regulation of glycoprotein iib-iiia receptor function ...fitc-pac1 binding to csb-9-permeabilized...

THE JOURNAL 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

OF BIOLOGICAL CHEMISTRY Vol ,267, No. 26, Issue of September 15, pp. 18424-18431,1992 Printed in U.S.A.

Regulation of Glycoprotein IIb-IIIa Receptor Function Studied with Platelets Permeabilized by the Pore-forming Complement Proteins CSb-9”

(Received for publication, March 2, 1992)

Sanford J. ShattilSQ, Michael Cunningham$, Therese WiedmerT, J i Zhaoll, Peter J. SimsT, and Lawrence F. Brass$ From the $Division of Hematology-Oncology, Departments of Medicine and Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 and the 70klahoma Medical Research Foundation, Oklahoma City, Oklahoma 73104

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore- forming complement proteins CSb-9. Platelet responses were then analyzed by flow cytometry. Non- lytic concentrations of CSb-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (F1TC)-labeled oli- gopeptides in a size-dependent manner. Under conditions of low external Ca2+, CSb-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to CSb-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 6’-0-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Ag- onist-induced GP 1%-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, CSb-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serinelthreonine kinases, and tyrosine kinases.

* These studies were supported by funds from the National Insti- tutes of Health Grants PO1 HL40387, HL36061, HL36946, and HL40796 and by funds from Cytogen Corporation. This work was presented in part at the 1991 Annual Meeting of the American Society of Hematology, Denver, CO and published in abstract form (Shattil, S. J., Wiedner, T., Sims, P. J., Zhao, J., Cunningham, M., and Brass, L. F.(1991) Blood 78, 183a). The costs of publication of this article were defrayed in part by the payment of page charges. This 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 Division of He- matology-Oncology, Silverstein 7, Hospital of the University of Penn- sylvania, 3400 Spruce St., Philadelphia, PA 19104. Tel.: 215-662- 3910; Fax: 215-662-7617.

The platelet plasma membrane glycoprotein (GP)’ IIb-IIIa complex (integrin a I m P s ) mediates platelet aggregation and platelet spreading on subendothelial surfaces (1-4). GP IIb and IIIa each contain a relatively large extracellular domain, a single transmembrane domain, and a short cytoplasmic tail (5,6). Although GP IIb-IIIa is present on the surface of both resting and activated platelets, it is able to bind soluble adhesive ligands such as fibrinogen and von Willebrand factor only after platelet activation, suggesting that cell activation evokes a conformational change that exposes ligand binding site(s) (7). Evidence that this conformational change actually occurs comes from the development of monoclonal antibodies that bind to GP IIb-IIIa in an activation-dependent manner (8-10) and, more recently, from the use of fluorescence reso- nance energy transfer techniques to demonstrate changes in the spatial relationships of the extracellular domains of the two glycoproteins during platelet activation (11).

If the exposure of ligand binding sites on the extracellular domains of GP IIb-IIIa is activation-dependent, then information about the state of platelet activation must be trans- mitted to the complex, either across the cell surface or via the cytoplasmic and transmembrane domains of one or both glycoproteins. The involvement of the cytoplasmic domains in this process is suggested by recent observations with mu- tant forms of GP IIb-IIIa. Co-transfection of Chinese hamster ovary cells with normal GP IIIa cDNA along with GP IIb cDNA in which the cytoplasmic tail had been deleted resulted in the formation of GP IIb-IIIa complexes that bound adhesive ligands constitutively (12). In addition, platelets from a variant form of Glanzmann’s thrombasthenia associated with a defect in fibrinogen receptor exposure have been shown to contain a point mutation in the cytoplasmic tail of GP IIIa (13). Thus, the conformation of the extracellular portions of GP IIb-IIIa can be affected by changes in the cytoplasmic tails, providing a potential mechanism whereby events within the cytoplasm of the platelet can affect ligand binding to the cell surface.

In previous studies, we have used platelets permeabilized with the detergent saponin to identify second messengers produced during platelet activation that might trigger the exposure of ligand binding sites on GP IIb-IIIa. Using radio- labeled PAC1, a monoclonal antibody specific for the activated conformation of GP IIb-IIIa, we found that the addition

The abbreviations used are: GP, glycoprotein; G protein, GTP- binding protein; PMA, phorbol myristate acetate; FITC, fluorescein isothiocyanate; GTPyS, guanosine 5’-O-(thiotriphosphate); PIPES, 1,4-piperazinediethanesulfonic acid; HEPES, 4-(2-hydroxyethyl)-l- piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethyl- enenitri1o)ltetraacetic acid.

