Reversible Translocation of Phosphoinositide 3-Kinase to the Cytoskeleton of ADP-aggregated Human Platelets Occurs Independently of Rho A and without Synthesis of Phosphatidylinositol (3,4)-Bisphosphate*

(Received for publication, May 25, 1996, and in revised form, November 14, 1996)

Christian Gachet Dagger , Bernard Payrastre §, Christine Guinebault §, Cathy Trumel §, Philippe Ohlmann , Gérard Mauco §, Jean-Pierre Cazenave , Monique Plantavid § and Hugues Chap §

From the INSERM U.311, ETS, 10 rue Spielmann, 67065 Strasbourg and § INSERM U.326, Hôpital Purpan, 31059 Toulouse, France

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha -p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3,4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha  translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60c-src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.


INTRODUCTION

Phosphoinositide 3-kinase (PI 3-kinases)1 (1) are enzymes involved in growth factor signal transduction through association with receptor and nonreceptor tyrosine kinases and with G-protein-coupled receptors such as the fMet-Leu-Phe receptor in neutrophils or the thrombin receptor in platelets (1-3). Phosphoinositide kinases and their products have been implicated in the reorganization of the cytoskeleton, and PI 3-kinase is known to be directly involved in platelet-derived growth factor, insulin-like growth factor-1, and insulin-induced membrane ruffling (4, 5). Blood platelets also undergo morphological changes in response to stimulation, in particular shape change, extension of pseudopods, secretion of granule contents, aggregation, and contraction, all of which are linked to cytoskeletal modifications. Thrombin activation of human platelets leads to cytoskeletal translocation of the heterodimeric (p85alpha -p110) PI 3-kinase (PI 3-kinase alpha ) and accumulation of PtdIns(3,4)P2 in an integrin alpha IIbbeta 3-dependent manner (6, 7).

Integrins are transmembrane heterodimers mediating cell-matrix and cell-cell interactions. The platelet alpha IIbbeta 3 integrin serves as an activation-dependent receptor for the adhesive proteins fibrinogen, fibronectin, and von Willebrand factor. In patients with Glanzmann's thrombasthenia (8), an inherited hemorrhagic disorder where the alpha IIbbeta 3 integrin is absent, reduced, or abnormal, platelets are unable to bind fibrinogen upon activation and consequently do not aggregate. In platelets, as in other cells, integrin ligation triggers the assembly of specific cytoskeletal proteins and enzymes into structures termed focal adhesion sites (9, 10). These focal adhesion structures comprise proteins such as vinculin, talin, and the integrin alpha IIbbeta 3 itself, enzymes such as PI 3-kinase alpha , phospholipase C, protein kinase C, the tyrosine kinases pp60c-src, pp72SYK, and focal adhesion kinase (FAK) or the small GTP-binding proteins, RhoA and Cdc42Hs (11-16). Rho, Rac, and Cdc42Hs are all members of the Ras superfamily of small GTP-binding proteins. These molecules are important regulators of the cytoskeleton, and evidence is now accumulating that Rho promotes the formation of focal adhesions and their anchoring to stress fibers (17-19).

Depending on the cell type studied, the heterodimeric PI 3-kinase alpha  has been found to be activated by several pathways including interactions of the p85alpha regulatory subunit with phosphorylated receptor tyrosine kinases, tyrosine kinases of the src family (20), p21ras (21, 22), RhoA (23, 24), Cdc42Hs (25), or FAK (16). In platelets stimulated by thrombin, both RhoA and FAK could participate to the activation of PI 3-kinase alpha , the former presumably by an indirect mechanism (24) and the latter by a direct interaction with the SH3 domain of the p85alpha subunit (16). In addition, heterotrimeric G-protein beta gamma complexes may be involved in the stimulation of a second isoform of PI 3-kinase present in platelets. This form is immunologically related to a recently cloned monomeric PI 3-kinase, which was designated PI 3-kinase gamma  and found to be activated in vitro by beta gamma subunits (24, 26, 27).

A close relationship between integrin alpha IIbbeta 3-dependent p85alpha translocation to the cytoskeleton and PtdIns(3,4)P2 accumulation has been demonstrated in thrombin-stimulated platelets (16), suggestive of a role of the heterodimeric PI 3-kinase at this stage of 3-phosphoinositide synthesis. However, true assessment of the direct action of thrombin on platelets is difficult due to the presence of several mediators, in particular ADP and serotonin, released from the platelet-dense granules and of mediators such as thromboxane A2 resulting from activation of the arachidonic pathway, all of which interact with their own specific receptors on the platelet membrane.

