride lipases pathway does not appear to be essential in releasing arachidonate during thrombin stimulation.     

The decrease in phosphatidylinositol 4,5-bisphosphate in ADP-stimulated washed rabbit platelets is not primarily due to phospholipase C activation.

Addition of 10 micron-ADP to washed rabbit platelets caused platelet shape change and aggregation without release of the contents of the amine-storage granules, and caused a transient decrease (8.8% at 10 s) in the amount of phosphatidylinositol 4,5-bisphosphate (PIP2). By 20 s the decrease in PIP2 was no longer apparent, but by 60 s the amount of PIP2 was again decreased. Addition of thrombin (1 unit/ml), which causes platelet shape change, aggregation and the release of the contents of the amine-storage granules, caused a decrease in the amount of PIP2 (8.0% at 10 s); at 60 s the amount of PIP2 was not significantly different from that in controls. In platelets prelabelled with [3H]glycerol, the specific radioactivity of PIP2 was increased at 10 s in ADP-stimulated platelets, and unchanged in thrombin-stimulated platelets. In platelets prelabelled with [3H]inositol and incubated with 20 mM-Li+ to inhibit the degradation of the inositol phosphates to inositol, there was no increase in the labelling of inositol trisphosphate (IP3) upon stimulation with ADP. In contrast, stimulation with thrombin caused a significant increase in the labelling of IP3 at 10 s. These differences in the changes in polyphosphoinositide metabolism in ADP- and thrombin-stimulated platelets are consistent with the hypothesis that the decrease in PIP2 in ADP-stimulated platelets may be due not to degradation of PIP2 by phospholipase C, but rather to a shift in the equilibrium between PIP2 and phosphatidylinositol 4-phosphate (PIP). Increases in the labelling of phosphatidic acid at 10 s and of inositol bisphosphate and inositol phosphate after 20 s are consistent with phospholipase C being stimulated through some other mechanism that leads to the degradation of PIP and phosphatidylinositol; one possibility is that ADP causes an increase in cytoplasmic Ca2+.     

Elevation of cAMP, but not cGMP, inhibits thrombin-stimulated tyrosine phosphorylation in human platelets.

Platelets have abundant tyrosine kinase activities, and activation of platelets results in the increased tyrosine phosphorylation of numerous protein substrates. The stimulation of tyrosine phosphorylation elicited by thrombin can be completely inhibited by preincubation with 10nm prostacyclin (PGI2), 1 microM PGD2, or 1mM N2,2'-O-dibutyryl-cAMP. In contrast, incubation of platelets with agents that increase cGMP (sodium nitroprusside or with 1mM 8-Bromo-cGMP) was without effect. The inhibition by prostacyclin was dose dependent, with an IC50 of approximately 3nM, corresponding to the dose range necessary to inhibit other platelet activation processes. These results demonstrate a novel pathway by which agents which raise cAMP may inhibit platelet signal transduction and differential mechanism of action between compounds which raise cAMP and those which elevate cGMP.     

Receptor stimulated accumulation of phosphatidylinositol (3,4,5)-trisphosphate by G-protein mediated pathways in human myeloid derived cells.

Phosphoinositide 30H-kinase (PI3K) activities are thought to be critical regulatory enzymes in a new intracellular signalling pathway, the activation of which results in the rapid accumulation of a putative signalling molecule, phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5) P3]. To date, activation of PI3K has always correlated with its recruitment into complexes containing protein tyrosine kinases (PTK). Here we report that agonists which utilize G-protein mediated transduction pathways can stimulate very rapid and large accumulations of PtdIns(3,4,5)P3 via a novel mechanism, possibly involving direct coupling between the G-protein and a PI3K activity. In addition, some of these agonists also stimulate small increases in PI3K activity in anti-phosphotyrosine and anti-src-type PTK antibody directed immunoprecipitates, indicating activation of PI3K via a 'conventional' PTK mediated mechanism; these pathways however, play only a minor role in the initial, agonist sensitive production of PtdIns(3,4,5)P3 in myeloid derived cells.     

Dissociation of Ca2+ mobilization from breakdown of phosphatidylinositol 4,5-bisphosphate in activated human platelets

The early breakdown of phosphatidylinositol 4,5-bisphosphate in human platelets stimulated by a threshold concentration of either collagen or thrombin was inhibited by 5 mM NaF through its inhibition of phospholipase C activity. However, 5 mM NaF did not inhibit Ca2+ mobilization due to the stimuli from internal stores, but it did inhibit the influx of extracellular Ca2+ through its suppression of thromboxane A2 formation.     

