©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence for a Glycoprotein IIb-IIIa- and Aggregation-independent Mechanism of Phosphatidylinositol 3`,4`-Bisphosphate Synthesis in Human Platelets (*)

Mauro Torti (1)(§), Giuseppe Ramaschi (1), Nathalie Montsarrat (2), Fabiola Sinigaglia (3), Cesare Balduini (1), Monique Plantavid (2), Monique Breton (2), Hugues Chap (2), Gerard Mauco (2)

From the (1) Department of Biochemistry, University of Pavia, via Bassi 21, 27100 Pavia, Italy, (2) Institut National de la Santé et de la Recherche Médicale, Unité 326, Hopital Purpan, 31059 Toulouse, France, and the (3) Institute of Biological Chemistry, University of Genoa, viale Benedetto XV, 31059 Genoa, Italy

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The synthesis of phosphatidylinositol 3`,4`-bisphosphate (PtdIns(3,4)P) in P-labeled human platelets induced by the tetrameric lectin concanavalin A and the physiological agonist thrombin were compared. Like thrombin, concanavalin A stimulated a time-dependent accumulation of PtdIns(3,4)P, which reached maximal levels after 5 min of stimulation. However, while synthesis of PtdIns(3,4)P induced by thrombin was dependent on platelet aggregation, the production of PtdIns(3,4)P induced by concanavalin A was unchanged when aggregation was prevented by the omission of stirring or when fibrinogen binding to platelets was inhibited by the tetrapeptide RGDS. Accumulation of PtdIns(3,4)P was not observed in platelets stimulated with succinyl-concanavalin A, a dimeric derivative of the lectin that binds to the same receptors on the platelet surface but does not promote clustering of membrane glycoproteins. The synthesis of PtdIns(3,4)P induced by concanavalin A was also independent of the membrane glycoprotein IIb-IIIa, as normal accumulation of this lipid was observed in platelets from two patients affected by Glanzmann thrombasthenia. In contrast, thrombin showed a strongly reduced ability to stimulate PtdIns(3,4)P production in thrombasthenic platelets. Although concanavalin A was able to induce association of the regulatory subunit of the phosphatidylinositol 3-kinase with tyrosine-phosphorylated proteins, the tyrosine kinase inhibitor tyrphostin AG-213 did not inhibit the lectin-induced synthesis of PtdIns(3,4)P. These results demonstrate the existence of a novel mechanism of PtdIns(3,4)P synthesis in human platelets, which is independent of glycoprotein IIb-IIIa and aggregation, but requires clustering of membrane glycoproteins. As clustering events occur during platelet aggregation promoted by physiological agonists, this new mechanism may also be involved in the aggregation-dependent production of PtdIns(3,4)P in thrombin-stimulated platelets.


INTRODUCTION

Human platelets accumulate the 3-phosphorylated phosphoinositides phosphatidylinositol 3`,4`-bisphosphate (PtdIns(3,4)P) and phosphatidylinositol 3`,4`,5`-trisphosphate (PtdIns(3,4,5)P) in response to strong agonists such as thrombin and the thromboxane analogue U46619 (1-3).

The biochemical pathways and the regulatory mechanisms leading to the production of the 3-phosphorylated phosphoinositides are not completely understood. The enzyme mainly responsible for the formation of these lipids is the phosphatidylinositol 3-kinase (PtdIns 3-kinase)()(4) . The PtdIns 3-kinase is a heterodimer composed of a 110-kDa catalytic subunit (p110) and an 85-kDa regulatory subunit (p85). In growth factor-stimulated cells, p85 is tyrosine phosphorylated and interacts, through its SH2 domains, with receptor-associated and cytosolic protein-tyrosine kinases (4) . However, interaction between p85 and Src-like kinases could also be due to the association of SH3 domain of the kinase and a proline-rich sequence of p85 (5) . In human platelets stimulated with thrombin, the PtdIns 3-kinase was found associated with the tyrosine kinases pp60, pp59, and pp120(6, 7) , and the accumulation of PtdIns(3,4)P was reduced by preincubation of the cells with the tyrosine kinase inhibitor tyrphostin (8) . These evidences indicate that tyrosine phosphorylation events may regulate the synthesis of the 3-phosphorylated phosphoinositides. However, it has been recently demonstrated that the PtdIns 3-kinase may be activated by different mechanisms involving both -subunits of heterotrimeric G-proteins (9) and the low molecular weight G-proteins Ras and Rho (10, 11) .

