©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phosphoinositide 3-Kinase and p85/Phosphoinositide 3-Kinase in Platelets
RELATIVE ACTIVATION BY THROMBIN RECEPTOR OR beta-PHORBOL MYRISTATE ACETATE AND ROLES IN PROMOTING THE LIGAND-BINDING FUNCTION OF alphabeta(3) INTEGRIN (*)

(Received for publication, July 5, 1995; and in revised form, January 4, 1996)

Jun Zhang Jin Zhang Sanford J. Shattil (1)(§) Michael C. Cunningham (1) Susan E. Rittenhouse (¶)

From the Department of Pharmacology/Jefferson Cancer Institute and Cardeza Foundation for Hematologic Research, Philadelphia, Pennsylvania 19107 Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Platelets exposed to thrombin or thrombin receptor agonist peptide (SFLLRN) activate phospholipase C and protein kinase C (PKC), and accumulate 3-phosphorylated phosphoinositides (3-PPI) as a function of the activation and relocalization of two cytoskeletally-associated phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-K. We now report that exposure of platelets to PKC-activating beta-phorbol myristate acetate (betaPMA) does not stimulate PI 3-K, but rather stimulates p85/PI 3-K, which associates with the cytoskeleton. Wortmannin is an inhibitor of both PI 3-Ks, known to act with more potency on p85/PI 3-K. betaPMA-stimulated 3-PPI accumulation is more sensitive to wortmannin (IC = 1.3 nM) than is SFLLRN- or thrombin-stimulated 3-PPI accumulation (IC = 10 nM). The activity of p85/PI 3-K in immunoprecipitates or in cytoskeletal fractions is inhibited more potently by exposure of platelets to wortmannin than is the activity of PI 3-K. betaPMA or SFLLRN promotes the conversion of platelet integrin alphabeta(3) into a fibrinogen-binding form required for platelet aggregation. Activation of alphabeta(3) in response to betaPMA or SFLLRN is inhibited by wortmannin with an IC of 1 nM in each case. Wortmannin inhibits neither activation of alphabeta(3) by ligand-induced binding site antibody (anti-LIBS6 Fab) nor anti-LIBS6 Fab-induced platelet aggregation in the presence of fibrinogen, indicating that this type of ``outside-in'' signaling by alphabeta(3) is largely PI 3-K-independent. We conclude that p85/PI 3-K, in preference to PI 3-K, contributes to activation of alphabeta(3) when the thrombin receptor or PKC is stimulated.


INTRODUCTION

We have reported that activation of the thrombin receptor on human platelets leads to the accumulation of PtdIns(3,4,5)P(3)(^1)and PtdIns(3,4)P(2) as a function of the stimulation of two phosphoinositide 3-kinases (PI 3-K): p85/PI 3-K and PI 3-K, both of which are dependent upon GTP-binding proteins(1, 2) . Each PI 3-K contributes to the production of both 3-PPIs. The former PI 3-K, a heterodimer containing a regulatory noncatalytic p85 subunit, is activated via the small G-protein Rho(1, 3) , whereas the latter, lacking p85, is activated via the beta subunit of dissociated heterotrimeric G-proteins(1, 4) . Phospholipase C (PLC) and protein kinase C (PKC) are also activated under the same conditions, and inhibition of PKC in permeabilized platelets by a pseudosubstrate peptide partially inhibits the accumulation of 3-PPI in platelets exposed to thrombin or the direct G-protein agonist GTPS(5) . Tumor-promoting beta-phorbol esters are known to activate PKC in platelets(6, 7) , as in other cells, and earlier studies with such a phorbol ester (2) indicated that 3-PPI accumulation could be stimulated, although not as strongly as by thrombin.

In view of findings about the two species of PI 3-K that are activated by thrombin(1) , we have now re-examined the activation of PI 3-K by phorbol esters. We reasoned initially that betaPMA, which has been shown in some cells to stabilize some heterotrimeric G-proteins(8) , would be unlikely to promote the release of activating Gbeta, and therefore be unable to activate PI 3-K. This would perhaps account for some of the weaker effects of betaPMA versus thrombin in stimulating 3-PPI accumulation. It has been reported more recently, however, that, when added to platelets, betaPMA causes phosphorylation of the alpha-subunit of G(z)(9) , and that such phosphorylation inhibits association of alpha(z) with beta(10) . The possibility of a release of beta from G(z) provided us with an additional reason to evaluate the activation of PI 3-K in response to betaPMA. Wortmannin, a fungal metabolite with hemorrhagic effects in animals, is an irreversible inhibitor of PI 3-K(11) , and p85/PI 3-K in neutrophils is more sensitive to wortmannin than is PI 3-K(12) . Therefore, in this study we examined the relative susceptibility to inhibition by wortmannin of betaPMA- and thrombin receptor-stimulated 3-PPI production as well as the effects of wortmannin on p85/PI 3-K and PI 3-K activities in platelets.

A crucial question for investigators studying signal transduction is ``what role(s) do PI 3-Ks (and 3-PPIs) play in the functions of a cell?'' Prior to its recognition as a PI 3-K inhibitor, wortmannin was described as an inhibitor of betaPMA-induced platelet aggregation(13) . Aggregation is the major hemostatic function of the platelet in vivo. Integrin alphabeta(3) mediates the aggregation process by binding fibrinogen after platelet activation by agonists(15, 16) . Accordingly, we have been interested in the role of the two PI 3-Ks in regulating the conversion of platelet integrin alphabeta(3) during platelet activation to a form that is competent to bind fibrinogen, a process often referred to as ``inside-out'' signaling(1, 14) . Therefore, in the present study we investigated the relative inhibitory effects of wortmannin on alphabeta(3) activation induced by a peptide stimulator of the thrombin receptor or by betaPMA. As a control, we studied the effects of wortmannin on induction of alphabeta(3) binding function by an antibody Fab fragment (anti-LIBS6 Fab) that binds to beta(3), thereby activating alphabeta(3)(17) . This antibody also afforded us the opportunity to study PI 3-K activation in direct response to fibrinogen binding and platelet aggregation (``outside-in'' signaling).


EXPERIMENTAL PROCEDURES

Materials

FITC-PAC1, FITC-SSA6, and biotin-PAC1 were prepared as described previously(18, 19, 20) . Antibody to CDC42Hs was contributed by Dr. Richard Cerione (Cornell University). Gbeta subunits isolated from bovine brain were the gifts of Dr. Peter Downes (University of Dundee) and Dr. Paul Sternweis (University of Texas, Southwestern). Anti-LIBS6 Fab was kindly donated by Dr. Mark Ginsberg (Scripps Research Institute). Purified fibrinogen was from Kabi Diagnostica and Calbiochem. Antibody to RhoA was purchased from Upstate Biotechnology. Wortmannin was purchased from Sigma and stored as a 10 mM stock solution in fresh Me(2)SO in the dark, frozen. Since wortmannin is unstable (and poorly soluble) in physiologic aqueous buffers, it was diluted in aqueous media just before addition to platelets. alphaPMA and betaPMA were purchased from BioMol. Other reagents were as described previously(1) .

