COMMUNICATION
A Novel Integrin-activated Pathway Forms PKB/Akt- stimulatory Phosphatidylinositol 3,4-Bisphosphate via Phosphatidylinositol 3-Phosphate in Platelets*

Hrvoje Banfic', Xiu-wen TangDagger , Ian H. BattyDagger , C. Peter DownesDagger , Ching-shih Chen§, and Susan E. Rittenhouse

From the Kimmel Cancer Institute and Cardeza Foundation for Hematologic Research, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, the Dagger  Department of Biochemistry, University of Dundee, Dundee Scotland DD1 4HN, United Kingdom, and the § Division of Medicinal Chemistry and Pharmaceutics, The University of Kentucky, Lexington, Kentucky 40536

    ABSTRACT
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Abstract
Introduction
Procedures
Results & Discussion
References

The aggregation of human platelets is an important physiological hemostatic event contingent upon receptor-dependent activation of the surface integrin alpha IIbbeta 3 and subsequent binding of fibrinogen. Aggregating platelets form phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2), which has been reported to stimulate in vitro the activity of the proto-oncogenic protein kinase PKB/Akt, as has phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3). It has been assumed that PtdIns(3,4)P2 is synthesized by either 5-phosphatase-catalyzed hydrolysis of PtdIns(3,4,5)P3 produced by phosphoinositide 3-kinase (PI3K) or phosphorylation by PI3K of PtdIns4P. We investigated the route(s) by which PtdIns(3,4)P2 is formed after directly activating alpha IIbbeta 3 with anti-ligand-induced binding site Fab fragment and report that aggregation does not lead to the generation of PtdIns(3,4,5)P3, but to transient formation of PtdIns3P and generation of PtdIns(3,4)P2, the latter primarily by PtdIns3P 4-kinase. Both this novel pathway and the activation of PKB/Akt are inhibited by the PI3K inhibitor, wortmannin, and the calpain inhibitor, calpeptin, constituting the first evidence that PtdIns(3,4)P2 can stimulate PKB/Akt in vivo in the absence of PtdIns(3,4,5)P3. Integrin-activated generation of the second messenger PtdIns(3,4)P2 thus depends upon a route distinct from that known to be utilized initially by growth factors. This pathway is of potential general relevance to the function of integrins.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results & Discussion
References

Human platelets have provided a model system for a variety of signal transduction events, including integrin-based signaling. Platelets can be activated by agents that include agonists for the thrombin receptor (THR-R),1 leading to a change in integrin alpha IIbbeta 3 conformation to one that binds plasma fibrinogen (FIB) and results in aggregation. The change in integrin is dependent partially upon the activation of an 85 KD subunit-containing form of PI3K (1, 2), which acts in vivo on PtdIns(4,5)P2 and rapidly generates PtdIns(3,4,5)P3 and PtdIns(3,4)P2, but not PtdIns3P (3, 4). Late (post-aggregation) accumulations of PtdIns(3,4)P2, but not the levels of PtdIns(3,4,5)P3 (5), have been found to be regulated by extracellular Ca2+ and binding of FIB to alpha IIbbeta 3 (5, 6). Other work has shown that THR-R-dependent accumulation of PtdIns(3,4)P2 can be impaired by calpeptin, an inhibitor of the Ca2+-dependent protease calpain, which is activated under these conditions (7-9). Norris et al. (9) have suggested that calpain hydrolytically inactivates PtdIns(3,4)P2 4-phosphatase, thereby elevating PtdIns(3,4)P2. The rise in PtdIns(3,4)P2 that follows THR-R stimulation has been correlated kinetically with the regulation of the serine-threonine kinase PKB/Akt (10), although a role for the earlier elevation in PtdIns(3,4,5)P3 levels could not be discounted by these studies. Indeed, both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 are potent stimuli for PDK1, which phosphorylates PKB/Akt and thereby activates it (11). Another report has also described a PKB/Akt-activating kinase that is stimulated by PtdIns(3,4,5)P3 (12). Two crucial questions naturally arise: 1) is the mechanism of integrin (aggregation)-induced formation of PtdIns(3,4)P2 different from that used by other receptors?; 2) can PKB/Akt be activated in platelets in the absence of PtdIns(3,4,5)P3 formation? To address these questions, we have by-passed the need for THR-R stimulation to promote alpha IIbbeta 3/FIB-dependent aggregation by taking advantage of the findings that exposure of platelets to the Fab fragment of an antibody directed to beta 3 (LIBS) favors the conformation of FIB-binding alpha IIbbeta 3 (13) and leads to the accumulation of PtdIns(3,4)P2 (14).