Synthesis of PtdIns(3,4)P and PtdIns(3,4,5)P are differently regulated in human platelets stimulated with thrombin. PtdIns(3,4,5)P accumulates rapidly, while PtdIns(3,4)P synthesis occurs in a late phase of platelet activation (12) . The increase of PtdIns(3,4)P is enhanced by extracellular Ca, which, in contrast, has no effect on the accumulation of PtdIns(3,4,5)P(12) . It has been demonstrated that in thrombin-stimulated platelets, synthesis of PtdIns(3,4)P but not PtdIns(3,4,5)P was inhibited by the tetrapeptide RGDS, which blocks interaction of fibrinogen with its membrane receptor, the glycoprotein IIb-IIIa (GP IIb-IIIa) (12, 13) . Moreover, a strongly reduced accumulation of PtdIns(3,4)P in response to thrombin was observed in platelets from patients affected by Glanzmann thrombasthenia, which lack GP IIb-IIIa and fail to aggregate (13). These evidences indicate a regulatory role of GP IIb-IIIa and platelet aggregation on the synthesis of PtdIns(3,4)P.

The GP IIb-IIIa belongs to the integrin family of receptors for adhesive proteins (integrin ) and plays an essential role in platelet physiology (14) . Platelet stimulation with thrombin results in the activation of GP IIb-IIIa, which then binds released fibrinogen and triggers platelet aggregation (15). Recently, it has been demonstrated that GP IIb-IIIa may also act as a transducer of signals into the platelets that can influence platelet function. An increasing number of events observed in activated cells have been found to be inhibited by blocking the receptor activity of GP IIb-IIIa: activation of the Na/H exchanger (16) , intracellular Ca increase (17) , tyrosine phosphorylation of several proteins (18, 19) , activation of protein phosphotyrosine phosphatases (20) , interaction of signaling molecules, such as pp60 and the low molecular weight G-protein rap2B, with the cytoskeleton (21, 22) , and synthesis of PtdIns(3,4)P(13) .

The understanding of the exact role played by GP IIb-IIIa in regulating these events is complicated by the fact that the classical experimental strategies used to block its receptor activity (specific monoclonal antibodies, RGD-containing peptides, thrombasthenic platelets, etc.) also result in the consequent inhibition of platelet aggregation. Therefore, under these conditions, it is not clear whether a certain event is directly controlled by GP IIb-IIIa or is linked to other events occurring during platelet aggregation. It is known that secondary events, following fibrinogen binding, occur on the cell surface during platelet aggregation. These events include clustering of GP IIb-IIIa (23) , the stabilization of the platelet aggregates by binding of secreted thrombospondin to its surface receptor (24, 25) , and the interaction of GP IIb-IIIa and other glycoproteins, such as the platelet-endothelial cell adhesion molecule 1 (PECAM-1), with the intracellular cytoskeleton (26, 27) .

Looking for an experimental system able to discriminate between the influence of GP IIb-IIIa and platelet aggregation on the production of PtdIns(3,4)P, we performed studies using the lectin concanavalin A (ConA). ConA is a strong platelet agonist able to induce phospholipase C activation (28) , protein-tyrosine phosphorylation (29) , and secretion and aggregation (30) . Although ConA binds to several mannose-containing glycoproteins, the main platelet receptor for this lectin has been identified as GP IIb-IIIa (31, 32) . Unlike physiological agonists, ConA can induce in nonaggregated platelets events that are usually observed only in aggregated cells. For instance, ConA promotes clustering of membrane glycoproteins, especially clustering of GP IIb-IIIa (33) , and induces the association of GP IIb-IIIa with the intracellular cytoskeleton (30) .

Here, we show that ConA stimulates the accumulation of PtdIns(3,4)P in human platelets and that this effect does not require fibrinogen binding to GP IIb-IIIa and platelet aggregation but is dependent on the lectin-induced clustering of membrane glycoproteins.