Incubations with Intact P-Labeled Platelets

Fresh human platelets from normal donors were prepared as described(1) . Platelet-rich plasma was incubated with 1 mM ASA for 20 min at room temperature to inactivate cyclooxygenase prior to washing of platelets and labeling with [P]P(i)(1) . Labeled platelets (2 times 10^9/ml), 250 µl, were incubated in buffer containing 1 mM [Ca] for 2 min at 37 °C with 0-1000 nM betaPMA, 1000 nM alphaPMA, or 0-10 µM SFLLRN. In addition, labeled platelets were incubated with buffer, betaPMA (200 nM) ± RGDS (0.5 mM), or SFLLRN (10 µM) for up to 5 min. Incubations were terminated with chloroform/methanol/HCl (2) and lipids extracted, resolved, deacylated, separated by high performance liquid chromatography, and quantitated as described(1, 2) . Furthermore, platelets were incubated for 5 min at 37 °C with Me(2)SO/buffer or 1-100 nM wortmannin just prior to addition of betaPMA (200 nM), SFLLRN (10 µM), or thrombin (5 units/ml) for 2 min, followed by lipid extraction. In some studies, platelets were incubated with Me(2)SO/buffer or 2 nM wortmannin for 5 min prior to addition of buffer or SFLLRN (10 µM) for up to 5 min. In other studies, platelets (250 µl), were incubated with 100 µM [Ca] at 37 °C with stirring (600 rpm) in siliconized aggregometer tubes, in the presence of CP (5 mM)-CPK (40 units/ml) to decrease effects of ADP. Alternatively, ADP (10 µM) was added for 5 min to unstirred platelets in the presence or absence of RGDS (1 mM; to block any effects of traces of fibrinogen in washed platelet preparations). Anti-LIBS6 Fab (192 µg/ml) was added for 5 min, followed by addition of fibrinogen (400 µg/ml) and 5 min further incubation. Incubations were terminated and lipids extracted and quantitated.

Incubations with Saponin-permeabilized Platelets

Platelets were incubated with [-P]ATP, saponin, and betaNAD with or without EDIN (Rho ADP-ribosylating enzyme; 20 µg/ml) as described (1) for 5 min prior to the addition of buffer, betaPMA (100 nM), or SFLLRN (10 µM) for 2 min, followed by extraction and quantification of 3-PPI.

Measurement of PI 3-K Activity in Platelet Fractions

Washed, ASA-treated platelets (2 times 10^9/ml) were incubated with Me(2)SO/buffer or 2 nM wortmannin for 5 min prior to incubation with buffer, thrombin (5 units/ml), alphaPMA (200 nM), or betaPMA (200 nM) for 60 or 120 s at 37 °C before lysis with an equal volume of 2% Triton X-100 buffer and isolation of the insoluble cytoskeletal fraction(1) . The remaining p85/PI 3-K was removed from the Triton-soluble 100,000 times g fraction by immunoprecipitation with p85-directed antibodies(1) : protein A-Sepharose had been loaded the previous night with anti-mouse rabbit IgG at 4 °C, and the excess removed. The Triton supernatant (1 volume) was then mixed with anti-p85 antibodies and protein A/IgG for 2 h at 4 °C, and the p85-immunoprecipitate washed and resuspended in 2 volumes of buffer A, pH 7.3(2) , for assay. Triton was removed in 1 h from the post-immunoprecipitation supernatant using Extractigel (1) and buffer A, yielding a dilution of 4 volumes. These procedures were designed to minimize exposure of lysed platelets to wortmannin. In some studies, the cytosolic fraction of unstimulated, sonicated platelets was exposed to immunoprecipitating antibodies to p85(1) , and the washed immunoprecipitate as well as the post-immunoprecipitation supernatant were incubated ± 100 nM betaPMA for 5 min prior to assay of PI 3-K activity. The presence of PKC (alpha, beta, ) was monitored by Western blotting. Washed cytoskeletal fractions, p85/PI 3-K immunoprecipitates, and Triton-free post-immunoprecipitation supernatants were also assayed for PI 3-K activity. PI 3-K assays (50 µl) were performed using equal quantities (280 µg/ml) of washed cytoskeletal protein or equal amounts, adjusted for dilution of post-immunoprecipitation supernatants or immunoprecipitates(1) . PtdIns(4,5)P(2) was present as substrate. PtdIns(4,5)P(2) and phosphatidylserine, 0.1 mg/ml, were preincubated on ice for 5 min with or without Gbeta (1 µM), betaARK-PH (5 µM; a pleckstrin homology domain-containing, non-catalytic, C-terminal fragment of beta-adrenergic kinase that binds Gbeta), or GTPS (10 µM) in buffer containing 40 mM HEPES, 2 mM EGTA, 0.2 mM EDTA, 1 mM dithiothreitol, 0.1 M NaCl, 4 mM MgCl(2), 200 µM ATP, 10 µCi/ml [-P]ATP, pH 7.4. Incubations were initiated by addition of enzyme fractions and continued for 3 or 5 min at 37 °C to obtain a linear response with respect to time. Lipids were extracted and quantitated as described(1) . In other experiments, equal amounts of cytoskeletal proteins were applied to 7.5% SDS-polyacrylamide gels and Western blotted as described, using a anti-p85alpha and beta monoclonal antibodies, antibody to RhoA, or antibody to CDC42Hs, followed by ECL detection and scanning densitometry in the linear range of the response(1) .

Aggregometry

ASA-treated platelets (10^9/ml) were stirred (600 rpm) with 100 µM Ca and with fibrinogen (400 µg/ml), or with anti-LIBS6 Fab (192 µg/ml) for 5 min followed by fibrinogen, in the presence of CP-CPK. In some cases, platelets were incubated with 100 nM wortmannin for 5 min at 37 °C, prior to washing, and incubated with anti-LIBS6 Fab/fibrinogen as above.

Flow Cytometric Studies

PAC1 is an antibody that detects the activated form of alphabeta(3), allowing quantitation by flow cytometry when PAC1 is conjugated to FITC or biotin(18, 20) . FITC-SSA6 binds to and detects exposed total alphabeta(3)(19) , and can thus monitor whether total surface expression is altered by agonists or inhibitors.