Platelet aggregation by ADP plays a key role in the development and extension of arterial thrombosis (28). Specific inhibitors of the ADP activation pathway such as the anti-aggregatory thienopyridine compounds ticlopidine and clopidogrel (29) markedly prolong the bleeding time and are used clinically as antithrombotic drugs. Furthermore, a rare congenital bleeding disorder with impairment of ADP-induced platelet aggregation (30, 31) strikingly resembles the acquired thrombopathy resulting from ticlopidine or clopidogrel intake (32). Contained at very high concentrations in the platelet-dense granules, ADP is released when platelets are stimulated by other aggregating agents such as thrombin or collagen and thus contributes to and reinforces platelet aggregation. Low concentrations of ADP also potentiate or amplify the effects of all other agents, even weak agonists such as epinephrine or serotonin. Addition of ADP to washed human platelet suspensions results in shape change, exposure of the fibrinogen binding site on the alpha IIbbeta 3 integrin, and in contrast to other agonists such as thrombin, reversible aggregation in the presence of fibrinogen and physiological concentrations of Ca2+. At the intracellular level, platelet activation following ADP binding to its receptor leads to a transient rise in free cytoplasmic Ca2+, resulting from both Ca2+ influx and mobilization of internal stores, without apparent activation of phospholipase C or D-myo-inositol 1,4,5-trisphosphate (33, 34). ADP also inhibits stimulated adenylyl cyclase (35). On the basis of its agonist selectivity and signaling properties, the platelet receptor for ADP has been classified as a P2T receptor of the P2 purinoceptor family (36). Although the biochemical structure of this receptor remains unknown, it may belong to the seven transmembrane domain G-protein-coupled receptor family since ADP has been found to activate the Gi2 subtype of the heterotrimeric G-protein family (37, 38).

The aim of the present study was to evaluate the direct effect of ADP on PI 3-kinase activation and to compare PtdIns(3,4)P2 accumulation during reversible and irreversible aggregation. ADP was found to induce a reversible modification of the cytoskeleton which paralleled aggregation. The regulatory subunit p85alpha of PI 3-kinase alpha  and FAK reversibly translocated to the cytoskeleton, and this effect was dependent on the presence of alpha IIbbeta 3 and on the binding of fibrinogen to its receptor. However, significant accumulation of PtdIns(3,4)P2 did not occur, indicating that although translocation of the heterodimeric PI 3-kinase alpha  occurred, activation did not. Moreover, in contrast to thrombin stimulation, the small GTP-binding protein RhoA was not significantly translocated to the cytoskeleton when platelets were stimulated with ADP, adding further support to a functional relationship between RhoA and PI 3-kinase.


EXPERIMENTAL PROCEDURES

Materials

The rabbit anti-p85alpha antibody was from Upstate Biotechnology Inc. (Lake Placid, NY), and rabbit anti-FAK and anti-RhoA antibodies were from Tebu (Santa Cruz Biotechnology Inc., Santa Cruz, CA), and an affinity purified sheep polyclonal antibody against pp60c-src was from Cambridge Research Biochemicals Inc. (Cambridge, UK).

Preparation of Washed Human Platelets

Human blood was collected from a forearm vein (6 blood volumes into 1 volume of acid/citrate/dextrose anticoagulant), and twice-washed platelet suspensions were prepared as described previously (39). In some experiments, platelets were labeled with sodium [32P]orthophosphate (200 µCi/ml) for 1 h at 37 °C during a first washing step in Tyrode's buffer containing no phosphate. The final resuspending medium, pH 7.35, was Tyrode's buffer containing 2 mM Ca2+, 1 mM Mg2+, 0.35% human serum albumin (Etablissement de Transfusion Sanguine, Strasbourg, France), and apyrase (2 µg/ml, a concentration which converted 0.25 µM ATP to AMP within 2 min at 37 °C). Platelets were stored at 37 °C throughout experiments, and cell count was adjusted in the final suspension to 7.5 × 105/µl using a Sysmex 100 particle counter (Merck Clevenot, Nogent-sur-Marne, France).