18424

Intracellular Regulation of GP Ilb-IIIa 18425

of GTPrS to permeabilized platelets stimulated expression of fibrinogen receptors (14). Since this nonhydrolyzable guanine nucleotide is known to activate G proteins (15), this suggested the involvement of one or more G proteins in GP IIb-IIIa activation. However, interpretation of those experiments was limited in at least two respects by the techniques used. The first limitation arose because saponin is a membrane detergent which causes a time- and concentration-dependent loss of GP IIb-IIIa function. Under the best of conditions, this placed a limit of approximately 30 s on the time in which molecules of interest could be incorporated into the platelet before fibrinogen or PACl binding was adversely affected. For some agonists and intracellular mediators, this was too little time, particularly when the effects of potential inhibitors were also being tested. The second limitation of this technique was that radioligand binding studies with PACl did not allow a distinction to be made between permeabilized platelets and any remaining intact platelets.

Faced with these limitations, one of the goals of the present study was to develop an alternative experimental system for platelet permeabilization in which the function of GP IIb-IIIa would be better preserved, and the permeabilized platelets would be distinguishable from intact platelets. Previous studies have shown that under the appropriate conditions, platelets treated with the pore-forming complement proteins, C5b- 9, become permeable to monovalent and divalent ions without undergoing lysis (16) and without losing their ability to aggregate or undergo secretion in response to extracellular agonists (17). Therefore, we reasoned that when compared with saponin, controlled permeabilization of the platelet plasma membrane with C5b-9 might better preserve the structural and functional integrity of cell surface GP IIb-IIIa and the intracellular molecules responsible for GP IIb-IIIa activation. Additionally, the use of fluorescein isothiocyanate (F1TC)- labeled PACl and a fluorescent marker of membrane permeabilization might provide a means to quantitate GP IIb-IIIa activation in single, permeabilized platelets by flow cytometry.

EXPERIMENTAL PROCEDURES

Materials-The fluorescent tracer sulforhodamine B (S1307) was obtained from Molecular Probes, Inc. (Eugene, OR). Phorbol myristate acetate, cyclic AMP, dibutyryl cyclic AMP, and FITC-dextrans were from Sigma. GTPyS was from Boehringer Mannheim (West Germany). Tyrphostin-23 was obtained from BIOMOL Research Laboratories, Plymouth Meeting, PA. Buffer solutions: solution I (145 mM NaCl, 4 mM KC1, 0.5 mM MgCl,, 0.5 mM sodium phosphate, 0.1% (w/v) glucose, 0.1% bovine serum albumin, 5 mM PIPES, pH 6.8); solution 11 (137 mM NaCl, 4 mM KCl, 0.5 mM MgCl, 0.5 mM sodium phosphate, 0.1% glucose (w/v), 0.1% bovine serum albumin, 20 mM HEPES, pH 7.4). All buffers were made up with water that had been passed over a Chelex 100 ion exchange column (Bio-Rad) in order to achieve a free eaz+ concentration estimated at 1-10 p~ (18).

Peptides, Monoclonal Antibodies, and Complement Proteins-The preparation and characterization of a peptide (SFLLRN) derived from the primary sequence of a platelet thrombin receptor (19) are described elsewhere (20). RFARKGALRQKNV, a peptide derived from the pseudo-substrate region of protein kinase C (21), was syn- thesized by the Molecular Biology Resource Facility of the Saint Francis Hospital of Tulsa Medical Research Institute (Oklahoma City, OK) by an Fmoc (9-fluoromethyloxycarbonyl) solid phase pro- cedure (22). Other peptides used in the experiment shown in Fig. 2 were gifts from Drs. John Lambris and David Manning, University of Pennsylvania. Peptides were purified by reverse phase high per- formance liquid chromatography on a C18 column equilibrated with 0.1% trifluoroacetic acid, H20 and eluted by a linear gradient of 0.09% trifluoroacetic acid, acetonitrile. PACl is an IgM monoclonal antibody specific for an epitope on GP IIb-IIIa that is expressed only upon platelet activation (9). A2A9 is an IgG monoclonal antibody specific for the GP IIb-IIIa complex (23). Ab 62, an IgG monoclonal antibody that binds to an epitope on GP IIIa and induces the exposure

of the fibrinogen binding site within GP IIb-IIIa, was a gift from Dr. Mark Ginsberg, Scripps Research Institute, La Jolla, CA (24). S12, an IgG monoclonal antibody specific for the platelet a-granule membrane protein, P-selectin, was a gift from Dr. Rodger P. McEver, University of Oklahoma Health Sciences Center, Oklahoma City, OK (25). Antibodies were purified from mouse ascites as described (9, 26). Human complement proteins C5b6, C7, C8, and C9 were purified and analyzed for functional activity according to methods described previously (27). Concentrations of unlabeled proteins were estimated by their known extinction coefficients (27). Antibodies and peptides were labeled with fluorescein using FITC-Celite (28). After labeling, peptides were repurified and their concentrations determined by BCA microassay (Pierce Chemical Co.). Concentrations of labeled antibodies were determined by dye binding assay (Bio-Rad).