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results & Discussion
References

Wortmannin, FIB, apyrase, and miscellaneous chemicals were purchased from Sigma. Anti-ligand-induced binding site antibody 6 was the generous gift of Dr. Mark Ginsberg (Scripps Research Institute, La Jolla, CA). Anti-LIBS6 Fab fragment ("LIBS") was prepared as described (13) and was used in all experiments. Calpeptin was purchased from NovaBiochem and Biomol, and calpastatin was from Calbiochem. [3H]Ins(1,3,4)P3 was from NEN Life Science Products and [gamma -32P]ATP from ICN. DiC16PtdIns3P was purchased from Matreya, Inc. (Pleasant Gap, PA) or, as for diC16PtdIns(3,4)P2 and diC16PtdIns(3,4,5)P3, was synthesized (15, 16). Synthetic phosphoinositides were characterized by 1H and 31P NMR, and fast atom bombardment mass spectrometry and no appreciable impurities were detected.

Intact Platelets-- Platelets were isolated as described from fresh human plasma treated with 1 mM aspirin to block prostanoid-induced activation of platelets (2). Washed human platelets (2 × 109/ml) were incubated to equilibrium (90 min) with [32P]Pi (3, 4) and exposed in the presence of apyrase (10 units/ml, to remove platelet-activating ADP) and Ca2+ (0.5 mM) to FIB (400 µg/ml) ± LIBS (4 µM) for varied periods ±stirring at 37 °C. Where present, LIBS was added to platelets 5 min prior to FIB. Lipids were extracted, resolved, and phosphoinositides quantitated (3, 4). In one experiment, diC16PtdIns3P (0.2 mM) was included in incubations, added 3 min after FIB, to determine whether there was platelet lysis resulting in access to PtdIns3P 4-kinase. In some cases, labeled platelets were preincubated with wortmannin (20 nM), the cell-penetrating calpain inhibitor calpeptin (100 µg/ml), or the cell-impermeable calpain inhibitor calpastatin. In other cases, platelets were incubated with SFLLRN (25 µM), a peptide agonist for the platelet THR-R, in place of LIBS + FIB, for 20 or 120 s. PtdIns(3,4,5)P3 mass, in extracts from nonlabeled platelets, was assayed as described (17). For non-equilibrium labeling, platelets were exposed to [32P]Pi for 2 min prior to the addition of FIB or 10 min prior to THR or SFLLRN. Incubations were terminated 6 min (LIBS + FIB) or 60 s (THR or SFLLRN) after exposure to agonists. Lipids were extracted and incorporation of 32P into phosphoinositides assessed. 32P-PtdIns(3,4)P2 was digested for positional analysis of label as described (4).

Platelet Fractions-- Cytosol was prepared from unstimulated platelets (19). Triton X-100-insoluble CSK (18, 19) was isolated from LIBS + FIB- or FIB-treated platelets after 5.5 min of stirring. EGTA (20 mM) and, in some cases, calpeptin (200 µg/ml), were included in Triton buffer for lysis. Washed CSK (50 µg) was incubated with 1 mg/ml mixtures (19) of diC16PtdIns3P, PtdIns, PtdIns4P, PtdIns(4,5)P2, or diC16PtdIns(3,4)P2 and phosphatidylserine, for assays of lipid kinase activity (2). CSK fractions were also assayed for [32P]PtdIns(3,4)P2-phosphatase activity at 37 °C for 10 min, utilizing substrate synthesized by the above kinase reaction and extracted. In other studies, unlabeled platelets were incubated ±calpeptin or wortmannin, followed by FIB or LIBS + FIB and stirring for various periods, or with SFLLRN (25 µM) for 2 min. CSK and Triton-soluble fractions were obtained (2, 19). PKB was immunoprecipitated from Triton-soluble fractions, and its activity assayed with "Crosstide" peptide as described (20). The presence of PKB in CSK fractions was also determined by Western blot (20).