Blood was obtained from normal donors and anti-coagulated with 0.15 volumes of NIH formula A acid-citrate dextrose solution supplemented with 1 µM prostaglandin E(1) and 1 unit/ml apyrase. Platelet-rich plasma was obtained (20) and incubated for 10 min at 37 °C with either 100 nM wortmannin or 0.5% Me(2)SO. Platelets were then gel-filtered into an incubation buffer containing 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl(2), 5.6 mM glucose, 1 mg/ml bovine serum albumin, 3.3 mM NaH(2)PO(4), and 20 mM HEPES, pH 6.5, adjusted to 2 times 10^8/ml, and supplemented with 10 µM indomethacin, a cyclooxygenase inhibitor. Aliquots of platelets (5 µl) were added to incubation buffer (45 µl), pH 7.4, such that the final mixture contained 10 µM indomethacin, 40 µg/ml FITC-PAC1, and agonist (0-10 µM ADP, 0.2 µM betaPMA, 50 µM SFLLRN, or 150 µg/ml anti-LIBS6 Fab). After 5 min in the dark at room temperature, samples were diluted with 200 µl of incubation buffer and FITC-PAC1 binding to 10^4 platelets/sample was analyzed by flow cytometry (18) . In some experiments, 10 µg/ml FITC-SSA6 was present as well as 40 µg/ml biotin-PAC1 (in place of FITC-PAC1) and phycoerythrin-streptavidin (1:100). After incubation and dilution as above, binding was quantitated by dual color flow cytometry.

In experiments to study wortmannin dose-dependence, platelet-rich plasma was incubated with 1 mM ASA for 20 min, and no wortmannin or Me(2)SO was added. Platelets were concentrated, applied to a Sepharose column (20) in buffer at pH 7.4, and diluted to 6 times 10^8/ml in buffer lacking bovine serum albumin (final [bovine serum albumin] = 0.04%). Platelets (45 µl) were added to 5 µl of wortmannin (0-100 nM, final) and incubated for 5 min at 37 °C, followed by addition of 5 µl of buffer, SFLLRN (10 µM, final) or betaPMA (200 nM, final), and incubation at 37 °C for 2 min. Incubations with SFLLRN alternated with those for betaPMA at each concentration of wortmannin, 60 s apart, and were terminated by the addition of 500 µl of 0.3% bovine serum albumin/buffer at room temperature. Of this, 20 µl were mixed with 20 µl of 80 µg/ml FITC-PAC1, and kept in the dark. After 5 min, 160 µl of phosphate-buffered saline was added, and the fluorescence signal read.


RESULTS

Stimulated 3-PPI Accumulation in Platelets

SFLLRN, a thrombin receptor-directed agonist peptide, and betaPMA, a potent agonist for PKC, each stimulated the accumulation of 3-PPI in a dose- and time-dependent manner (Fig. 1, A-C), whereas alphaPMA, a phorbol ester that is ineffective in activating PKC, was also without effect in this regard. After an initial increase, by 5 min of exposure to either agonist, the levels of PtdIns(3,4,5)P(3) had declined, as had those of PtdIns(3,4)P(2) when betaPMA was used as an agonist. Based on these results, 10 µM SFLLRN and 200 nM betaPMA and 2-min incubations were chosen for future studies. Notably, P-labeled PtdOH was formed in response to SFLLRN only (Fig. 1, A and B), indicating that PLC is not activated by direct stimulation of PKC. PMA is also known not to stimulate platelet cytosolic phospholipase A(2)(21, 22) , an enzyme reported to be inhibited by low concentrations of wortmannin (23) . Furthermore, RGDS, at concentrations that block binding of fibrinogen and inhibit late-stage accumulations of PtdIns(3,4)P(2) in response to thrombin(24) , did not affect the levels of 3-PPI accumulating in response to betaPMA (not shown).


Figure 1: Accumulation of P-labeled 3-PPI and PtdOH in response to SFLLRN or PMA. Washed ASA-treated platelets were labeled with P(i) as described under ``Experimental Procedures'' and incubated for 120 s with 0-1000 nM betaPMA (A), 1000 nM alphaPMA (A*), or up to 10 µM SFLLRN (B). Other incubations (C) were for up to 5 min with SFLLRN (10 µM; solid symbols) or betaPMA (200 nM; open symbols). Incubations were terminated with acidic chloroform/methanol, and extracted lipids were resolved and quantitated. Results are expressed as % of basal (agonist-free) values ± range of duplicates (contained within symbols). Basal values: A and B, respectively, PtdIns(3,4,5)P(3) (circle) = 1738 ± 67 dpm, 2870 ± 240 dpm; PtdIns(3,4)P(2) (bullet) = 2068 ± 26 dpm, 4187 ± 93 dpm; PtdOH (Delta) = 12,382 ± 132 dpm, 22,731 ± 221 dpm.



PI 3-K Activities in Triton-soluble and -insoluble Platelet Fractions

When cytoskeletal fractions from platelets activated either by thrombin receptor-directed agonists or PMA were examined, differences in specific activity of PI 3-K were observed. These differences held true whether platelets were stimulated for 60 (not shown) or 120 s (Fig. 2A). SFLLRN (or thrombin, not shown) and betaPMA, but not alphaPMA, stimulated an increase in specific activity over basal levels. More conspicuously, only thrombin receptor stimulation resulted in cytoskeletally associated PI 3-K activity that was activable by Gbeta and inhibitable by Gbeta-binding betaARK-PH, indicative of the presence of PI 3-K(1) . Cytoskeleton from betaPMA-activated platelets thus displayed no recognizable PI 3-K activity. To determine whether activated PI 3-K might have remained Triton-soluble, the Triton supernatants were examined after the removal of Triton and >94% of p85/PI 3-K by immunoprecipitation (Fig. 2B). Activity of this supernatant for betaPMA-exposed platelets was no different from that for unstimulated controls, whereas Triton supernatants from SFLLRN-exposed platelets contained increased PI 3-K activity, inhibitable (62-75%) by betaARK-PH. All of the Triton-soluble fractions were stimulatable by Gbeta, indicating the presence of PI 3-K activity, although this fraction from betaPMA-exposed platelets was significantly less activated by Gbeta than were fractions from control platelets. When p85/PI 3-K-depleted platelet cytosol that still contained PKC was exposed to 100 nM betaPMA for 5 min, PI 3-K activity was inhibited 45%, whereas immunoprecipitated p85/PI 3-K activity was not affected (not shown).