Platelet Aggregation Studies

Aggregation was measured at 37 °C by a turbidimetric method in a dual-channel Payton aggregometer (Payton Associates, Scarborough, Ontario, Canada). A 1.45-ml aliquot of nonlabeled or 32P-labeled platelet suspension was stirred at 1,100 rpm and activated by addition of ADP in the absence or presence of human fibrinogen (0.8 mg/ml), or of thrombin in the absence of fibrinogen. The extent of aggregation was estimated quantitatively by measuring the maximum curve height above base-line level. At predetermined times, the reaction was stopped by addition of 1 ml of chloroform/methanol (v/v) for lipid extraction and analysis or by addition of an equal volume of CSK buffer (50 mM Tris-HCl, pH 7.4, 10 mM EGTA, 1 mM Na3VO4, 4 µg/ml aprotinin, 4 µg/ml leupeptin, 100 µg/ml phenylmethylsulfonyl fluoride, 2% (v/v) Triton X-100).

Lipid Extraction and Analysis

Lipids were extracted and analyzed as described previously (2).

Cytoskeleton Extraction

Unlabeled platelets, activated or nonactivated, were mixed with 1 volume of CSK buffer and incubated successively for 5 min at room temperature and for 10 min at 4 °C under shaking. Cytoskeletal material was collected by centrifugation (12,000 × g, 10 min, 4 °C), washed once with 2 volumes of CSK buffer containing 1% (v/v) Triton X-100, and then washed five times with 2 volumes of CSK buffer containing no Triton.

Gel Electrophoresis and Immunoblotting

Cytoskeletal proteins were solubilized in electrophoresis buffer (10 mM Tris-HCl, pH 6.8, 15% (v/v) glycerol, 25 mM dithiothreitol, 3% SDS), boiled for 5 min, and separated on 7.5% SDS-PAGE gels. The protein bands were blotted onto nitrocellulose, and immunodetection was performed with relevant antibodies as described.


RESULTS

Reversible Modification of the Cytoskeleton

Washed human platelets were stimulated with 10 µM ADP in the presence of fibrinogen or with 1 unit/ml thrombin in the absence of added fibrinogen. Typical aggregation curves were obtained (Fig. 1A), ADP inducing reversible and thrombin irreversible aggregation. In some experiments, the reaction was stopped at predetermined time points, the cytoskeleton was extracted and cytoskeletal proteins were solubilized, separated by SDS-PAGE, and stained with Coomassie Blue. Small amounts of actin binding protein (250 kDa), alpha -actinin (100 kDa), and F-actin (45 kDa) were found in the cytoskeleton of resting platelets. After stimulation with ADP, actin binding protein, myosin (200 kDa), and actin were reversibly translocated to the cytoskeleton, maximum incorporation corresponding to the maximum amplitude of platelet aggregation (Fig. 1B, left panel). Myosin was only weakly incorporated into the cytoskeleton of ADP-stimulated platelets. When platelets were stimulated with thrombin, translocation of actin binding protein, myosin, and actin to the cytoskeleton was not reversible (Fig. 1B, right panel). Actin polymerization induced by ADP was reversible and followed a course parallel to the amplitude of aggregation (Fig. 1C).


Fig. 1. Differential aggregation and cytoskeletal reorganization induced by ADP and thrombin. A, platelet aggregation was induced by ADP (10 µM) in the presence of fibrinogen or by thrombin (1 unit/ml) in the absence of added fibrinogen and followed as described under "Experimental Procedures." Curves are representative of five independent experiments giving very similar results. B, in parallel, cytoskeletons were isolated from ADP- (10 µM + fibrinogen) or thrombin (1 unit/ml) -stimulated platelets at the indicated times. Cytoskeletal proteins (corresponding to 7.5 × 107 platelets) were separated by SDS-PAGE (7.5%) and revealed by Coomassie Blue staining, actin, and the major actin-binding proteins being identified on the right side of the gel. Data are representative of two independent experiments giving very similar results. C, the F-actin content of ADP- or thrombin-stimulated platelets was quantified by densitometric analysis (ScanMaker IIHR, Microtek, Germany) of the Coomassie Blue-stained gel.
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Reversible Translocation of the Regulatory p85alpha Subunit of PI 3-Kinase alpha  without [32P]PtdIns(3,4)P2 Accumulation during Platelet Aggregation