EXPERIMENTAL PROCEDURES

Materials

Concanavalin A, thrombin, the tetrapeptide RGDS, leupeptin, and aprotinin were obtained from Sigma. Succinyl-ConA was from Vector Laboratories. Tyrphostin AG-213 was a gift from Dr. A. Levitzki (Hebrew University, Jerusalem, Israel). [P]Orthophosphate and enhanced chemiluminesce reagents were from Amersham Corp. Protein A-Sepharose was from Pharmacia Biotech Inc. Nitrocellulose was from Schleicher and Schuell. Anti-p85 polyclonal antiserum and anti-phosphotyrosine monoclonal antibodies were from UBI. Prestained molecular weight markers, peroxidase- and alkaline phosphatase-conjugated antibodies were obtained from Bio-Rad.

Platelet Preparation and Labeling

Human platelets were prepared from platelet concentrates as previously described (3) . Platelets were resuspended at the concentration of 10 cells/ml and labeled with [P]orthophosphate (0.3 mCi/ml) in 135 mM NaCl, 2.7 mM KCl, 12 mM NaHCO, 0.16 mM NaHPO, 2 mM MgCl, 0.2 mM EGTA, 5.5 mM glucose, 0.34% bovine serum albumin, pH 6.5 (buffer A), for 90 min at room temperature. Platelets were then washed with buffer A without glucose and EGTA (buffer B) and finally resuspended at the concentration of 10 cells/ml in buffer B, pH 7.4, containing 2.5 mM CaCl. Thrombasthenic platelets were prepared from freshly drawn blood and labeled as described above. In parallel, platelets from healthy donors were prepared and used as controls.

Platelet Incubation, Lipid Extraction, and HPLC Analysis

Platelet samples (1 ml, 10 cells/ml) were equilibrated at 37 °C. RGDS (1 mM, final concentration), tyrphostin (100 µM, final concentration), or buffer were added 2 min before stimulation with 100 µg/ml ConA or 1 unit/ml thrombin. As tyrphostin was stored as a 100 mM stock solution in dimethyl sulfoxide, an equal volume of dimethyl sulfoxide was added to control samples. Stimulation with the agonists was usually performed for 5 min at 37 °C with or without stirring as indicated. Lipids were extracted, deacylated by methylamine treatment, separated, and identified by HPLC on a Whatman Partisphere strong anion exchange column as previously described (3) .

Immunoprecipitation and Immunoblotting

Platelet stimulation was stopped by addition of 1 volume of 40 mM Tris/HCl, pH 7.4, 200 mM NaCl, 20 µg/ml leupeptin, 20 µg/ml aprotinin, 0.2 mM phenylmethylsulfonyl fluoride, 2 mM NaVO, 2% Triton X-100. Samples were vortexed, placed on ice for 10 min, and then centrifuged at 13,000 g for 5 min. Lysates were incubated with 5 µl of anti-p85 polyclonal antiserum, anti-phosphotyrosine antibodies, or control serum for 2 h at 4 °C. Immunocomplexes were recovered with protein A-Sepharose (80 µl of a 50 mg/ml stock suspension), washed three times with 1 ml of 20 mM Tris/HCl, pH 7.4, 100 mM NaCl, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM NaVO, 1% Triton X-100, and finally resuspended in SDS-sample buffer (100 mM Tris/HCl, pH 8.0, 10% glycerol, 25 mM dithiothreitol, 3% SDS). Samples were boiled 10 min, and proteins were separated by SDS-polyacrylamide gel electrophoresis on 7.5% polyacrylamide gels and then transferred to nitrocellulose. Immunoblotting with anti-p85 polyclonal antiserum (1:500 dilution) or anti-phosphotyrosine antibodies (1:1000 dilution) was performed as previously described (34) . Appropriated secondary antibodies conjugated to alkaline phosphatase or horseradish peroxidase were used. Immunoreactive proteins were visualized with 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium reaction or by the chemiluminescent reaction.