Platelet Preparation and Permeabilization with C5b-9-Whole blood was obtained from healthy volunteers and anticoagulated with a 1:6 volume of ACD-A anticoagulant. Platelet-rich plasma ,was collected by centrifugation at 180 X g for 15 min and incubated for 5 min with 1 p M prostaglandin El and 1 unit/ml apyrase (Sigma). Platelets were then isolated by sedimentation at 500 X g for 15 min and resuspended in 5 ml of solution I supplemented with ACD (66.7 pl/ml), prostaglandin E1 (1 pM), and apyrase (1 unit/ml). After further sedimentation at 500 X g for 15 min, platelets were resuspended at a concentration of 3 X 109/ml in solution I1 supplemented with 1 mM leupeptin.

Washed platelets were permeabilized with C5b-9 by adding the complement components in stages at 37 “C, without stirring. In the first stage, platelets were incubated for 2 min with C5b6 (8.8 pg/lOs platelets), after which C7 (1.65 pg/108 platelets) was added for 5 min. The platelet concentration at this point was 2 X 109/ml. Platelets were then transferred to reaction tubes containing C8 (10 pg/108 platelets), C9 (4-8 pg/108 platelets), the fluorescent tracer, sulforhodamine B (also known as S1307; 8 pg/ml), EGTA (10 p ~ ) , leupeptin (1 mM), and either FITC-PAC1 (30-40 pg/ml) or FITC-S12 (10 pg/ ml). EGTA at 10 p~ did not adversely affect the integrity or ligand binding function of GP IIb-IIIa. Where indicated, GTPyS was added either at the same time as C8 and C9 or up to 10 min later. Other platelet agonists were added 2-10 min after the C8 and C9. Incuba- tions with C8 and C9 were typically for 10 min in subdued light. Then 30-pl aliquots were transferred to final reaction tubes containing the agonists or a diluent control, sulforhodamine B (8 pg/ml) and leupeptin (1 mM) in a final volume of 50 pl. After a further 20-min incubation in the dark, samples were diluted with 450 p1 of phosphate- buffered saline containing 10 mg/ml bovine serum albumin, and the platelets were analyzed immediately by flow cytometry.

Flow Cytometry-Flow cytometry was carried out using either a Becton-Dickinson FACStar or FACSCAN flow cytometer formatted for two color analysis as described (28, 29). Light scatter and fluorescence channels were set at logarithmic gain. After acquisition of data for 10,000 particles/sample, a gate was set around the platelets for analysis of particle forward and right angle light scatter and fluorescence, using a 530/30 band pass filter for FL1 fluorescence and a 585/ 42 filter for FL2 fluorescence. Platelet-associated FITC fluorescence was measured in the FL1 channel and sulforhodamine B fluorescence in the FL2 channel. Electronic compensation settings eliminated any contribution of sulforhodamine B fluorescence to the FL1 channel and FITC fluorescence to the FL2 channel. Data were analyzed on a Hewlett-Packard 217 computer equipped with Consort 30 software (version F). Further details of the analysis, including the method for discriminating between permeabilized and intact platelets, are pro- vided under “Results.”

Platelet Protein Phosphorylation-Platelets were labeled with 32Pi and analyzed for protein phosphorylation as described elsewhere (30).

RESULTS

Characteristics of Platelets Permeabilized with C5b-9-A goal of the present study was to develop a system for permea- bilking platelets that could be used to study regulation of the ligand binding activity of platelet GP IIb-IIIa. As a first step, we tested the ability of the pore-forming complement proteins, C5b-9, to allow incorporation of small molecules into platelets under nonlytic conditions. As described under “Experimental Procedures,” C5b-9 complexes were assembled on the platelet membrane in the presence of 1 mM leupeptin to inhibit calcium-dependent proteolysis, 10 PM EGTA to prevent plate-

18426 Intracellular Regulation of GP Ilb-IIIa

let activation caused by Ca influx (31), and sulforhodamine B, a small (558 Da) water-soluble fluorescent tracer. Inclusion of sulforhodamine B allowed the identification of permeabilized platelets by virtue of their increased fluorescence in the FL2 channel of the flow cytometer (Fig. 1). Using uptake of sulforhodamine B to define a permeabilized platelet, the per- centage of permeabilized cells in the following experiments was typically 40-50% of the total population of platelets, but ranged from 20 to 80%.