Using a polyclonal antibody, we found an increase in amounts of the regulatory subunit p85alpha in the cytoskeleton during both ADP- and thrombin-induced platelet aggregation (Fig. 2A). In the case of ADP-stimulated platelets, translocation was reversible and followed the amplitude of aggregation. However, 20 and 40 s after stimulation, levels of p85alpha in the cytoskeleton were identical using either ADP or thrombin, and PI 3-kinase activity measured in the cytoskeleton followed the same time course (not shown). In order to measure [32P]PtdIns(3,4)P2 accumulation during platelet aggregation, 32P-labeled washed human platelets were stimulated with 10 µM ADP in the absence or presence of fibrinogen or with 1 unit/ml thrombin in the absence of added fibrinogen. Thrombin stimulation gave rise to the expected time-dependent accumulation of [32P]PtdIns(3,4)P2 (Fig. 2B, right panel), whereas ADP did not induce any significant synthesis of [32P]PtdIns(3,4)P2 (Fig. 2B, left panel). In 5 of 7 experiments, where 32P incorporation into lipids was especially high, we could detect only transient trace amounts of radioactivity in both PtdIns(3,4)P2 and PtdIns(3,4,5)P3 at short times (20 s) following ADP stimulation. Addition of fibrinogen did not increase this labeling. Similar results were obtained using 100 µM ADP (data not shown).


Fig. 2. Reversible translocation of p85alpha to the cytoskeleton without PtdIns(3,4)P2 accumulation in ADP-stimulated platelets as compared with time-dependent PtdIns(3,4)P2 accumulation after thrombin stimulation. A, cytoskeletons were extracted from ADP- (10 µM + fibrinogen) or thrombin (1 unit/ml) -stimulated platelets at the indicated times. Proteins of the cytoskeleton (corresponding to 7.5 × 107 platelets) were separated by SDS-PAGE (7.5%), blotted onto nitrocellulose, and tested for reactivity with an anti-p85alpha antibody using enhanced chemiluminescence. B, the time course of PtdIns(3,4)P2 accumulation in washed platelets stimulated by ADP (10 µM + fibrinogen) or thrombin (1 unit/ml) was followed as indicated under "Experimental Procedures."
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p85alpha Translocation Requires Integrin alpha IIbbeta 3 and Fibrinogen Binding

ADP-induced translocation of PI 3-kinase alpha  to the cytoskeleton was dependent on the presence of the integrin alpha IIbbeta 3, since this effect was not detectable using platelets from a type I Glanzmann's thrombasthenia patient (40) (Fig. 3, right panels). Translocation was also dependent on the binding of fibrinogen to its receptor and was clearly reduced when fibrinogen was omitted (Fig. 3, left panels). The residual translocation observed is probably due to secreted fibrinogen.


Fig. 3. Integrin alpha IIbbeta 3 and fibrinogen binding dependence of p85alpha translocation. Control and type I Glanzmann's thrombasthenia (GT) platelets were stimulated with ADP (10 µM) in the presence or absence of added fibrinogen. At the indicated time points, the reaction was stopped and translocation of p85alpha was evaluated as in Fig. 2.
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Translocation of pp60c-src, FAK, and RhoA to the Cytoskeleton

Washed human platelets were stimulated with 10 µM ADP in the presence of fibrinogen or with 1 unit/ml thrombin in the absence of added fibrinogen; the cytoskeleton was extracted at indicated time points and analyzed by Western blotting. The tyrosine kinases pp60c-src and FAK were translocated to the cytoskeleton in a similar manner to the PI 3-kinase regulatory subunit p85alpha (Fig. 4). In contrast, although thrombin induced clearly detectable translocation of RhoA to the cytoskeleton (Fig. 4, right panel), ADP did not (Fig. 4, left panel), and we could distinguish only a faint band at 20 s.


Fig. 4. Reversible translocation of pp60c-src and FAK but not RhoA to the cytoskeleton of ADP (10 µM + fibrinogen) -stimulated platelets, as compared with irreversible translocation of all three proteins after thrombin stimulation. Platelets were stimulated with ADP (10 µM + fibrinogen) or thrombin (1 unit/ml) for increasing periods as indicated on the figure. Reactions were then stopped; the cytoskeletons were immediately extracted, and cytoskeletal proteins (corresponding to 7.5 × 107 platelets) were separated by SDS-PAGE (7.5% for p125FAK and pp60c-src or 12% for RhoA), blotted onto nitrocellulose, and examined for reactivity with the indicated antibodies. Alkaline phosphatase detection was used for pp60c-src analysis and the enhanced chemiluminescence system for p125FAK and RhoA.
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DISCUSSION