RESULTS

Production of PtdIns(3,4)P in ConA-stimulated Platelets

The deacylated lipids from P-labeled human platelets, unstimulated and aggregated for 5 min with 1 unit/ml thrombin or 100 µg/ml ConA under constant stirring, were separated by strong anion exchange HPLC (Fig. 1). As previously reported (3) , thrombin induced a marked increase of GroP, corresponding to the deacylated form of phosphatidic acid, which is produced by the sequential action of phospholipase C and diacylglycerol kinase, and the appearance of a new peak corresponding to deacylated PtdIns(3,4)P (GroPIns(3,4)P), which was not detected in resting platelets. The HPLC profile of the deacylated lipids from P-labeled platelets stimulated with ConA was very similar to that from thrombin-stimulated platelets, indicating that, like thrombin, ConA induced both phospholipase C activation and accumulation of PtdIns(3,4)P. We also observed a small amount of GroPIns(3)P in resting platelets, which did not significantly change upon treatment with the agonists. The PtdIns(3,4,5)P was not considered, since, in most of our experiments, it was not efficiently separated from an unknown peak eluting at about 68 min.


Figure 1: HPLC profiles of the deacylated lipids from resting, thrombin-, and ConA-stimulated platelets. P-Labeled platelets (10 cells) were incubated at 37 °C in the absence or in the presence of 1 unit/ml thrombin or 100 µg/ml ConA for 5 min under constant stirring. Lipids were extracted, deacylated, and analyzed by strong anion exchange HPLC. Profiles are representative of more than 10 separate experiments. GroP, sn-glycero-3-phosphate; GroPIns(3)P, sn-glycero-phosphoinositol-3`-phosphate; GroPIns(4)P, sn-glycero-phosphoinositol-4`-phosphate; GroPIns(3,4)P, sn-glycero-phosphoinositol-3`,4`-bisphosphate; GroPIns(4,5)P, sn-glycero-phosphoinositol-4`,5`-bisphosphate.



Fig. 2 shows the time course of GroP and GroPIns(3,4)P production in ConA-stimulated platelets. In contrast to thrombin, which is known to induce activation of phospholipase C within seconds (35) , increase of GroP in ConA-treated platelets was evident only after 2 min of stimulation. This is in agreement with previously reported results measuring the formation of soluble inositol phosphates in [H]inositol-labeled platelets (28) . The synthesis of GroPIns(3,4)P showed a similar kinetic, being detectable between 1 and 2 min after the addition of the agonist. Maximal accumulation of GroPIns(3,4)P was measured at 5 min.


Figure 2: Time course of the formation of phosphatidic acid and PtdIns(3,4)P in ConA-stimulated platelets. P-Labeled platelets were incubated at 37 °C with 100 µg/ml ConA under constant stirring for the indicated times. Lipids were extracted, deacylated, and separated by HPLC. A, time course of GroP accumulation; B, time course of sn-glycero-phosphoinositol-3`,4`-bisphosphate synthesis. Data are representative of three similar experiments.



Synthesis of PtdIns(3,4)P in ConA-stimulated Platelets Did Not Require Platelet Aggregation

To investigate the role of platelet aggregation in the synthesis of PtdIns(3,4)P, P-labeled platelets were stimulated at 37 °C with 1 unit/ml thrombin and 100 µg/ml ConA for 5 min with and without constant stirring. Both thrombin and ConA-treated platelets did not aggregate when stirring was omitted (data not shown). As shown in Fig. 3A, the production of PtdIns(3,4)P in thrombin-stimulated platelets was dramatically decreased in the absence of aggregation (about 60% inhibition). In contrast, a similar amount of PtdIns(3,4)P was measured in ConA-stimulated platelets independently from the occurrence of aggregation. The aggregation-independent synthesis of PtdIns(3,4)P in ConA-stimulated platelets was confirmed by using the tetrapeptide RGDS. It was reported that preincubation of platelets with 1 mM RGDS caused about 60% inhibition of PtdIns(3,4)P synthesis induced by thrombin (13) . However, as shown in Fig. 3B, treatment with 1 mM RGDS did not modify the ability of ConA to induce accumulation of PtdIns(3,4)P in human platelets.