To determine whether the size of the pores created by C5b- 9 under the conditions of these experiments was sufficient to permit the entry of small peptides into the cells, platelets were permeabilized for 10 min with C5b-9 in the presence of one of 14 FITC-labeled peptides ranging in size from 8 to 21 amino acids and in molecular mass from 984 to 2466 Da. Based on the difference in FITC fluorescence between platelets permeabilized with C5b-9 and intact platelets not treated with C5b-9, each of the FITC-labeled peptides became incorporated into the C5b-g-treated platelets (Fig. 2). This is consistent with permeabilization of the plasma membrane due to the complement pore. A smaller compound, sulforhodamine B (558 Da), exhibited even greater uptake than the peptides, whereas FITC-labeled dextrans with average molecular masses of 4,000,10,000, and 20,000 Da exhibited progressively restricted access into the platelets (Fig. 2). A 50-kDa Fab fragment of a monoclonal antibody to Src, an intracellular protein tyrosine kinase, failed to gain entry into the permeabilized platelets (not shown). The scatter in the peptide data shown in Fig. 2 suggests that factors in addition to molecular size influence peptide uptake. There was no obvious relation- ship between uptake and net peptide charge. To test whether the association of the peptides with permeabilized platelets might be due simply to nonspecific association with the surface of unintentionally activated platelets, measurements were also performed with intact platelets deliberately activated with a combination of collagen and peptide SFLLRN. This peptide is derived from the extracellular domain of a platelet thrombin receptor, and it activates platelets by binding to that receptor (20). It was chosen as an agonist because, unlike thrombin, SFLLRN-induced platelet activation was

No CSb-9 Treatment

0.6%

C5b9 Treatment

I 43.1%

,oo , 6 1 A 2 ~~ I I 1

Forward Light Scatter (arbitrary units)

FIG. 1. Identification of permeabilized platelets after treatment with CSb-9. Washed platelets were incubated with a cell- impermeable fluorescent dye, sulforhodamine B (S1307; 8 Fg/ml), in the absence (left panel) or presence (right panel) of the C5b-9 proteins. After 25 min, samples were analyzed by flow cytometry, which allowed a distinction to be made between platelets that had taken up the dye and those which had not. Each figure is a contour plot of 10,OOO particles. Forward light scatter, an indicator of particle size, is shown on the x axis and dye uptake is shown on the y axis. Both axes are on a log scale. An arbitrary line was drawn just above the upper limit of particle fluorescence for the platelets not treated with C5b-9. Using this line as the threshold for defining permeabilized platelets, 43% of the platelets treated with C5b-9 underwent permeabilization in this experiment.

"1 d

1 """

1

T

"""" "Q"". 04 7

100 1,000 10,000 100, Molecular Mass (Da)

00

FIG. 2. Uptake of FITC-labeled peptides and other fluorescent compounds into platelets permeabilized with CSb-9. Washed platelets were incubated with C5b-9 or buffer in the presence of either sulforhodamine B (558 Da), various FITC-labeled peptides ranging in molecular mass from 984 to 2466 Da, or FITC-dextrans (4,000-20,000 Da). All fluorescent compounds were at 5 p ~ . After 10 min, the association of these compounds with the platelets was assessed by flow cytometry and expressed as the relative fluorescence of the C5b-9-permeabilized platelets compared with that of intact platelets not treated with C5b-9. The horizontal line depicts a relative fluorescence of 1, the ratio that would be expected if a compound did not become selectively incorporated into permeabilizedplatelets. Data represent means & S.E. of five-nine experiments. The specific compounds are as follows. Compounds: 1, sulforhodamine B; 2, HKDMQLGR (single-letter amino acid code); 3, KKRAARATS; 4, ANNLRGCGLY: 5. NPNDKYEPF: 6. QLNLKEYNLV: 7. RMH LRQYELL; 8, ALAQAPPVYLDVLG; 6, RFARKGALRQKNV; 10, FSLLRNPNDKYEPF; 1 1 , KKQRFRHRNRKGY; 12, GPLS PSKDCGSPKYAYFNGC; 13, LFAHFIQPSAQKSPTSPLNC; 14, SEDNADDEVDTRPASFWETC; 15, CNYITELRRQHARASHLG LAR; 16, dextran, average mass 4000 Da; 17, dextran, 10,000 Da; 18, dextran, 20,000 Da. Peptides 5 and 10 were chosen for these studies because they have no discernible effect on platelet function (20). Peptide 9, an inhibitor of protein kinase C, was chosen to see if it could become incorporated into permeabilized platelets for subsequent use in the experiments shown in Figs. 7 and 8. All other peptides were tested because of availability, and they have no known effects on platelets.

unaffected by the leupeptin present in the reaction mixture. No significant binding of FITC-labeled peptides to these agonist-stimulated platelets was observed (not shown).