As is now well established (7, 16), thrombin activation of washed human platelets resulted in translocation of the heterodimeric PI 3-kinase alpha  to the actin-rich cytoskeleton, together with production and accumulation of PtdIns(3,4)P2 (Fig. 2B, right panel). These effects of thrombin are dependent on the presence of functional alpha IIbbeta 3 integrin (6, 41). In general terms, coordinated signaling through agonist receptors and integrins results in reorganization of the cytoskeleton and formation of focal adhesion structures with translocation and activation of signaling proteins and enzymes (9). The aim of our study was to assess the specific role of ADP in these events and to investigate PI 3-kinase alpha  activation during reversible aggregation. So far, the molecular mechanisms leading to platelet aggregation by ADP and its typical feature of reversibility are not well understood. Our results showed an increase in levels of the regulatory subunit p85alpha in the cytoskeleton of ADP-stimulated platelets equivalent to the increase observed in thrombin-stimulated platelets (Fig. 2A). This translocation was reversible and followed a course parallel to the amplitude of aggregation. Furthermore, this effect of ADP was dependent on the presence of the integrin alpha IIbbeta 3 and on the binding of fibrinogen to its receptor (Fig. 3), clearly indicating an "outside-in" signaling event involving the "ADP-activated" integrin. Reversibility of the association of transduction proteins with the cytoskeleton of platelets activated by a thrombin receptor agonist peptide (TRAP) has already been reported to occur 15 min after stimulation in a Ca2+-dependent but aggregation-independent manner (42). Our results demonstrate the rapidly reversible association of these proteins in a manner differing according to the agonist used and the aggregation response.

Surprisingly, PtdIns(3,4)P2 did not accumulate even at the maximum amplitude of aggregation, although PI 3-kinase alpha  was translocated to the cytoskeleton in amounts comparable with those found in thrombin-aggregated platelets. This result demonstrates that translocation of the enzyme is an aggregation-dependent event but is not sufficient for activation of PI 3-kinase. At least three pathways of activation of PI 3-kinases have been reported in platelets, involving the small GTP-binding protein RhoA (23, 24) and the tyrosine kinase FAK (16) for the (p85alpha -p110) enzyme, or the beta gamma subunit complex of heterotrimeric G-proteins for the p110 PI 3-kinase gamma  (24, 26, 27). In the case of ADP-induced platelet aggregation, we found pp60c-src and FAK to be reversibly translocated to the cytoskeleton in a manner similar to p85alpha , whereas RhoA was not. These observations deserve several comments. (i) Gi2 proteins involved in the ADP signaling pathway do not seem to provide PI 3-kinase activating beta gamma subunits which would otherwise have activated such an enzyme. (ii) The clear-cut difference between ADP and thrombin in inducing translocation of RhoA suggests that PI 3-kinase alpha  could be regulated by this small G-protein as previously reported (23, 24). Interestingly, using lysophosphatidic acid as an agonist of a G-protein-coupled receptor, evidence has been provided that Rho-dependent assembly of an actin-based signaling complex linked to integrins was stimulated downstream of Gq (43, 44). In contrast to thrombin, ADP activates only Gi2 with no effect on Gq (38). Our data would fit with this scheme, underlying a possible role of RhoA in regulating PI 3-kinase activity and stabilization of the cytoskeleton. (iii) One cannot exclude a direct role of FAK, since its translocation to the cytoskeleton does not necessarily imply activation. This point could be clarified by looking at tyrosine phosphorylation of FAK during ADP-induced platelet aggregation. Nevertheless, the ADP scavenger apyrase has been shown to prevent tyrosine phosphorylation of FAK and the spreading of platelets on immobilized fibrinogen, which suggests that ADP in fact induces this phosphorylation (45).