Figure 2: PI 3-K activity in cell fractions derived from platelets activated with PMA or SFLLRN. ASA-treated platelets were incubated for 120 s with buffer, alphaPMA (200 nM), or betaPMA (200 nM). Incubations were terminated with cold Triton lysis buffer and spun at 15,000 times g times 4 min. A, equal amounts of washed cytoskeletal protein were assayed for PI 3-K activity, using PtdIns(4,5)P(2) as substrate, in the absence (open bars) or presence of betaARK-PH (5 µM; cross-hatched bars) or Gbeta (1 µM; diagonally striped bars). In other studies (B), p85/PI 3-K was removed (>94%) by immunoprecipitation (with antibodies to p85(alpha,beta)) of p85/PI 3-K, followed by removal of Triton X-100 from the post-immunoprecipitation supernatant(1) . PI 3-K activity in the post-immunoprecipitation supernatant was assayed in the absence (open bars) or presence of betaARK-PH (cross-hatched bars) or Gbeta (diagonally striped bars) as above. Results are the mean ± range of a representative experiment of two performed in duplicate. Similar results were observed when thrombin replaced SFLLRN as agonist (60 s), or when platelets were exposed to SFLLRN for 60 s.



In contrast to the findings for PI 3-K, a specific increase (increase/µg of cytoskeletal protein) in the immunologically detectable p85 subunits of PI 3-K (p85alpha and p85beta) was observed in cytoskeletal fractions when either SFLLRN (or thrombin) or betaPMA was the agonist. Whereas alphaPMA did not alter the amount of p85 in cytoskeleton, exposure to betaPMA for 60 s increased p85alpha 2.0-fold and p85beta 2.1-fold. Similarly, exposure to SFLLRN increased p85alpha 2.0-fold and p85beta 2.2-fold. The ratio of p85alpha/p85beta = 6/1. There was also an increase in the presence of the small GTP-binding proteins Rho and CDC42Hs (2-fold), which are known to promote p85/PI 3-K activity(1, 3, 25) , in SFLLRN- and betaPMA-stimulated platelet cytoskeletal fractions. In keeping with activation of p85/PI 3-K already reported (1) for platelets exposed to thrombin, ADP-ribosylation of Rho was found to inhibit the accumulation of 3-PPI in permeabilized platelets stimulated with either SFLLRN or betaPMA, although the degree of inhibition of SFLLRN-induced 3-PPI (63-75%) was greater than that of betaPMA-induced 3-PPI (40-44%). A role for additional factors is implied by the smaller inhibition observed when betaPMA, a sustained, non-localized agonist for PKC, was used.

Effects of Wortmannin of PI 3-K Activities and Activation of alphabeta(3)

Wortmannin inhibited 3-PPI accumulation in response to SFLLRN or betaPMA, exhibiting an 8 times greater potency with respect to betaPMA's effects (Fig. 3, A and B). The IC values of wortmannin for betaPMA- and SFLLRN-activated PtdIns(3,4,5)P(3) accumulation were 1.3 and 10 nM, respectively. Exposure of intact unstimulated platelets to 2 nM wortmannin lead to a selective inhibition of p85/PI 3-K versus PI 3-K, examined in fractions of Triton lysates (Fig. 4A). This also proved to be the case for platelets exposed to wortmannin prior to stimulation by SFLLRN for 2 min and isolation of the cytoskeletal fraction (Fig. 4B), whereas recruitment to the cytoskeleton of p85(alpha or beta)/PI 3-K was unaffected by exposure of platelets to low-dose wortmannin (Fig. 4, parentheses and brackets).


Figure 3: Sensitivity to wortmannin of 3-PPI accumulation in platelets exposed to betaPMA or SFLLRN. P-Labeled platelets (as in Fig. 1) were incubated for 5 min at 37 °C with various concentrations of wortmannin, followed by 120 s with buffer, betaPMA (200 nM; bullet), or SFLLRN (10 µM; circle). After termination of the incubation, resolved lipids were quantitated as described in the legend to Fig. 1. [P]PtdOH levels were unaffected by wortmannin. Basal values are subtracted from stimulated values. Data are expressed in terms of stimulated values, as a % of wortmannin-free controls, and are the mean ± S.D. of the results of three experiments in duplicate. Results for thrombin (5 units/ml; one experiment in duplicate, not shown) yielded the same IC (10-15 nM) as did those for SFLLRN. A, PtdIns(3,4)P(2); B, PtdIns(3,4,5)P(3). In a representative experiment, stimulated PtdIns(3,4,5)P(3) values (dpm) were 9293 ± 838(SFLLRN) and 4686 ± 52(PMA) and stimulated PtdIns(3,4)P(2) values (dpm) were 27,029 ± 1754(SFLLRN) and 6567 ± 144(PMA).




Figure 4: Effects of low-dose wortmannin on p85/PI 3-K and PI 3-K activities in platelet fractions. A, preparations of ASA-treated platelets were exposed to Me(2)SO/buffer (open bars) or 2 nM wortmannin (diagonally striped bars) for 5 min in duplicate, followed directly (no SFLLRN added) by ice-cold Triton lysis and centrifugation at 100,000 times g times 60 min at 4 °C. The Triton-soluble fraction (which contained >99% of p85/PI 3-K and PI 3-K and the same amount of protein ± wortmannin) was exposed to immunoprecipitating antibodies to p85/PI 3-K for 2 h at 4 °C. Triton was removed from the post-immunoprecipitation supernatant, containing virtually all of PI 3-K(1) . Washed p85/PI 3-K immunoprecipitates and post-immunoprecipitation supernatants (PI 3-K) were assayed in duplicate for PI 3-K activity in the absence or presence of GTPS (10 µM, former) or Gbeta (1 µM, latter). Basal activities were subtracted from stimulated activities. Results are the mean activity ± S.D. for two experiments in duplicate. B, platelets, as in A, were incubated ± wortmannin followed, however, by incubation with SFLLRN (10 µM) or buffer for 120 s. Cytoskeletal fractions (15,000 times g times 4 min at 4 °C = ``CSK'') were obtained rapidly after Triton lysis. The yields of cytoskeletal protein were the same ± wortmannin, but increased 26% + SFLLRN. Equal amounts of washed cytoskeletal protein preparations (containing the majority of activated p85/PI 3-K and PI 3-K, post-SFLLRN; 1) were assayed with or without betaARK-PH (5 µM) for PI 3-K activity. Results were then adjusted for total cytoskeletal protein yield. Activities were calculated for samples with (cross-hatched bars) and without (open bars) wortmannin treatment. Total activated PI 3-K activity = total activity -total activity. Total activated PI 3-K activity = total activity - total activity. Total activated p85/PI 3-K activity = total activated PI 3-K activity - total activated PI 3-K activity. Results are the mean activity ± S.D. for two duplicate preparations. Numbers in parentheses, -fold increase in specific p85alpha/PI 3-K; numbers in brackets, -fold increase in specific p85beta/PI 3-K (activated/resting cytoskeleton) quantitated after Western blotting, where the same amount of protein was applied to resolving gels. The ratio of alpha/beta-p85 was 6.2/1.