The functional integrity of platelets permeabilized with C5b-9 was studied by measuring their ability to undergo a- granule secretion in response to agonists. Granule secretion was detected in the flow cytometer using FITC-S12, an antibody specific for the a-granule membrane protein, P-selectin, which is expressed on the platelet surface after granule secretion (25). In the studies shown in Fig. 3, platelets were incubated for 2 min with or without C5b-9. Then phorbol myristate acetate (PMA), a plasma membrane-permeable activator of protein kinase C, or GTP-/& a membrane-impermeable guanine nucleotide analog that stimulates the activity of platelet G proteins, was added. As in Fig. 1, sulforhodamine B was included to distinguish permeabilized from nonpermeabilized platelets, and the horizontal dotted line defines the upper limit of background dye fluorescence of platelets not

Intracellular Regulation of GP IIb-IIIa 18427

A. No agonist

0 lo3 1-

C - - m C5b-9 Treatment

X F. PMA E. G T P 6 D. No agonist m

A. No agonist

3 P

1 No C5b-9 Treatment

1.

C .- - .E C5b-9 Treatment ; D No agonist F. PMA E. G T P 6

100 ' I1 I I I 100 I b l Ib2 I b 3 100 I b l I 2 I b 3 100 Ibl I b P I b 3

P-Selectin Expression (FITC-S12 Fluorescence)

FIG. 3. Surface expression of P-selectin in platelets permeabilized by C5b-9. Platelets were incubated with buffer (A-C) or C5b-9 (D-F) for 2 min in the presence of sulforhodamine B (8 pg/ ml) and the P-selectin antibody FITC-S12 (10 pglml). Then buffer ( N o agonist), 200 p~ GTP-& or 0.1 p~ PMA was added for 20 min, and platelet fluorescence due to sulforhodamine B uptake and FITC- S12 binding was measured by flow cytometry. The data are plotted on log scales as contour plots relating FITC-S12 fluorescence on the x axis to sulforhodamine B uptake on the y axis. This experiment is representative of three so performed.

treated with C5b-9. Since fluorescence due to FITC-S12 binding is shown along the x axis, permeabilized platelets that undergo a-granule secretion would be expected to appear in the upper right quadrant of each contour plot. Consistent with this interpretation, the mean FITC-S12 fluorescence of the sulforhodamine B-positive platelets was 95 arbitrary fluorescence units in the presence of PMA (Fig. 3F), compared with 40 units in the absence of agonist (Fig. 30). The secre- tory response of the permeabilized platelets was more heterogeneous than that of intact platelets, with some permeabilized cells undergoing full a-granule release and others little or none (cf. Fig. 3, C and F). The permeabilized platelets also underwent a-granule release in response to GTPyS (133 fluorescence units; Fig. 3E) . As expected, induction of a- granule release by GTPyS, which requires plasma membrane permeabilization to gain entry to the cytoplasm, was not observed for those platelets that had either not been exposed to C5b-9 (Fig. 3B) or had been treated with C5b-9 but not successfully permeabilized (e.g. the platelets below the dotted line in Fig. 3E).

Collectively, these experiments establish that flow cytometry can be used to distinguish between intact platelets and platelets permeabilized by C5b-9. Furthermore, they indicate that complement-permeabilized platelets can take up small organic molecules, such as GTPyS and synthetic peptides, while remaining responsive to agonists, including those that directly activate platelet G proteins and protein kinase C.

GP Ilb-IIIa Activation in Platelets Permeabilized with C5b- 9-To study the effect of platelet permeabilization with C5b- 9 on the ligand binding function of GP IIb-IIIa, GTPyS was added at various times after the addition of C5b-9. Fibrinogen receptor expression was then quantitated using the activation- dependent antibody FITC-PAC1 (9). As is shown in Fig. 4E, when GTP-yS was added 2 min after C5b-9, FITC-PAC1 binding to permeabilized platelets increased (platelets in the upper right quadrant of the contour plots). In contrast, platelets that were not exposed to C5b-9 (Fig. 4B) or those that were treated with C5b-9 but not successfully permeabilized (below the dotted line in Fig. 4E) failed to respond to GTPyS.

r"--l C. PMA

100 I I1 100 I b 2 A 3 100 Ibl 1b2 , 6 3 100 I b l Ib2 I b 3

GP Ilb-llla Activation (FTTC-PAC1 Fluorescence)

FIG. 4. GP I&-IIIa activation in platelets permeabilized with C5b-9. Platelets were incubated with buffer (A-C) or C5b-9 (D-F) for 2 min in the presence of sulforhodamine B (8 pg/ml) and FITC-PAC1 (40 pg/ml). Then buffer (No agonist), 200 p~ GTP-yS, or 0.1 p~ PMA was added for 20 min, and platelet fluorescence due to sulforhodamine B uptake and FITC-PAC1 binding was measured by flow cytometry. The data are plotted on lop scales. This experiment is representative of 15 so performed.