Figure 3: Effect of platelet aggregation on the production of PtdIns(3,4)P. A, P-Labeled platelets were stimulated with 1 unit/ml thrombin or 100 µg/ml ConA at 37 °C for 5 min with or without stirring as indicated. Formation of PtdIns(3,4)P was determined by HPLC analysis of the deacylated lipids. Synthesis of PtdIns(3,4)P in stimulated platelets under constant stirring (4308 ± 1112 dpm in thrombin-stimulated platelets and 2357 ± 626 dpm in ConA-stimulated platelets) is reported as 100% of production. Statistic significances by Student's t test are as follows: thrombin with stirring versus thrombin without stirring, 0.01 < p < 0.02; ConA with stirring versus ConA without stirring, not significant (p > 0.3). B, P-labeled platelets were incubated at 37 °C for 2 min with buffer or with 1 mM RGDS before stimulation with 100 µg/ml ConA for 5 min under constant stirring. Results are expressed as percentage of the production of PtdIns(3,4)P observed in ConA-stimulated platelets. Student's t test indicates that the difference between ConA and RGDS + ConA is not statistically significant (0.1 < p < 0.2).



Accumulation of PtdIns(3,4)P in ConA-treated Platelets Was Triggered by Clustering of Membrane Glycoproteins Different from GP IIb-IIIa

The lectin ConA is a tetrameric ligand able to induce clustering of its membrane receptor (33) . We investigated the role of platelet glycoproteins clustering in ConA-induced synthesis of PtdIns(3,4)P by using a dimeric derivative of the lectin, the succinyl-ConA (S-ConA). S-ConA maintains the same glycoprotein specificity as the native lectin and can recognize the same components on the platelet surface (36) . However, unlike ConA, S-ConA does not induce clustering of its receptor (37) . Fig. 4shows that S-ConA, unlike the tetrameric ConA, did not induce accumulation of PtdIns(3,4)P in human platelets and did not behave as a platelet agonist. This indicates that the clustering of the ConA receptor by the native lectin rather than the mere binding to the membrane glycoproteins can activate the signaling pathways leading to PtdIns(3,4)P accumulation.


Figure 4: Succinyl-ConA did not induce synthesis of PtdIns(3,4)P. P-Labeled platelets were incubated with buffer (Resting), with 100 µg/ml ConA (ConA) or 100 µg/ml S-ConA at 37 °C for 5 min with constant stirring. Typical HPLC profiles, representative of several experiments, are reported. Abbreviations are as in Fig. 1.



As GP IIb-IIIa is the main platelet receptor for ConA, which becomes clustered upon binding of the lectin (33) , we analyzed the ConA-induced accumulation of PtdIns(3,4)P in platelets from two patients affected by type I Glanzmann thrombasthenia. Platelets from both patients did not possess immunologically detectable GP IIb-IIIa (data not shown). shows that the amount of PtdIns(3,4)P observed in ConA-treated thrombasthenic platelets was only slightly reduced when compared to that observed in ConA-treated normal platelets. We also tested the effect of thrombin on platelets from one thrombasthenic patient. As previously described (13), we confirmed that synthesis of PtdIns(3,4)P in thrombin-stimulated thrombasthenic platelets was greatly reduced when compared with normal platelets ().

Protein-tyrosine Phosphorylation Did Not Mediate ConA-induced Synthesis of PtdIns(3,4)P

It has been previously shown that ConA induces tyrosine phosphorylation of several proteins in human platelets (29) . Since a proposed mechanism of PtdIns 3-kinase activation involves the tyrosine phosphorylation of the regulatory subunit p85 (4) , we analyzed whether the regulatory subunit of the PtdIns 3-kinase, p85, was tyrosine phosphorylated in ConA-stimulated platelets. Lysates from resting and ConA-treated platelets were immunoprecipitated either with anti-p85 antiserum or with a preimmune serum. Immunoprecipitated proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and probed with anti-phosphotyrosine antibodies. As shown in Fig. 5 , no tyrosine-phosphorylated proteins with molecular mass corresponding to the regulatory subunit of the PtdIns 3-kinase were detected in the anti-p85 immunoprecipitates. In contrast, p85 was clearly detected in immunoprecipitates obtained with anti-phosphotyrosine antibodies from ConA-treated platelets but not from resting platelets (Fig. 5). This indicates that ConA induces the association of nonphosphorylated p85 with tyrosine-phosphorylated proteins. A similar behavior was also reported in thrombin-stimulated platelets (8) .