A membrane-permeant ester of phosphatidylinositol 3,4, 5-trisphosphate (PIP(3)) is an activator of human neutrophil migration

Activity of phosphatidylinositol (PI) 3-kinase is required for optimal migration of human neutrophils [Niggli and Keller (1999) Eur. J. Pharmacol. 335, 43-52]. We have tested the direct effect of a product of PI 3-kinase, phosphatidylinositol 3,4,5-trisphosphate (PIP(3)), on neutrophil migration. To this end, a membrane-permeant ester of PIP(3), dilauroyl phosphatidylinositol 3,4, 5-trisphosphate-heptakis-(acetooxymethyl)ester (PIP(3)/AM) was used. PIP(3)/AM (ED(50): 10-17 microM) induced development of polarity and accumulation of F-actin in the leading lamellae in up to 70% of the cells. These cells exhibited stimulated random migration, comparable to that observed in uniform concentrations of chemotactic peptide. Evidence is provided for a role of Rho-kinase and for activation of PI 3-kinase in a positive feedback loop in PIP(3)/AM-induced motility.     

Rapid accumulation of phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate correlates with calcium mobilization in salt-stressed arabidopsis

The phosphoinositide phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is a key signaling molecule in animal cells. It can be hydrolyzed to release 1,2-diacyglycerol and inositol 1,4,5-trisphosphate (IP(3)), which in animal cells lead to protein kinase C activation and cellular calcium mobilization, respectively. In addition to its critical roles in constitutive and regulated secretion of proteins, PtdIns(4,5)P(2) binds to proteins that modify cytoskeletal architecture and phospholipid constituents. Herein, we report that Arabidopsis plants grown in liquid media rapidly increase PtdIns(4,5)P(2) synthesis in response to treatment with sodium chloride, potassium chloride, and sorbitol. These results demonstrate that when challenged with salinity and osmotic stress, terrestrial plants respond differently than algae, yeasts, and animal cells that accumulate different species of phosphoinositides. We also show data demonstrating that whole-plant IP(3) levels increase significantly within 1 min of stress initiation, and that IP(3) levels continue to increase for more than 30 min during stress application. Furthermore, using the calcium indicators Fura-2 and Fluo-3 we show that root intracellular calcium concentrations increase in response to stress treatments. Taken together, these results suggest that in response to salt and osmotic stress, Arabidopsis uses a signaling pathway in which a small but significant portion of PtdIns(4,5)P(2) is hydrolyzed to IP(3). The accumulation of IP(3) occurs during a time frame similar to that observed for stress-induced calcium mobilization. These data also suggest that the majority of the PtdIns(4,5)P(2) synthesized in response to salt and osmotic stress may be utilized for cellular signaling events distinct from the canonical IP(3) signaling pathway.     

Lung albumin accumulation is spatially heterogeneous but not correlated with regional pulmonary perfusion.

The contribution of pulmonary perfusion heterogeneity to the development of regional differences in lung injury and edema is unknown. To test whether regional differences in pulmonary perfusion are associated with regional differences in microvascular function during lung injury, pigs were mechanically ventilated in the prone position and infused with endotoxin (Escherichia coli 055:B5, 0.15 microg. kg(-1). h(-1); n = 8) or saline (n = 4) for 4 h. Extravascular albumin accumulation and perfusion were measured in multiple approximately 0.7-ml lung regions by injecting pigs with radiolabeled albumin and radioactive microspheres, respectively. Extravascular albumin accumulation was spatially heterogeneous but not correlated with regional perfusion. Extravascular albumin accumulation was greater in dorsal than ventral regions, and regions with similar albumin accumulation were spatially clustered. This spatial organization was less evident in endotoxemic than control pigs. We conclude that there are regional differences in lung albumin accumulation that are spatially organized but not mediated by regional differences in pulmonary perfusion. We speculate that regional differences in microvascular pressure or endothelial function may account for the observed distribution of extravascular albumin accumulation.     

Formation and metabolism of inositol 1,4,5-trisphosphate in human platelets.

1. myo-[3H]Inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], when added to lysed platelets, was rapidly converted into [3H]inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], which was in turn converted into [3H]inositol 1,3,4-trisphosphate [Ins(1,3,4)P3]. This result demonstrates that platelets have the same metabolic pathways for interconversion of inositol polyphosphates that are found in other cells. 2. Labelling of platelets with [32P]Pi, followed by h.p.l.c., was used to measure thrombin-induced changes in the three inositol polyphosphates. Interfering compounds were removed by a combination of enzymic and non-enzymic techniques. 3. Ins(1,4,5)P3 was formed rapidly, and reached a maximum at about 4 s. It was also rapidly degraded, and was no longer detectable after 30-60 s. 4. Formation of Ins(1,3,4,5)P4 was almost as rapid as that of Ins(1,4,5)P3, and it remained detectable for a longer time. 5. Ins(1,3,4)P3 was formed after an initial lag, and this isomer reached its maximum, which was 10-fold higher than that of Ins(1,4,5)P3, at 30 s. 6. Comparison of the intracellular Ca2+ concentration as measured with fura-2 indicates that agents other than Ins(1,4,5)P3 are responsible for the sustained maintenance of a high concentration of intracellular Ca2+. It is proposed that either Ins(1,3,4)P3 or Ins(1,3,4,5)P4 may also be Ca2+-mobilizing agents.     

Production of phosphatidylinositol 3,4,5-trisphosphate and phosphatidic acid in platelet rafts: evidence for a critical role of cholesterol-enriched domains in human platelet activation.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.