Interestingly, when the effects of wortmannin on exposure of activated alphabeta(3) (as measured by binding of PAC1 antibody) in response to the two agonists were compared (Fig. 5A), the IC values were found to be equal. Furthermore, they were the same as for inhibition of betaPMA-induced PtdIns(3,4,5)P(3) accumulation, i.e. 1 nM. A comparison of the effects of low-dose wortmannin (i.e. 1 and 2 nM, in the range giving close to maximal inhibition of PAC-1 binding) on 3-PPI accumulation in response to betaPMA or SFLLRN (Fig. 5B), revealed two points: 1) whereas the % inhibition by wortmannin of SFLLRN-induced 3-PPI accumulation was much less than that of betaPMA-induced accumulation (see also Fig. 3), the total decrease in labeled 3-PPI was very similar for the two agonists, and 2) when (at 2 nM wortmannin) 3-PPI formation in response to SFLLRN had decreased only 20%, the amount of 3-PPI remaining was greater than the amount of 3-PPI produced by betaPMA in the absence of wortmannin; yet, this amount of 3-PPI was apparently unable to ``support'' activation of alphabeta(3) (Fig. 5A). The degree of inhibition by 2 nM wortmannin of 3-PPI accumulation in response to SFLLRN did not vary appreciably over the course of a 5-min incubation with agonist (not shown).


Figure 5: Sensitivity to wortmannin of alphabeta(3) reorganization and 3-PPI accumulation for platelets activated by betaPMA or SFLLRN. Preparations of ASA-treated platelets were incubated for 5 min at 37 °C with 0-100 nM wortmannin (A) prior to exposure for 120 s to buffer, betaPMA (200 nM, bullet) or SFLLRN (10 µM, circle), alternating at each concentration of wortmannin between betaPMA and SFLLRN, as was true for labeled platelets (Fig. 3). FITC-PAC1 binding was assayed as described under ``Experimental Procedures.'' Results are representative of two experiments (each comparing platelet responses to SFLLRN or betaPMA using the same preparation of platelets) performed in duplicate, and are shown as arbitrary fluorescence units±range. Basal fluorescence is indicated by Delta*. In both cases, wortmannin's IC for betaPMA as agonist = IC for SFLLRN as agonist, i.e. <2 nM. In studies with P-labeled platelets (B), platelet suspensions were incubated as in Fig. 3with 0-2 nM wortmannin for 5 min prior to exposure to buffer, SFLLRN (10 µM, open bars), or betaPMA (200 nM, diagonally striped bars) for 90 s and labeled 3-PPI were quantitated. The same preparation of labeled platelets was incubated with agonists or buffer in order to assure common responsiveness and common baselines values, so that absolute changes could be compared. Results are for one of two experiments performed in duplicate and a third experiment (±2 nM wortmannin), presented as the average % of unstimulated (``Basal'') values ± range. In all experiments, the amount of residual 3-PPI generated by platelets exposed to SFLLRN + 2 nM wortmannin exceeded that produced by platelets exposed to betaPMA-wortmannin.



The magnitude of the SFLLRN-induced PAC1-binding response was slightly less than that in other (eg., Fig. 6) experiments because a lower concentration of SFLLRN was used (in keeping with 3-PPI studies). The inhibitory effect of wortmannin was limited to the PAC1-binding conformation of alphabeta(3), since the total surface expression of this integrin, monitored with SSA6 antibody in the same experiment, was unchanged by wortmannin (Fig. 6). More specifically, the inhibition was limited to agonist-induced conversion of alphabeta(3), since enhancement of PAC1 binding by anti-LIBS6-Fab, which promotes a direct conversion of alphabeta(3) to the activated state, was not impaired by wortmannin (Fig. 7). For comparison, the effects of wortmannin on PAC1 binding after platelets were exposed to three other agonists for platelet aggregation are shown.


Figure 6: Wortmannin affects the conformation, not the total surface expression, of alphabeta(3). Platelets were incubated with wortmannin (100 nM) or 0.5% Me(2)SO for 5 min at 37 °C prior to incubation with buffer, betaPMA, or SFLLRN. Binding of FITC-SSA6 (A, total expression) or biotin-PAC1 (B, active conformation) was assayed, as described, by dual color flow cytometry. Each data bar represents the mean ± S.E. of three independent experiments.




Figure 7: Effect of wortmannin on agonist-induced reorganization of alphabeta(3) fibrinogen receptors. Platelets were incubated with 100 nM wortmannin or Me(2)SO prior to gel filtration and incubation for 5 min in the presence of the different agonists, indomethacin (10 µM; inhibiting thromboxane A(2) synthesis), and FITC-PAC1 as described under ``Experimental Procedures.'' Each bar represents the mean ± S.D. of triplicate values from a single experiment representative of two performed.



As is true for betaPMA and thrombin/SFLLRN, ADP, a known platelet agonist, induces activation of alphabeta(3) (Fig. 7) and fibrinogen-dependent aggregation, which are also inhibited by wortmannin. This ADP response is of significance, since washed and stirred platelets frequently contain and release traces of ADP, which can complicate interpretations of aggregation results. We confirmed, as reported before, that ADP (10 µM) stimulates the accumulation of [P]PtdOH in P-labeled platelets (26) and further observed a modest increase in 3-PPI in ASA-treated platelets (ASA included to block potentiating effects of thromboxane A(2); (27) ) in 5 min (increase in PtdOH = 2.65 ± 0.05-fold; PtdIns(3,4,5)P(3) = 1.83 ± 0.04-fold; PtdIns(3,4)P(2) = 2.01 ± 0.06-fold). RGDS, added to block the effects of any traces of fibrinogen, was without effect on this response. Anti-LIBS6 Fab and fibrinogen, added to ASA-treated platelets in the presence of an enzyme system (CP-CPK) to decrease ADP, caused a small increase in labeled PtdIns(4,5)P(2) (1.39 ± 0.01-fold). An increase in PtdIns(3,4,5)P(3) (1.14 ± 0.03-fold) was barely detectable. PtdIns(3,4)P(2) increased 1.99 ± 0.04-fold. Basal values for PtdIns(4,5)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4)P(2) were 5.31 ± 0.01 times 10^6 dpm, 4.78 ± 0.09 times 10^3 dpm, and 1.54 ± 0.11 times 10^3 dpm, respectively. It thus appears that, in the absence of other receptor-directed agonists, fibrinogen binding leads to an ``outside-in'' stimulation of more than one phosphoinositide-directed kinase. These changes are all quite small, however, in comparison with the effects of thrombin/SFLLRN or betaPMA. When the effects of wortmannin were evaluated with respect to anti-LIBS6-Fab/fibrinogen-induced aggregation of ASA- and CP-CPK-treated platelets, no inhibition was observed (not shown).