    RESULTS AND DISCUSSION
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Abstract
Introduction
Procedures
Results & Discussion
References

We observed that exposure of 32P-labeled platelets to LIBS + FIB caused a time-dependent, transient accumulation of [32P]PtdIns3P, preceding the maximum accumulation of [32P]PtdIns(3,4)P2, both of which were accelerated by stirring (Fig. 1), which also accelerated aggregation (not shown). Apparently clustering of platelets in an aggregate was required for a full response. Neither LIBS nor FIB alone was effective, with or without stirring. No increase in the amount of PtdIns(3,4,5)P3 was detectable in response to LIBS + FIB, as determined either by 32P labeling or mass measurements, assayed as described recently (17). In contrast, SFLLRN caused an increase in PtdIns(3,4,5)P3 mass of 4-6-fold in 20 s; a 4-fold increase was observed for [32P]PtdIns(3,4,5)P3. SFLLRN-activated platelet levels of PtdIns(3,4,5)P3 were 35 pmol/5 × 108 platelets. Preincubation of platelets with 20 nM wortmannin or 100 µg/ml calpeptin inhibited the accumulations of PtdIns3P/PtdIns(3,4)P2 by 80/91 and 92/95%, respectively, whereas neither calpeptin nor wortmannin inhibited LIBS + FIB-induced aggregation. The presence of exogenous diC16PtdIns3P had no effect on the amount of [32P]PtdIns(3,4)P2 that accumulated, whether or not wortmannin was present, which implied that the platelets were intact (see below). When platelets were not labeled to equilibrium, but were labeled briefly with [32P]Pi before activation by stirring with LIBS + FIB, again increased radioactivity was observed in PtdIns(3,4)P2 and PtdIns3P versus LIBS-free controls. LIBS had only a minor (+10%) effect on incorporation of 32P into PtdIns(4,5)P2, which also argued against LIBS + FIB-induced aggregation causing platelet lysis and thereby increased uptake of 32Pi. When the relative amounts of 32P on the 1, 3, and 4 positions of the inositol ring of PtdIns(3,4)P2 (the last site to be labeled being the "hottest" phosphate) were analyzed, the 4 position contained the majority of 32P (Fig. 2). In contrast, for platelets exposed briefly to either thrombin or SFLLRN (Fig. 2), the 3-phosphate was the hottest, in confirmation of our earlier findings (4) and of reports, utilizing similar protocols, for activated neutrophils (21) and growth factor-stimulated 3T3 cells (22). Thus, most of PtdIns(3,4)P2 synthesized in response to aggregation by LIBS + FIB is generated via PtdIns right-arrow PtdIns3P right-arrow PtdIns(3,4)P2, whereas early generation of PtdIns(3,4)P2 caused by THR-R stimulation proceeds primarily via PtdIns(4,5)P2 right-arrow PtdIns(3,4,5)P3 right-arrow PtdIns(3,4)P2, and/or PtdIns4P right-arrow PtdIns(3,4)P2.


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Fig. 1.   Production of PtdIns3P and PtdIns(3,4)P2 in LIBS + FIB-stimulated platelets. Platelets, labeled to equilibrium with 32P, were preincubated with (broken lines) or without (solid lines) calpeptin and exposed to LIBS + FIB without (A) or with (B) stirring for varied periods. [32P]PtdIns3P (open symbols) and [32P]PtdIns(3,4)P2 (closed symbols) were quantitated in comparison with FIB-exposed controls and expressed as fold control ± S.D., representing two to three experiments. Calpeptin had no effect on basal levels of [32P]phosphoinositides. Average basal radioactivities for two separate experiments were: PtdIns(3,4,5)P3 = 1466 ± 34 dpm; PtdIns(3,4)P2 = 2781 ± 403 dpm; PtdIns3P = 7276 ± 1076 dpm. For all figures, error bars are contained within symbols, if not shown.


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Fig. 2.   Distribution of 32P on the inositol ring of PtdIns(3,4)P2. Non-equilibrium-labeled, stirred platelets were exposed to LIBS + FIB (open bars), SFLLRN (closed bars), or thrombin (diagonally striped bars). The amount of 32P in each position of the inositol ring of PtdIns(3,4)P2 was analyzed and shown as a percent of the total. Results are the average ± range of duplicates (SFLLRN, thrombin) or the mean ± S.D., n = 6 (LIBS + FIB).

