From the Kimmel Cancer Institute and Cardeza Foundation for
Hematologic Research, Thomas Jefferson University, Philadelphia,
Pennsylvania 19107, the 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
The aggregation of human platelets is an
important physiological hemostatic event contingent upon
receptor-dependent activation of the surface integrin
IIb
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
IIb
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 IIb
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
IIb
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
IIb
3/FIB-dependent
aggregation by taking advantage of the findings that exposure of
platelets to the Fab fragment of an antibody directed to
3 (LIBS) favors the conformation of FIB-binding
IIb
3 (13) and leads to the accumulation
of PtdIns(3,4)P2 (14).
![]() |
EXPERIMENTAL PROCEDURES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 [-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 PtdIns3P
PtdIns(3,4)P2, whereas early generation of
PtdIns(3,4)P2 caused by THR-R stimulation proceeds
primarily via PtdIns(4,5)P2
PtdIns(3,4,5)P3
PtdIns(3,4)P2, and/or PtdIns4P
PtdIns(3,4)P2.
|
|
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
IIb
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.
|
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).
|
We conclude that a novel pathway is employed during
IIb
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 PI3K
(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. Banfi, S. Volinia, and S. E. Rittenhouse, manuscript in preparation.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|