100 No agonist

0 2 4 6 8 1 0 Minutes of Permeabilization

Before Addition of GTP$

FIG. 5. Effect of time of platelet incubation with C5b-9 on subsequent GP IIb-IIIa activation by GTPyS. C5b-9 was assembled on washed platelets in the presence of FITC-PAC1 (40 pg/ml). Time 0 is defined as the time at which C5b67 platelets were incubated with C8 and C9 to initiate C5b-9 pore assembly. GTP-yS (200 p ~ ) or buffer was added to the incubations at the indicated times. Thirty min later, FITC-PAC1 binding to platelets was determined by flow cytometry. The black circles indicate platelets successfully permeabilized with C5b-9 and treated with GTP-yS. The shaded area represents C5b-9-permeabilized platelets treated with buffer rather than GTPyS. Error bars and the shaded area represent the means f S.E. of five experiments.

The interval between the addition of C5b-9 and the addition of GTPyS proved to be important. When the interval was 2 min, the average increase in FITC-PAC1 binding to permeabilized platelets was 10-fold. When the interval was length- ened to 5 or 10 min, the increase in FITC-PAC1 binding was a more modest 7.5- and 6-fold, respectively (Fig. 5). This time- dependent loss of FITC-PAC1 binding was also observed when PMA or the thrombin receptor peptide, SFLLRN, was used as agonists. It was probably not due to major damage to the extracellular portion of GP IIb-IIIa, since neither the binding of the antibody A2A9 to its "complex-dependent'' epitope on GP IIb-IIIa nor the ability of the anti-GP IIIa

18428 Intracellular Regulation of GP IIb-IIIa antibody, Ab 62, to directly induce fibrinogen receptor expression was impaired (data not shown) (24). Of note, the decay in agonist-induced PACl binding exhibited by C5b-9-permeabilized cells was much less than that of platelets permeabilized with 10-15 pg/ml saponin (not shown). In subsequent experiments, platelet agonists were added within 2 min of C5b-9 permeabilization.

FITC-PAC1 binding in response to other agonists was also evaluated. Both the thrombin-receptor agonist peptide, SFLLRN, and PMA caused an increase in FITC-PAC1 binding to C5b-9-permeabilized platelets. However, as had been observed for a-granule secretion, PACl binding to permeabilized platelets was more heterogeneous than observed with intact platelets (for example, compare Fig. 4, C with F ) . The experiments presented so far indicate that platelets permeabilized with C5b-9 represent a suitable model system to study mechanisms of GP IIb-IIIa activation. Furthermore, the dem- onstration that GTPyS causes fibrinogen receptor expression in permeable but not intact platelets suggests that GP IIb- IIIa activation can proceed through one or more G protein- mediated events.

Regulation of G P IIb-IIIa Activation in Platelets Permeabil- ized with C5b-9-Additional experiments were performed with C5b-9-treated platelets to evaluate intracellular factors that might regulate fibrinogen receptor expression. First, permeabilization was carried out in the presence of either cyclic AMP, which cannot enter intact platelets, or its membrane- permeable analog, dibutyryl cyclic AMP. By activating protein kinase A, platelet cyclic AMP inhibits agonist-induced fibrinogen binding and platelet aggregation (32). As antici- pated, dibutyryl CAMP inhibited agonist-induced FITC-PAC1 binding to both nonpermeabilized and permeabilized platelets. In contrast, cyclic AMP inhibited only platelets that had become permeabilized by C5b-9, consistent with the require- ment for a membrane pore for this compound to gain entry to the cytoplasm (Fig. 6). These results indicate that fibrinogen receptor expression can be inhibited by cyclic AMP under conditions that permit the selective entry of this mediator into the cytoplasm of C5b-g-permeabilized platelets.

The response of platelets in Fig. 4 to PMA supports previous studies with this compound, suggesting that protein kinase C is involved in GP IIb-IIIa activation (14). One limitation of previous studies has been the use of relatively

Permeabillzed Platelets Non-Permeabilized Platelets I , 1-

i800 -

. . .