A PtdIns3P 4-kinase has been reported to be present in the cytosolic fraction of platelets and erythrocytes, although its activity was not compared with that of PtdIns4P 3-kinase (23). We compared the two activities in platelet cytosol and found the 4-kinase to be at least as active as the 3-kinase, but the former activity was unimpaired by 20 nM wortmannin, whereas the latter was inhibited more than 90%. No PtdIns(3,4)P2 5-kinase activity was detectable. The Triton-insoluble fraction of THR-R-activated platelets has been found to contain focal adhesion-like aggregates of alpha IIbbeta 3, CSK proteins, and PI3Ks (18, 19, 24), as well as calpain (7). When platelets were exposed to LIBS + FIB or FIB (stirred controls), and then CSK was obtained, the specific activity and total activity of PtdIns3P 4-kinase were found to increase 2-fold (Fig. 3) and 3-6-fold, respectively, with LIBS + FIB. Similarly, the specific activities of PtdIns 3-kinase and PtdIns4P 3-kinase also increased 2-3-fold, whereas that of PtdIns(4,5)P2 3-kinase or PtdIns 4-kinase did not increase. The increases were blocked completely by preincubation of platelets with calpeptin, but not by calpastatin or calpeptin added only with the platelet lysis buffer (not shown). Wortmannin inhibited the increase in CSK PI3K activities by up to 96%, whereas PtdIns3P 4-kinase was inhibited by only 4-10%. Hydrolysis of [32P]PtdIns(3,4)P2 by CSK phosphatase (not shown) was 40-41%, was decreased 92% by 10 µM unlabeled diC16PtdIns(3,4)P2, and did not differ for FIB- versus LIBS + FIB-treated platelets; therefore, the increased production of PtdIns(3,4)P2 in CSK fractions is due to increased kinase activity, rather than decreased phosphatase activity.


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Fig. 3.   Changes in specific activity of CSK phosphoinositide kinases. Platelets, with or without (open bars) exposure to wortmannin (solid bars) or calpeptin (diagonally striped bars) were incubated with LIBS + FIB or FIB, lysed, and Triton-insoluble CSK isolated. Equal amounts of CSK protein were assayed for various phosphoinositide kinase activities (indicated below figure). Assay results were divided by values for control (FIB) CSK and are shown for two to three experiments ± S.D.

Other experiments were performed to determine whether platelet exposure to LIBS + FIB would affect PKB/Akt activity. PKB activity was increased up to 3-fold in comparison with FIB controls, paralleling the rise in PtdIns(3,4)P2 levels, and this was inhibited 90-97% by preincubation of platelets with either wortmannin or calpeptin (Fig. 4). SFLLRN, causing a 6-fold rise in PtdIns(3,4)P2 in 2 min, and a 3-5-fold (32P or mass assay) rise in PtdIns(3,4,5)P3 in 20 s, stimulated PKB by 10-fold (2 min), in agreement with the increase in PKB activity reported previously (10). PKB, which was immunologically undetectable in CSK from unstimulated platelets, increased substantially in CSK of LIBS + FIB-exposed cells (not shown).


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Fig. 4.   Activation of PKB/Akt in platelets. Platelets were incubated with LIBS + FIB or FIB for different periods with stirring. Wortmannin (+W) or calpeptin (+C) with LIBS + FIB-exposed platelets were also studied for 12 min. PKB/Akt was immunoprecipitated from Triton-soluble fractions and assayed for kinase activity. Control values did not vary with time. Results shown are for two experiments, expressed as activity divided by control (FIB) value.