Inhibition of PI 3-kinase by wortmannin or LY294002 has been reported to reverse the platelet aggregation induced by agonists such as TRAP (46). These authors suggested that PI 3-kinase activation may be necessary for prolonged alpha IIbbeta 3 activation and irreversible platelet aggregation. However, using wortmannin up to 100 nM, we were unable to reverse the irreversible aggregation induced by thrombin, even though the aggregates were smaller (data not shown). Aggregation was also found to be necessary for the late accumulation of PtdIns(3,4)P2 measured 3-5 min after thrombin addition (16). Furthermore, previous studies have clearly established that thrombin-induced translocation to the cytoskeleton of several signaling proteins including alpha IIbbeta 3, pp60c-src, PI 3-kinase alpha  (12, 16), as well as FAK tyrosine phosphorylation (47) require platelet to platelet contacts. Hence, the current data suggest that irreversible aggregation is necessary to initiate a "mechanical" transduction pathway leading to PI 3-kinase alpha  activation, whereas reversible aggregation would appear to be insufficient. Thrombospondin, a large trimeric adhesive molecule released from the alpha  granules of thrombin- but not ADP-stimulated platelets, could contribute to such a late signaling event by binding to the plasma membrane through its receptor CD 36, which has been shown to be linked to tyrosine kinases of the src family (48). On the other hand, when platelets were stimulated with lysophosphatidic acid (49) or concanavalin A (50), aggregation did not appear to be necessary for the synthesis of PtdIns(3,4)P2. Identification of the PI 3-kinase isoforms involved in these processes would contribute to a better understanding of the different pathways and stages of 3-phosphoinositide synthesis in platelets. Nevertheless, the physiological significance of the synthesis of PtdIns(3,4)P2 depending on irreversible platelet aggregation remains to be established. The microvesiculation and clot retraction occurring at this stage of platelet activation are controlled by mechanisms involving reorganization of the membrane and cytoskeleton, which could be regulated by 3-phosphoinositide synthesis.

Alternatively, the results presented here are also consistent with the hypothesis that the typical reversibility of ADP-induced platelet aggregation finds its origin in the lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 formation. Recent data on the effects of wortmannin on platelet aggregation induced by TRAP (46) may indicate that PI 3-kinase products are necessary to stabilize platelet aggregates. Moreover, it has been demonstrated that PtdIns(3,4,5)P3 is able to bind to the SH2 domains of several proteins including p85alpha (51), thereby blocking the binding of PI 3-kinase to tyrosine phosphorylated proteins, which would suggest direct involvement of these D3-phosphorylated phosphoinositides. Whether PtdIns(3,4)P2, among other components of assembled signaling complexes, contributes directly to the stability of platelet aggregates is not known. The differential effects of ADP and thrombin on platelet activation and aggregation may thus provide a physiological model leading to the improvement of our understanding of PI 3-kinase pathways, at least in platelets.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed: INSERM U.311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomatériaux, Etablissement de Transfusion Sanguine, B.P. no. 36, 10, rue Spielmann, 67065 Strasbourg Cédex, France. Tel.: (33) 3 88 21 25 25; Fax: (33) 3 88 21 25 21; E-mail: christian.gachet{at}etss.u-strasbg.fr.
1    The abbreviations used are: PI 3-kinase, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol; PtdIns(3,4)P2, phosphatidylinositol (3,4)-bisphosphate; FAK, focal adhesion kinase; TRAP, thrombin receptor agonist peptide; PAGE, polyacrylamide gel electrophoresis.

Acknowledgments

We thank C. Viala for technical assistance, J. N. Mulvihill for reviewing the English of the manuscript.