1500 - 1200 -

900 -

600 - T

30: u No GTP6

I

T T t6000

NO GTP+ PMA ." agonist

, . . . . . . agonist

FIG. 6. Effect of cyclic AMP and dibutyryl cyclic AMP on GP IIb-IIIa activation in platelets permeabilized with CSb-9. Platelets were incubated with C5b-9 in the presence of 5 mM cyclic AMP (shaded bars), 5 mM dibutyryl cyclic AMP (striped bars), or buffer (black bars). Ten min later, platelets were supplemented with GTP-yS (200 pM), PMA (5 nM), or additional buffer (No agonist). After 20 additional min, FITC-PAC1 binding to platelets was measured. Data in the left panel were obtained with platelets permeabilized with C5b-9. Data on the right panel were obtained with platelets that had been treated with C5b-9 but had not incorporated sulforhodamine B. The data represent means & S.E. of four experiments.

nonspecific inhibitors of protein kinase C to prove a role for the enzyme in platelet function. To confirm a specific role for protein kinase C in GP IIb-IIIa activation, platelets were permeabilized with C5b-9 in the presence of RFARK- GALRQKNV, a 13-amino acid peptide derived from the pseudo-substrate region of protein kinase C. This peptide is a specific inhibitor of protein kinase C when studied in cell- free systems (21). RFARKGALRQKNV was also one of the peptides shown in Fig. 2 to gain entry into platelets permeabilized with C5b-9. For the studies shown in Fig. 7, platelets were incubated with C5b-9 for 2 min in the presence of 0.5 mM RFARKGALRQKNV and then stimulated for an additional 2 min with either GTPyS or the thrombin receptor agonist peptide SFLLRN. In three experiments, RFARK- GALRQKNV caused a 57% inhibition of GTPyS-induced FITC-PAC1 binding to permeabilized platelets and a 53% inhibition of SFLLRN-induced binding ( p < 0.01). Several unrelated peptides of similar size had no such effect (not shown). RFARKGALRQKNV also inhibited GTPyS-induced phosphorylation of p47, the major protein kinase C substrate in platelets (Fig. 8). In contrast, RFARKGALRQKNV had no effect on FITC-PAC1 binding to intact platelets stimulated with SFLLRN (Fig. 7). Taken together, these experiments with a thrombin receptor agonist, with GTP-yS, with a peptide inhibitor of protein kinase C and with cyclic AMP suggest that activation of GP IIb-IIIa can be mediated in a sequential fashion by agonist occupancy of a thrombin receptor, activation of G protein(s), and phosphorylation of protein substrate(s) by protein kinase C. Cyclic AMP may be a negative regulator at one or more steps of this pathway.

Protein kinase C is a serinelthreonine kinase. In addition to protein phosphorylation on serinelthreonine residues, stimulation of platelets by agonists also results in the phosphorylation of several unidentified proteins on tyrosine residues, a reaction presumably mediated by one or more Src- related protein tyrosine kinases present in platelets (33, 34). To examine whether protein tyrosine phosphorylation might be involved in the regulation of GP IIb-IIIa, agonist-induced FITC-PAC1 binding was studied in the presence of 50 pM tyrphostin-23, a selective inhibitor of protein tyrosine kinases

Perrneabillzed Platelets

T

Agonist No G T V SFLLRN

Intact Plalelels

300

Agonist N o S F L L R N

FIG. 7. Effect of the peptide, RFARKGALRQKNV, an inhibitor of protein kinase C, on GP IIb-IIIa activation. Washed platelets containing FITC-PAC1 (40 pg/ml) were incubated with buffer or permeabilized with C5b-9 for 2 min, either in the absence (black bars) or presence (shaded bars) of 0.5 mM RFARK- GALRQKNV. Then GTP-yS (200 p ~ ) , thrombin receptor peptide SFLLRN (50 p ~ ) , or buffer ( N o agonist) was added and FITC-PAC1 binding measured 2 min later by flow cytometry. In this particular experiment, the relatively short incubation time after the addition of agonists was based on data illustrated in Fig. 8 that the inhibitory effect of RFARKGALRQKNV on protein kinase C-mediated protein phosphorylation was maximal 1-2 min after addition of agonist. Data in the left panel are from platelets permeabilized with C5b-9; data in the right panel are from intact platelets not treated with C5b-9. The data represent means +. S.E. of three experiments.


"PKC 19-31 + PKC 19-31

47 kDa +

"""""m

0 1 2 3 0 1 2 3 Time Imln)

FIG. 8. Effect of the peptide, RFARKGALRQKNV (PKC Z9-3Z) . on phosphorylation of platelet proteins. Platelets were laheled with ".'I', and then permeahilized with C5h-9 for 2 min in the absence or presence of 0.5 mM RFARKGALRQKNV. Two min later, platelets were stimulated with 200 PM GTPyS. Incuhations were then carried out for the indicated periods of time and the samples processed for SDS-gel electrophoresis and autoradiography. The arrow points to t,he major 47-kDa platelet protein phosphorylated hy protein kinase C.