We conclude that a novel pathway is employed during alpha IIbbeta 3 integrin-dependent signaling in platelets, in which calpain activation may play a crucial role. To our knowledge, this is the first indication of stimulatory control of PtdIns3P synthesis in any cell. Calpain is known to hydrolyze several cytoskeletally associated proteins. Given the inhibition of PtdIns3P and PtdIns(3,4)P2 accumulations and of increased CSK PI3K activity by preincubation with calpeptin, but not by calpastatin or by calpeptin added in lysis buffer, it seems likely that calpain hydrolyzes a PI3K or attacks a CSK protein that affects PI3K. Further direct studies with calpain and platelet PI3Ks should help to clarify this issue. The LIBS + FIB-activated PI3K apparently cannot utilize PtdIns(4,5)P2 as a substrate, but acts primarily on PtdIns, as for several recently described PI3Ks from yeast, Drosophila, and mammalian cells. Possible candidates for this role, in addition to a form of p85/PI3K or PI3Kgamma (both present in platelets; Refs. 19 and 24) that may have been altered, are: 1) a mammalian version of VPS34 (25), which is known in yeast to be involved in vesicle trafficking, and is sensitive to wortmannin, acts only on PtdIns and is present in platelets,2 and 2) two C2 domain-containing PI3Ks, whose messages are found in U937 (26) and CHRF cells, are present in platelets,3, >4 and the cloned and expressed versions of which utilize PtdIns and PtdIns4P in great preference to PtdIns(4,5)P2 (26). It is noteworthy that the activation of PtdIns3P 4-kinase is also calpeptin-sensitive. Under our conditions, the stimulated, calpeptin-inhibitable increase in PtdIns(3,4)P2 accumulation is attributable to PtdIns 3-kinase and PtdIns3P 4-kinase, rather than to inhibited 4-phosphatase activity (9), which inhibition would cause a decrease in PtdIns3P levels. Instead, we observe a calpeptin-inhibitable increase in PtdIns3P and no effect of LIBS + FIB on CSK phosphatase activity with respect to PtdIns(3,4)P2 substrate.

These findings may have important implications for integrin-linked signaling in a variety of cells. Furthermore, the stimulated increase in PtdIns(3,4)P2, without a rise in PtdIns(3,4,5)P3, strongly suggests that the former functions as a signal in its own right. Indeed, the PtdIns(3,4)P2 that accumulates as a result of the increased, coupled activity of PtdIns 3-kinase and PtdIns3P 4-kinase is capable of activating PKB/Akt in the platelet, and PtdIns(3,4)P2 (and possibly PtdIns3P) may prove to have additional targets in this system. These data would imply that, for example, stimulation of matrix-cultured cells by agonists that do not promote PtdIns(3,4,5)P3 accumulation or activation of conventional PI3Ks, but do cause wortmannin-inhibitable activation of p70 S6 kinase and PKB/Akt (27) might be re-examined with respect to PtdIns(3,4)P2 and the route of its formation.

    ACKNOWLEDGEMENTS

We thank Dr. Mark Ginsberg for generously contributing LIBS6 antibody, Dr. Ernest Dow (NEN Life Science Products) for providing [3H]Ins(1,3,4)P3, Dr. Dario Alessi for providing anti-PKB antibody, and the Jefferson Commons pool facility for the opportunity to swim and think.

    FOOTNOTES

* This work was supported by NHLBI Grant HL 38622 (to S. E. R.), NATO Grant 950672 (to S. E. R.), by the NIHGM (to C.-s. C.), and by awards from the Medical Research Council and Leukemia Research Fund (to C. P. D.).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.

To whom correspondence should be addressed. Tel.: 215-503-1289; Fax: 215-923-7145.

1 The abbreviations used are: THR-R, thrombin receptor; PI3K, phosphoinositide 3-kinase; PtdIns, phosphatidylinositol (locants of other phosphates on the myoinositol ring are indicated); diC16PtdIns, 1-O-(1,2-di-O-palmitoyl-sn-glycerol-3-phosphoryl)-D-myoinositol (other locants as above); PDK1, phosphoinositide-dependent kinase 1; FIB, fibrinogen; LIBS, anti-ligand-induced binding site 6 antibody Fab fraction; CSK, cytoskeleton; PKB/Akt, protein kinase B related to AKR mouse T-cell lymphoma-derived oncogenic product.

2 J. Zhang and S. E. Rittenhouse, unpublished results.

3 J. Zhang, J. Domin, S. Volinia, S. E. Rittenhouse, manuscript in preparation.

4 J. Zhang, H. Banfic', S. Volinia, and S. E. Rittenhouse, manuscript in preparation.

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Abstract
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Results & Discussion
References

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