Figure 5: ConA-induced association of p85 with tyrosine-phosphorylated proteins. Lysates from resting (basal (Bas)) or ConA-stimulated platelets (100 µg/ml ConA, 5 min at 37 °C) were immunoprecipitated with anti-p85 polyclonal antiserum, anti-phosphotyrosine antibodies, or preimmune serum as indicated under ``Experimental Procedures.'' Immunoprecipitated proteins and a sample of total platelet lysate (PLT) were transferred to nitrocellulose and probed with anti-phosphotyrosine antibodies (A) or anti-p85 polyclonal antiserum (B) as indicated. Molecular mass markers are indicated on the left. The position of p85 is indicated on the right.



The role of protein-tyrosine phosphorylation events in the synthesis of PtdIns(3,4)P induced by ConA was further investigated using the specific tyrosine kinase inhibitor tyrphostin AG-213. This compound was known to inhibit both aggregation and PtdIns(3,4)P synthesis in thrombin-stimulated platelets (8). We found that, when added to tyrphostin-treated platelets, ConA was able to induce accumulation of PtdIns(3,4)P similar to that observed in control platelets (Fig. 6). On the other hand, we confirmed that in our experimental condition tyrphostin was able to strongly decrease both aggregation and accumulation of PtdIns(3,4)P in thrombin-stimulated platelets (data not shown).


Figure 6: Effect of tyrphostin AG-213 on the production of PtdIns(3,4)P in ConA-stimulated platelets. P-Labeled platelets were incubated for 2 min at 37 °C with 100 µM tyrphostin AG-213 or an equal volume of dimethyl sulfoxide, and then buffer or ConA (100 µg/ml, final concentration) was added. After 5 min, lipids were extracted and deacylated, and the production of PtdIns(3,4)P was determined by HPLC analysis. Data are expressed as percentage of the PtdIns(3,4)P production in ConA-stimulated platelets.




DISCUSSION

In this study, we demonstrate that synthesis of PtdIns(3,4)P in human platelets stimulated with the lectin ConA does not require platelet aggregation. Prevention of aggregation by the omission of stirring or by preincubation of platelets with the tetrapeptide RGDS did not impair the ability of ConA to stimulate the accumulation of PtdIns(3,4)P. These evidences are particularly interesting when compared with the results obtained using thrombin as platelet agonist. Previous works demonstrated that inhibition of fibrinogen binding by RGD-containing peptides resulted in an inhibition of PtdIns(3,4)P synthesis induced by thrombin (12, 13) , and a strong reduction of accumulation of PtdIns(3,4)P was observed in platelets from patients affected by Glanzmann thrombasthenia, which lack the fibrinogen receptor and fail to aggregate (13) . This evidence supported a role for GP IIb-IIIa in regulating the activation of PtdIns 3-kinase in human platelets. It should be noted, however, that the use of RGD-containing peptides or thrombasthenic platelets actually interferes with two closely related but clearly different events occurring in activated cells: fibrinogen binding to GP IIb-IIIa and platelet aggregation. Both events require a functional GP IIb-IIIa, as binding of fibrinogen is strictly necessary to start aggregation. However, while fibrinogen binding to GP IIb-IIIa fails to produce aggregation if platelets are not stirred, when stimulated platelets are simultaneously stirred, aggregation ensues, and secondary events on the cell surface occur: the GP IIb-IIIa is clustered (23) , several glycoproteins, including the GP IIb-IIIa itself and the PECAM-1, interact with the intracellular cytoskeleton (26, 27) , and other adhesive proteins, such as released thrombospondin, bind to their receptors on the cell surface to stabilize platelet aggregates (25, 26) . As inhibition of fibrinogen binding necessarily results in the prevention of platelet aggregation and related events, the available data actually do not clarify if the synthesis of PtdIns(3,4)P is directly regulated by GP IIb-IIIa or by other events occurring during platelet aggregation. In this paper, we show that stimulation of platelets with thrombin in the absence of stirring, in such a way to allow fibrinogen binding to GP IIb-IIIa, but not aggregation resulted in a marked decrease of PtdIns(3,4)P synthesis, which is similar to the decrease previously described in thrombin-stimulated, RGDS-treated platelets (12, 13). This indicates that the regulatory role of GP IIb-IIIa on PtdIns(3,4)P production is mainly played through platelet aggregation. When ConA was used as platelet agonist, we observed accumulation of PtdIns(3,4)P independent of both fibrinogen binding and platelet aggregation (i.e. in the absence of stirring and in the presence of the peptide RGDS). Therefore, our results identify an excellent simplified experimental system to study the mechanisms regulating the PtdIns 3-kinase in human platelets.