REFERENCES

  1. Fry, M. J. (1994) Biochim. Biophys. Acta 1226, 237-268 [Medline] [Order article via Infotrieve]
  2. Sultan, C., Breton, M., Mauco, G., Grondin, P., Plantavid, M., and Chap, H. (1990) Biochem. J. 269, 831-834 [Medline] [Order article via Infotrieve]
  3. Kucera, G. L., and Rittenhouse, S. E. (1990) J. Biol. Chem. 265, 5345-5348 [Abstract/Free Full Text]
  4. Kotani, K., Yonezawa, K., Hara, K., Ueda, H., Kitamure, Y., Sakane, H., Ando, A., Chavanieu, A., Calas, B., Grigorescu, F., Nishiyama, M., Waterfield, M., and Kasuza, M. (1994) EMBO J. 13, 2313-2321 [Abstract]
  5. Wennström, S., Siegbahn, A., Yokote, K., Ardvisson, A., Heldin, C., Mori, S., and Claesson-Welek, C. (1994) Oncogene 9, 651-660 [Medline] [Order article via Infotrieve]
  6. Sultan, C., Plantavid, M., Bachelot, C., Grondin, P., Breton, M., Mauco, G., Lévy-Toledano, S., Caen, J. P., and Chap, H. (1991) J. Biol. Chem. 266, 23554-23557 [Abstract/Free Full Text]
  7. Zhang, J., Fry, M. J., Waterfield, M. D., Jaken, S., Liao, L., Fox, J. E. B., and Rittenhouse, S. E. (1992) J. Biol. Chem. 267, 4686-4692 [Abstract/Free Full Text]
  8. Bray, P. F. (1994) Thromb. Haemostasis 72, 492-502 [Medline] [Order article via Infotrieve]
  9. Shattil, S. J. (1995) Thromb. Haemostasis 74, 149-155 [Medline] [Order article via Infotrieve]
  10. Miyamoto, S., Teramoto, H., Coso, O. A., Gutkind, J. S., Burbelo, P. D., Akiyama, S. K., and Yamada, K. M. (1995) J. Cell Biol. 131, 791-805 [Abstract]
  11. Grondin, P., Plantavid, M., Sultan, C., Breton, M., Mauco, G., and Chap, H. (1991) J. Biol. Chem. 266, 15705-15709 [Abstract/Free Full Text]
  12. Oda, A., Druker, B. J., Smith, M., and Salzman, E. W. (1992) J. Biol. Chem. 267, 20075-20081 [Abstract/Free Full Text]
  13. Fox, J. E. B., Lipfert, L., Clark, E. A., Reynolds, C. C., Austin, C. D., and Brugge, J. S. (1993) J. Biol. Chem. 268, 25973-25984 [Abstract/Free Full Text]
  14. Tohyama, Y., Yanagi, S., Sada, K., and Yamamura, H. (1994) J. Biol. Chem. 269, 32796-32799 [Abstract/Free Full Text]
  15. Dash, D., Aepfelbacher, M., and Siess, W. (1995) J. Biol. Chem. 270, 17321-17326 [Abstract/Free Full Text]
  16. Guinebault, C., Payrastre, B., Racaud-Sultan, C., Mazarguil, H., Breton, M., Mauco, G., Plantavid, M., and Chap, H. (1995) J. Cell Biol. 129, 831-842 [Abstract]
  17. Ridley, A. J., and Hall, A. (1992) Cell 70, 389-399 [Medline] [Order article via Infotrieve]
  18. Ridley, A. J., and Hall, A. (1994) EMBO J. 13, 2600-2610 [Abstract]
  19. Nobes, C. D., and Hall, A. (1995) Cell 81, 53-62 [Medline] [Order article via Infotrieve]
  20. Shoelson, S. E., Sirvaraja, M., Williams, K. P., Hu, P., Schlessinger, J., and Weiss, M. A. (1993) EMBO J. 12, 795-802 [Abstract]
  21. Sjölander, A., Yamamoto, K., Huber, B. E., and Lapetina, E. (1992) Proc. Natl. Acad. Sci. U. S. A. 88, 7908-7912 [Abstract]
  22. Rodriguez-Viciana, P., Warne, P., Dhand, R., Vanhaesebroeck, B., Gout, I., Fry, M., Waterfield, M. D., and Downward, J. (1994) Nature 370, 527-532 [CrossRef][Medline] [Order article via Infotrieve]
  23. Zhang, J., King, W. G., Dillon, S., Hall, A., Feig, L., and Rittenhouse, S. E. (1993) J. Biol. Chem. 268, 22251-22254 [Abstract/Free Full Text]
  24. Zhang, J., Zhang, J., Benovic, J. L., Sugai, M., Wetzker, R., Gout, I., and Rittenhouse, S. E. (1995) J. Biol. Chem. 270, 6589-6594 [Abstract/Free Full Text]
  25. Zheng, Y., Bagrodia, S., and Cerione, R. A. (1994) J. Biol. Chem. 269, 18727-18730 [Abstract/Free Full Text]
  26. Stephens, L., Smrcka, A., Cooke, F. T., Jackson, T. R., Sternweis, P. C., and Hawkins, P. T. (1994) Cell 77, 83-93 [Medline] [Order article via Infotrieve]