DISCUSSION

Our results point to two major conclusions: 1) whereas the thrombin receptor activates two different PI 3-Ks in platelets, i.e. p85/PI 3-K and PI 3-K, stimulation of PKC leads to the activation of only p85/PI 3-K, and 2) the activation of p85/PI 3-K, rather than of PI 3-K, is involved in inside-out signaling affecting the conformational change in alphabeta(3) that is a prerequisite for fibrinogen binding and platelet aggregation.

Thrombin or SFLLRN, in activating heterotrimeric GTP-binding proteins, stimulates PLC and, via generated diacylglycerol, PKC. Apparently, this, in turn, promotes the activation of p85/PI 3-K in a Rho-dependent manner(1) . Liberated Gbeta binds to and activates PI 3-K, (^2)which is a Rho-insensitive event(1) . betaPMA does not activate PLC/diacylglycerol kinase (Fig. 1A), or free PLC- or PI 3-K-activating Gbeta (Fig. 2). Indeed, exposure of platelets to betaPMA may even be inhibitory for PI 3-K (Fig. 2B). (^3)Rather, in activating PKC, betaPMA stimulates a partially Rho-dependent p85/PI 3-K and an increased association of Rho and CDC42Hs (a direct agonist for p85/PI 3-K; (25) ), as well as p85/PI 3-K (both alpha- and beta-forms), with a cytoskeletal fraction. The activation of p85/PI 3-K that we have observed in response to betaPMA is not inhibited by RGDS, and is therefore independent of fibrinogen binding. This would be expected, since fibrinogen was not added, and betaPMA, unlike SFLLRN/thrombin, is a poor secretogogue and would not be expected to release stored fibrinogen. Consequently, we are focussing here purely on inside-out signaling responses.

It has been noted that p85/PI 3-K in neutrophils is more susceptible to inhibition by wortmannin than is PI 3-K(12) . This appears to be the case for platelets, as well (Fig. 4). Wortmannin is also an irreversible inhibitor of PI 3-Ks(12, 28, 29) . Therefore, it was important that short incubation periods be employed to optimize differences in sensitivities between the two enzymes. Presumably, if wortmannin, even at low concentrations, were stoichiometrically in excess of PI 3-K targets, long periods of incubation would eventually lead to complete inhibition at such concentrations were other factors equal. The permeability barrier of the intact cell lowers effective concentrations, and it is also possible that the arrangement of the enzymes in the intact platelet also permits differences in susceptibility to wortmannin to be detected more readily than in cytosolic fractions incubated directly. (^4)By incubating platelets with different wortmannin concentrations under the same conditions for studies of both 3-PPI generation and PAC1 binding (a more sensitive measure of activated alphabeta(3) exposure than is aggregation), we were able to compare the susceptibilities of these responses. It is clear from Fig. 3and Fig. 5that, although there is a wortmannin-attributable decrease of only 28% in the level of PtdIns(3,4,5)P(3) generated in response to the more potent agonist SFLLRN, the inhibitory effect of wortmannin on activated exposure of PAC1-binding alphabeta(3) is 80% of maximum. In contrast, the inhibitory effects of varied wortmannin concentrations on betaPMA-stimulated PtdIns(3,4,5)P(3) accumulation and activated alphabeta(3) exposure correlate well.

Surprisingly, the total amount of 3-PPI formed in the presence of 2 nM wortmannin + SFLLRN is greater than that formed in the presence of betaPMA, without wortmannin (Fig. 5B), yet this remaining SFLLRN-induced 3-PPI apparently is unable to promote conversion of alphabeta(3) (Fig. 5A). Of further interest, the total amount (rather than %) of decrease in 3-PPI generated in response to SFLLRN or betaPMA caused by 2 nM wortmannin is similar for the two agonists (Fig. 5B). These observations point to the importance, crucial for alphabeta(3) activation, of not only the mass of agonist-generated 3-PPI, but also of another factor, which probably involves localization. This additional factor appears to be related to the type of PI 3-K that generates 3-PPI. At the low concentrations of wortmannin that maximally inhibit alphabeta(3) activation in response to SFLLRN or betaPMA (Fig. 5A) and 3-PPI production in response to betaPMA ( Fig. 3and Fig. 5B), p85/PI 3-K is inhibited preferentially (Fig. 4). We calculate that 75% of the total wortmannin-induced decrease in SFLLRN-stimulated PI 3-K activity in the cytoskeletal fraction, resulting in about 80% of the maximum inhibited binding of PAC1 to alphabeta(3), is due to inhibition by wortmannin of p85/PI 3-K, whereas 25% of the inhibition of stimulated PI 3-K is due to inhibition of PI 3-K (Fig. 4B). Since 40% of the original activated cytoskeletal PI 3-K (without wortmannin treatment) is due to p85/PI 3-K, total stimulated PI 3-K activity is inhibited only about 30% by this concentration of wortmannin. Thus, about 70% of stimulated PI 3-K activity (the great majority now due to PI 3-K) is unimpaired by 2 nM wortmannin, yet most of the wortmannin-inhibitable PAC1 binding is, at this point, inhibited. These results point to a role for p85/PI 3-K, as opposed to PI 3-K, in promoting the activation or maintenance of active alphabeta(3). The selectivity of the effect may involve differences in localization of the two PI 3-Ks which we are unable to detect using crude Triton-insoluble fractions, since both p85/PI 3-K and PI 3-K are recruited to the Triton-insoluble cytoskeleton of thrombin/SFLLRN-activated platelets. Our findings also raise the issue of what function PI 3-K-generated 3-PPI might serve.

The route by which betaPMA (and PKC) activates p85/PI 3-K and causes increased association with the cytoskeletal fraction has not yet been elucidated, although it seems likely to involve tyrosine phosphorylation. Our data indicate that inhibition of p85/PI 3-K activity by low doses of wortmannin does not inhibit recruitment of p85/PI 3-K to the cytoskeletal fraction. PMA has been shown to stimulate tyrosine phosphorylation in human platelets(30) . We have examined p85 immunoprecipitates from activated platelets and found no evidence of tyrosine-phosphorylated p85, as has been noted by others (31) . It is known, however, that binding of appropriate tyrosine-phosphorylated peptides to the SH2 domains of p85 can activate p85/PI 3-K(32, 33, 34) . Furthermore, inhibition of tyrosine phosphatases in platelets promotes PAC1 binding, aggregation, and production of PtdIns(3,4)P(2)(35) , and inhibition of tyrosine kinases curtails thrombin-induced aggregation and PtdIns(3,4)P(2) accumulation(36) . Part of this activation, however, may be dependent upon interactions between p85 and p125. A recent interesting study (31) has demonstrated that p85/PI 3-K can be activated directly by interactions between the SH3 domain of p85 and the proline-rich region of p125, a tyrosine kinase that localizes with integrins at the platelet cytoskeleton. At the early, ``pre-aggregation'' stage of platelet activation, p125 does not become itself tyrosine-phosphorylated, since such phosphorylation appears to be dependent upon both alphabeta(3)-mediated platelet aggregation and PKC and Ca signals(37, 38) . It is conceivable that activation of PKC can affect the accessibility of the p125 proline domains to p85/PI 3-K.