The most intriguing consideration about the aggregation-independent synthesis of PtdIns(3,4)P in ConA-stimulated platelets is that, unlike other lectins, ConA reproduces in nonaggregated platelet events that usually occur only during aggregation when physiological agonists are used. For instance, ConA induces clustering of membrane glycoproteins including GP IIb-IIIa (33) , which is normally promoted by fibrinogen during aggregation (23) . Moreover, ConA promotes the association of the fibrinogen receptor with the intracellular cytoskeleton (30) , which, in thrombin-stimulated platelets, is normally a consequence of aggregation (26) . Here, we demonstrate that the clustering of membrane glycoproteins is a crucial event in ConA-induced synthesis of PtdIns(3,4)P. In fact, the dimeric derivative of the lectin, the succinyl-ConA, which binds to the same surface components as the native lectin but does not induce clustering of the receptors, is unable to induce the synthesis of PtdIns(3,4)P in human platelets. Although it cannot be ruled out that ConA and thrombin activate the PtdIns 3-kinase through different signaling pathways, these results may indicate that also in thrombin-aggregated platelets the signal leading to accumulation of PtdIns(3,4)P is generated by the fibrinogen-promoted clustering of membrane glycoproteins.

One of the membrane components clustered during platelet aggregation and upon binding of ConA is the GP IIb-IIIa (23, 33) . We used platelets from patients affected by Glanzmann thrombasthenia, which lack the GP IIb-IIIa, to clarify the role of clustering of the fibrinogen receptor. It was previously reported that thrombin displayed a reduced ability to induce synthesis of PtdIns(3,4)P in thrombasthenic platelets (13) . Here, we confirm this observation. However, when we compared the production of PtdIns(3,4)P in ConA-treated thrombasthenic platelets with that of ConA-treated normal platelets, we only observed a slight reduction, which was not comparable with the inhibition observed when thrombin was used as agonist. We conclude that the reduced synthesis of PtdIns(3,4)P in thrombin-treated thrombasthenic platelets was related not to the absence of GP IIb-IIIa but to the inability of these platelets to aggregate. The different behavior of ConA indicates that, when distinguished from platelet aggregation, GP IIb-IIIa is not an essential regulatory factor for PtdIns 3-kinase activation. It is thus clear that the signal transduction pathway initiated by the lectin ConA and leading to accumulation of PtdIns(3,4)P requires clustering of membrane glycoproteins different from the fibrinogen receptor.

We propose here a working model to explain the GP IIb-IIIa-dependent synthesis of PtdIns(3,4)P in thrombin-aggregated platelets. When platelets are activated, fibrinogen binds to GP IIb-IIIa, and in the presence of stirring, aggregation ensues. During aggregation, fibrinogen triggers clustering of GP IIb-IIIa. As aggregation proceeds, other membrane glycoproteins are included in these clusters, maybe facilitated by the action of other adhesive proteins. In this regard, it is interesting that a proposed model to explain the role of secreted thrombospondin in the stabilization of platelet aggregates involved the physical association between fibrinogen-bound GP IIb-IIIa and its putative receptor, the glycoprotein IV (25, 38) . The formation of these heterogenic clusters finally generates the signal leading to PtdIns(3,4)P accumulation. ConA, by binding directly to the membrane glycoproteins, may act bypassing the initial steps and directly promoting the signal-generating clusters. As ConA binds to several major and minor membrane glycoproteins, it is very difficult to identify the receptor responsible for the activation of the PtdIns 3-kinase. Moreover, we cannot rule out that the recruitment of different glycoproteins rather than a single class of molecules into the cluster is responsible for the activation of the PtdIns 3-kinase.