  27. Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubchenkov, M., Malek, D., Stoyanova, S., Vanhaesebroeck, B., Dhand, R., Nürnberg, B., Gierschik, P., Seedorf, K., Hsuan, J. J., Waterfield, M. D., and Wetzker, R. (1995) Science 269, 690-693 [Medline] [Order article via Infotrieve]
  28. Born, G. V. R. (1985) Circulation 72, 741-742 [Medline] [Order article via Infotrieve]
  29. Schrör, K. (1993) Platelets 4, 252-261
  30. Cattaneo, M., Lecchi, A., Randi, A. M., McGregor, J. L., and Mannuci, P. M. (1992) Blood 80, 2787-2796 [Abstract]
  31. Nurden, P., Savi, P., Heilmann, E., Bihour, C., Herbert, J. M., Maffrand, J. P., and Nurden, A. (1995) J. Clin. Invest. 95, 1612-1622 [Medline] [Order article via Infotrieve]
  32. Gachet, C., Cattaneo, M., Ohlmann, P., Lecchi, A., Hechler, B., Chevalier, J., Cassel, D., Mannucci, P., and Cazenave, J.-P. (1995) Br. J. Haematol. 91, 434-444 [Medline] [Order article via Infotrieve]
  33. Packham, M. A., Livne, A., Ruben, D. H., and Rand, M. (1993) Biochem. J. 290, 849-856 [Medline] [Order article via Infotrieve]
  34. Pulcinelli, F. M., Ashby, B., Gazzaniga, P. P., and Daniel, J. L. (1995) FEBS Lett. 364, 87-90 [CrossRef][Medline] [Order article via Infotrieve]
  35. Hourani, S. M. O., and Hall, D. A. (1994) Trends Pharmacol. Sci. 15, 103-108 [Medline] [Order article via Infotrieve]
  36. Dubyac, G. R., and El Moatassim, C. (1993) Am. J. Physiol. 265, C577-C606 [Abstract/Free Full Text]
  37. Gachet, C., Cazenave, J.-P., Ohlmann, P., Hilf, G., Wieland, T., and Jakobs, K. H. (1992) Eur. J. Biochem. 207, 259-263 [Abstract]
  38. Ohlmann, P., Laugwitz, K.-L., Spicher, K., Nurnberg, K., Schultz, G., Cazenave, J.-P., and Gachet, C. (1995) Biochem. J. 312, 775-779 [Medline] [Order article via Infotrieve]
  39. Cazenave, J.-P., Hemmendinger, S., Beretz, A., Sutter-Bay, A., and Launay, J. (1983) Ann. Biol. Clin. 41, 167-179 [Medline] [Order article via Infotrieve]
  40. De La Salle, C., Schwartz, A., Baas, M. J., Lanza, F., and Cazenave, J.-P. (1995) Thromb. Haemostasis 74, 990-991 [Medline] [Order article via Infotrieve]
  41. Sorisky, A., King, W. G., and Rittenhouse, S. E. (1992) Biochem. J. 286, 581-584 [Medline] [Order article via Infotrieve]
  42. Dash, D., Aepfelbacher, M., and Siess, W. (1995) FEBS Lett. 363, 231-234 [CrossRef][Medline] [Order article via Infotrieve]
  43. Moolenar, W. H. (1995) J. Biol. Chem. 270, 12949-12952 [Free Full Text]
  44. Hordijk, P. L., Verlaan, I., van Corven, E. J., and Moolenar, W. H. (1994) J. Biol. Chem. 269, 645-651 [Abstract/Free Full Text]
  45. Haimovich, B., Lipfert, L., Brugge, J. S., and Shattil, S. J. (1993) J. Biol. Chem. 268, 15868-15877 [Abstract/Free Full Text]
  46. Kovacsovics, T. J., Bachelot, C., Toker, A., Vlahos, C. J., Duckworth, B., Cantley, L. C., and Hartwig, J. H. (1995) J. Biol. Chem. 270, 11358-11366 [Abstract/Free Full Text]
  47. Lipfert, L., Haimovich, B., Schaller, M. D., Cobbi, B. S., Parsons, J. T., and Brugge, J. S. (1992) J. Cell Biol. 119, 905-912 [Abstract]
  48. Huang, M. M., Bolen, J. B., Barnwell, J. W., Shattil, S. J., and Brugge, J. S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 7844-7848 [Abstract]
  49. Zhang, J., and Rittenhouse, S. E. (1995) Biochem. Bioph. Res. Commun. 211, 484-490 [CrossRef][Medline] [Order article via Infotrieve]
  50. Torti, M., Ramaschi, G., Montserrat, N., Singaglia, P., Balduni, C., Plantavid, M., Breton, M., Chap, H., and Mauco, G. (1995) J. Biol. Chem. 270, 13179-13185 [Abstract/Free Full Text]
  51. Rameh, L. E., Chen, C. S., and Cantley, L. C. (1995) Cell 83, 821-830 [Medline] [Order article via Infotrieve]

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