While these studies were underway, it was reported that two different PI 3-K inhibitors, wortmannin and LY294002, inhibit both conversion of alphabeta(3) to a fibrinogen-binding form and the aggregation of platelets stimulated by thrombin receptor-activating peptide(39) . Our results are in partial agreement with those reported. In contrast to the previous report, however, we have found that concentrations of wortmannin (100 nM, 5 min) that completely inhibit PI 3-K activity (Fig. 3) do not impair exposure of activated alphabeta(3) in response to the Fab portion of an antibody known as anti-LIBS6 (Fig. 7), nor does it impair anti-LIBS6 Fab-induced platelet aggregation when fibrinogen is present in excess. We suggest that part of the reported inhibitory effects may relate to the presence of ADP and/or thromboxane A(2), each of which (present experiments; (2) ) can activate PI 3-K in platelet preparations that have not been treated with agents to remove or block synthesis of these agonists(39) . Furthermore, we have observed a slight stimulation of phosphoinositide kinase product accumulations by anti-LIBS6 Fab plus fibrinogen-induced platelet aggregation, but not as pronounced as reported. Thus, in the absence of an additional agonist, outside-in signaling via alphabeta(3)-mediated fibrinogen binding and aggregation does not seem to be as strong as the incremental PtdIns(3,4)P(2) accumulation observed when platelets are exposed to agonists such as thrombin (when fibrinogen binding and aggregation are not blocked; Refs. 24 and 40).

Granted that 3-PPI accumulation is important for agonist-induced alphabeta(3) conformational changes, one can only speculate at this point about how PtdIns(3,4,5)P(3) and/or PtdIns(3,4)P(2) exert effects. The activation by 3-PPI of protein kinase activity would be the most likely route for 3-PPI action, since the amounts of 3-PPI (especially PtdIns(3,4,5)P(3)) generated are rather small with respect to cytoskeletal proteins(41) , although highly localized stoichiometric effects cannot be excluded at present. We have observed in other studies (42) that PtdIns(3,4,5)P(3) (2 µM), added to saponin-permeabilized platelets, stimulates a kinase that phosphorylates pleckstrin and overcomes the inhibitory effects of wortmannin on pleckstrin phosphorylation. It is possible that a PtdIns(3,4,5)P(3)-activated protein kinase is also involved in the conversion of alphabeta(3).

A portion of agonist-induced conformational changes in alphabeta(3) is wortmannin-insensitive (Fig. 5Fig. 6Fig. 7), some of which may be attributable to wortmannin-insensitive generation of PtdOH. PtdOH has been shown to promote activation of alphabeta(3)(43) . Nevertheless, the wortmannin-sensitive component is a significant proportion of total alphabeta(3) activation, and therefore p85/PI 3-K activation should now be regarded as an important signal leading to functional responses in platelets.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL 38622 (to S. E. R.) and HL 40387 (to S. J. S.). 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.

§
Current address: Depts. of Vascular Biology and Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA 92037.

To whom correspondence should be addressed. Fax: 215-923-7145.

(^1)
The abbreviations used are: PtdIns, phosphatidylinositol (locants of other phosphates on myo-inositol ring are shown in parentheses), PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2); PI 3-K, phosphoinositide 3-kinase; p85, non-enzymatic subunit (both alpha and beta isoforms) of heterodimeric PI 3-K; PI 3-K, non-p85-containing PI 3-K that is activated by Gbeta; Gbeta, beta subunits of heterotrimeric GTP-binding proteins; 3-PPI, 3-phosphorylated phosphoinositides; PtdOH, phosphatidic acid; PLC, phospholipase C; PKC, protein kinase C; PMA, phorbol myristate acetate; SFLLRN, Ser, Phe, Leu, Leu, Arg, Asn; FITC-PAC1, fluorescein isothiocyanate-labeled antibody to activated conformation of alphabeta(3) integrin; FITC-SSA6, FITC-labeled antibody to active and inactive forms of alphabeta(3); biotin-PAC1, biotin-labeled PAC1 antibody; RGDS, Arg, Gly, Asp, Ser; GTPS, guanosine 5`-3-O-(thio)trisphosphate; ASA, acetylsalicylic acid (aspirin); CP-CPK, creatine phosphate-creatine phosphokinase; betaARK-PH, beta-adrenergic receptor kinase fragment containing the pleckstrin homology domain; EDIN, epidermal cell differentiation inhibitor that catalyzes ADP-ribosylation of Rho; ECL, enhanced chemiluminescence; Me(2)SO, dimethyl sulfoxide.

(^2)
Galpha subunits, unlike Gbeta, have not been found to stimulate purified platelet PI 3-K activity (X-W. Tang and C. P. Downes, personal communication).

(^3)
C. S. Abrams, J. Zhang, C. P. Downes, X-W. Tang, W. Zhao, and S. E. Rittenhouse, submitted for publication.

(^4)
J. Zhang and S. E. Rittenhouse, unpublished results.


ACKNOWLEDGEMENTS

We thank the blood drawing services of the Blood Center, Cardeza Foundation for Hematologic Research, Dr. Sandor S. Shapiro, for the use of his platelet aggregometer, Dr. Ivan Gout for p85-directed antibodies, Dr. Jeff Benovic for preparations of betaARK-PH, Dr. Moto Sugai for recombinant EDIN, and Dr. Peter Downes for sharing some of his results on purified platelet PI 3-K.