The signaling pathway activated by the cluster of membrane glycoproteins and leading to PtdIns(3,4)P accumulation is still unknown. We investigated the possible relationship between protein-tyrosine phosphorylation and activation of PtdIns 3-kinase. Although we found that ConA induced the association of the regulatory subunit of the enzyme with tyrosine-phosphorylated proteins, we did not observe any tyrosine phosphorylation of p85 under conditions producing maximal accumulation of PtdIns(3,4)P. Moreover, we found that the tyrosine kinase inhibitor tyrphostin did not affect the production of PtdIns(3,4)P in ConA-stimulated platelets. These evidences indicate that tyrosine phosphorylation events are not essential for PtdIns 3-kinase activation. Inhibition of PtdIns(3,4)P synthesis by tyrphostin was described in thrombin-stimulated platelets (8) . However, treatment of platelets with this tyrosine kinase inhibitor also resulted in the inhibition of platelet aggregation (8) . Therefore, it was questionable whether the effect of tyrphostin on PtdIns(3,4)P production was directly related to the inhibition of tyrosine kinases or mediated by the inhibition of platelet aggregation. The data presented in this work using an aggregation-independent model suggest that the observed inhibition of PtdIns(3,4)P synthesis by tyrphostin in thrombin-activated platelets was most likely related to the inhibition of platelet aggregation. Thus, the signaling pathway leading to PtdIns(3,4)P synthesis in human platelets seems to be different from that operating in nucleated cells stimulated with growth factors, where an important role is played by the protein-tyrosine kinases (4) . In a recent report, activation of PtdIns 3-kinase by the low molecular weight GTP-binding protein Rho was demonstrated in human platelets (11) . Rho is known to play a role in the cytoskeletal organization (39) , and it is interesting that the cytoskeleton of aggregated platelets interacts with both membrane glycoproteins and activated PtdIns 3-kinase (26, 40, 41) . The exact role of the platelet cytoskeleton and associated proteins in the stimulation of the synthesis of PtdIns(3,4)P remains to be investigated, but recent results by Guinebault et al.(7) suggest that interactions between pp120 and PtdIns 3-kinase in specific regions of the cytoskeleton may directly activate the PtdIns 3-kinase.

In conclusion, the aggregation-independent synthesis of PtdIns(3,4)P in ConA-stimulated platelets described in this paper represents an excellent experimental model used to clarify the role played by GP IIb-IIIa, membrane glycoproteins clustering, and protein-tyrosine kinases in the stimulation of PtdIns(3,4)P production in human platelets. This model can be usefully exploited in future investigations to provide further insights into the biochemical mechanisms regulating PtdIns(3,4)P synthesis in human platelets.

  
Table: Synthesis of PtdIns(3,4)P in thrombasthenic platelets

Platelets from healthy donors and from two patients affected by Glanzmann thrombasthenia (C. S. and M. L.) were labeled with P and stimulated at 37 °C for 5 min with 1 unit/ml thrombin or 100 µg/ml ConA. Production of PtdIns(3,4)P was analyzed by HPLC. Data are expressed as percentage of the production of PtdIns(3,4)P observed in stimulated normal platelets.



FOOTNOTES

*
This work was supported by grants from the INSERM-CNR agreement and from Ministero dell' Universit e della Ricerca Scientifica e Tecnologica (Italy). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 39-382-507238; Fax: 39-382-507240.

The abbreviations used are: PtdIns 3-kinase, phosphatidylinositol 3-kinase; PtdIns(3)P, phosphatidylinositol 3`-phosphate; PtdIns(3,4)P, phosphatidylinositol 3`,4`-bisphosphate; PtdIns(3,4,5)P, phosphatidylinositol 3`,4`,5`-trisphosphate; ConA, concanavalin A; S-ConA, succinyl-ConA; GP, glycoprotein; HPLC, high performance liquid chromatography.


ACKNOWLEDGEMENTS

We thank Dr. M. Poggio (Transfusion Unit, Hospital L. Sacco, Milan, Italy) for providing blood samples of thrombasthenic patients and Drs. B. Payrastre and C. Racaud-Sultan (INSERM U326, Toulouse, France) for helpful discussion.


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