REFERENCES

  1. 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]
  2. Kucera, G. L., and Rittenhouse, S. E. (1990) J. Biol. Chem. 265, 5345-5348 [Abstract/Free Full Text]
  3. 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]
  4. Stoyanov, B., Volinia, S., Hanck, T., Rubio, I., Loubtchenkov, Malek, D., Stoyanova, S., Vanhaesebroeck, B., Dhand, R., Nurnberg, B., Gierschik, P., K. Seedorf, K., Hsuan, J., Waterfield, M. D., and Wetzker, R. (1995) Science 269, 690-693 [Medline] [Order article via Infotrieve]
  5. King, W. G., Kucera, G. L., Sorisky, A., Zhang, J., and Rittenhouse, S. E. (1991) Biochem. J. 278, 475-480 [Medline] [Order article via Infotrieve]
  6. Takai, Y., Kaibuchi, K., Tsuda, T., and Hoshijima, M. (1985) J. Cell. Biochem. 29, 143-155 [Medline] [Order article via Infotrieve]
  7. Nishizuka, Y. (1989) Cancer 63, 1892-1903 [Medline] [Order article via Infotrieve]
  8. Choi, E. J., and Toscano, W. A., Jr. (1988) J. Biol. Chem. 263, 17167-17172 [Abstract/Free Full Text]
  9. Lounsbury, K. M., Casey, P. J., Brass, L. F., and Manning, D. R. (1991) J. Biol. Chem. 266, 22051-22056 [Abstract/Free Full Text]
  10. Fields, T. A., and Casey, P. J. (1995) J. Biol. Chem. 270, 23119-23125 [Abstract/Free Full Text]
  11. Yano, H., Nakanishi, S., Kimura, K., Hanai, N., Saitoh, Y., Fukui, Y., Nonomura, Y., and Matsuda, Y. (1993) J. Biol. Chem. 268, 25846-25856 [Abstract/Free Full Text]
  12. 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]
  13. Yatomi, Y., Hazeki, O., Kume, S., and Ui, M. (1992) Biochem. J. 285, 745-751 [Medline] [Order article via Infotrieve]
  14. Rittenhouse, S. E. (1995) Semin. Hematol. 32, 120-125 [Medline] [Order article via Infotrieve]
  15. Bennett, J. S., and Vilaire, G. (1979) J. Clin. Invest. 64, 1393-1401 [Medline] [Order article via Infotrieve]
  16. Sims, P. J., Ginsberg, M. H., Plow, E. F., and Shattil, S. J. (1991) J. Biol. Chem. 266, 7345-7352 [Abstract/Free Full Text]
  17. Frelinger, A. L., III, Du, X., Plow, E. F., and Ginsberg, M. H. (1991) J. Biol. Chem. 266, 17106-17111 [Abstract/Free Full Text]
  18. Shattil, S. J., Cunningham, M., Wiedmer, T., Zhao, J., Sims, P. J., and Brass, L. F. (1992) J. Biol. Chem. 267, 18424-18431 [Abstract/Free Full Text]
  19. Abrams, C. S., Ruggeri, Z., Taub, R. A., Hoxie, J. A., Nagaswami, C., Weisel, J. W., and Shattil, S. J. (1992) J. Biol. Chem. 267, 2775-2785 [Abstract/Free Full Text]
  20. Shattil, S. J., Hoxie, J. A., Cunningham, M., and Brass, L. F. (1985) J. Biol. Chem. 260, 11107-11114 [Abstract/Free Full Text]
  21. Halenda, S. P., Zavoico, G. B., and Feinstein, M. B. (1985) J. Biol. Chem. 260, 12484-12491 [Abstract/Free Full Text]
  22. Börsch-Haubold, A. G., Kramer, R. M., and Watson, S. P. (1995) J. Biol. Chem. 270, 25885-25892 [Abstract/Free Full Text]
  23. Cross, M. J., Stewart, A., Hodgkin, M. N., Kerr, D. J., and Wakelam, M. J. O. (1995) J. Biol. Chem. 270, 25352-25355 [Abstract/Free Full Text]
  24. Sorisky, A., King, W. G., and Rittenhouse, S. E. (1992) Biochem. J. 286, 581-584 [Medline] [Order article via Infotrieve]
  25. Zheng, Y., Bagrodia, S., and Cerione, R. A. (1994) J. Biol. Chem. 269, 18727-18730 [Abstract/Free Full Text]
  26. Lloyd, J. V., Nishizawa, E. E., and Mustard, J. F. (1973) Br. J. Haematol. 25, 77-99 [Medline] [Order article via Infotrieve]
  27. Banga, H. S., Simons, E. R., Brass, L. F., and Rittenhouse, S. E. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 9197-9201 [Abstract]
  28. Yano, H., Nakanishi, S., Kimura, K., Hanai, N., Saitoh, Y., Fukui, Y., Nonomura, Y., and Matsuda, Y. (1993) J. Biol. Chem. 268, 25846-25856 [Abstract/Free Full Text]
  29. Woscholski, R., Kodaki, T., McKinnon, M., Waterfield, M. D., and Parker, P. J. (1994) FEBS Lett. 342, 109-114 [CrossRef][Medline] [Order article via Infotrieve]
  30. Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 901-905 [Abstract]
  31. 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]
  32. Holt, K. H., Olson, L., Moye-Rowley, W. S., and Pessin, J. E. (1994) Mol. Cell. Biol. 14, 42-49 [Abstract]
  33. Escobedo, J. A., Navankasattusas, S., Kavanaugh, W. M., Milfay, D., Fried, V. A., and Williams, L. T. (1991) Cell 65, 75-82 [Medline] [Order article via Infotrieve]
  34. Thomason, P. A., James, S. R., Casey, P. J., and Downes, C. P. (1994) J. Biol. Chem. 269, 16525-16528 [Abstract/Free Full Text]
  35. Pumiglia, K. M., Lau, L-F., Huang, C-K., Burroughs, S., and Feinstein, M. B. (1992) Biochem. J. 286, 441-449 [Medline] [Order article via Infotrieve]
  36. Guinebault, C., Payrastre, B., Sultan, C., Mauco, G., Breton, M., LevyToledano, S., Plantavid, M., and Chap, H. (1993) Biochem. J. 292, 851-856 [Medline] [Order article via Infotrieve]
  37. Shattil, S. J., Haimovich, B., Cunningham, M., Lipfert, L., Parsons, J. T., Ginsberg, M. H., and Brugge, J. S. (1994) J. Biol. Chem. 269, 14738-14745 [Abstract/Free Full Text]
  38. Clark, E. A., Shattil, S. J., and Brugge, J. S. (1994) Trends Biochem. Sci. 19, 464-469 [CrossRef][Medline] [Order article via Infotrieve]
  39. 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]
  40. Sultan, C., Plantavid, M., Bachelot, C., Grondin, P., Breton, M., Mauco, G., Levy-Toledano, S., Caen, J. P., and Chap, H. (1991) J. Biol. Chem. 266, 23554-23557 [Abstract/Free Full Text]
  41. Rittenhouse, S. E. (1996) in Advances in Molecular and Cell Biology: The Platelet (Lapetina, E. G., ed) JAI Press, CT, in press
  42. Zhang, J., Falck., J. R., Reddy, K. K., Abrams, C. S., Zhao, W., and Rittenhouse, S. E. (1995) J. Biol. Chem. 270, 22807-22810 [Abstract/Free Full Text]
  43. Smyth, S. S., Hillery, C. A., and Parise, L. V. (1992) J. Biol. Chem. 267, 15568-15577 [Abstract/Free Full Text]

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