Agonist Agonist

FIG. 9. Effect of the tyrosine kinase inhibitor, tyrphostin- 23, on agonist-induced activation of platelet G P IIh-IIIa. 1'lat.elets were incuhated for 2 min with either huffer or C5b-9 in the absence (block burs) or presence (shaded bars) of 50 PM tyrphostin- 23 (also known as A23 or RG-50810). Platelets were incubated an addition81 28 min with either huffer ( N o agonist), a thromhin receptor peptide agonist (SFLLRN; 100 PM), GTPyS (200 P M ) , or PMA (0.1 P M ) . FITC-PAC1 binding was then measured by flow cytometry. Data represent means * S.E. of four to eight experiments.

at this concentration (35-37). Tyrphostin-23 partially inhih- i ted GTPyS- and SFLLRN-induced FITC-PAC1 binding to permeabilized platelets ( p < 0.03) (Fig. 9). Tyrphostin-23 also inhibited SFLLRN-induced FITC-PAC1 binding to intact platelets not treated with C5b-9, hut it did not inhibit the response of these platelets to PMA (Fig. 9). This suggests that protein tyrosine kinases may play a role in the activation of G P IIb-IIIa. Furthermore, the results with GTPyS seem to indicate that tyrosine phosphorylation may operate down- stream from the receptor-mediated activation of G proteins.

DISCUSSION

One of the key events of platelet activation is the conformational change in the glycoprotein IIh-IIIa complex that allows it to serve as an adhesion receptor. Although there is now substantial evidence that such a conformational change occurs, there is relatively little information ahout the intracellular events that trigger it. One of the obstacles that has

prevented the collection of such information is the lack of a suitable model system for studying activation of the receptor complex. Although cell lines transfected with cDNA encoding GP IIb and IIIa are able to assemble the heterodimer complex on t he cell surface, they do not appear to bind fibrinogen in response to extracellular agonists (12, 38). Similarly, mega- karyoblastic cell lines such as HEL cells, Dami cells and CHRF-288 cells form apparently normal G P IIb-IIIa complexes, but do not hind fihrinogen upon cell activation.? Sub- cellular fractionation of platelets has also proven to he an unsatisfactory approach, since G P IIh-IIIa complexes in isolated platelet membranes or inserted into liposomes hind fibrinogen with a low stoichiometry and cannot he activated by agonists to bind more fihrinogen' (39). At present, therefore, the only feasible approach has been to permeabilize the platelet plasma memhrane as selectively as possible with saponin (14). However, this approach is not entirely satisfac- tory, since saponin has detrimental effects on fihrinogen receptor expression that occur within 30-60 s, a time frame that coincides with the normal kinetics of platelet activation. thereby precluding analysis of the precise intracellular proc- esses that regulate G P IIh-IIIa.

In the present studies we have used the complement proteins, Cfib-9, as an alternative way to create pores in the platelet plasma membrane and have used flow cytometry to demonstrate that a suh-population of platelets treated with C5h-9 becomes selectively permeahle to organic compounds of potential utility for study of the intracellular regulation of fihrinogen receptor expression. After incubation with C5b-9, the permeahilized platelets could take up FITC-labeled peptides ranging in size from 8 to 21 amino acids (984-2466 Da) as well as small molecules that do not normally enter platelets, such as GTPyS and cyclic AMP. Although we do not. know the precise size exclusion limit for the permeahilized platelets. the uptake of FITC-labeled dextrans ranging in average molecular mass from 4,000-20,000 Da was progressively restricted (Fig. 2), and uptake of a SO-kDa antibody Fah frap- ment was not observed. In this study, the conditions of platelet permeabilization were optimized to strike a balance hetween the extent of permeabilization and the preservation of GP IIb-IIIa function. In fact, in contrast to saponin-permeahilized platelets, platelets permeabilized with C5b-9 proved capable of expressing fibrinogen receptors upon agonist stimulation for at least 10 min after permeabilization. Therefore, it was possible to incorporate potential mediators and inhibitors into the platelet and begin to define specific roles for molecules potentially involved in the regulation of G P IIb-IIIa.

Flow cytometry offers a distinct advantage over other methods to study platelet memhrane responses, hecause it c m detect sub-populations of platelets (28). This enahled us to distinguish between platelets that. had hecome permeahilized by C5b-9 and those that had not. A permeabilized platelet was defined as one that acquired increased "red" fluorescence due to uptake of the tracer dye sulforhodamine H. Although this definition might he considered arbitrary, the flow cytometric distinction between dye-positive and dye-nepative platelets was usually unambiguous and wm consistent with the results of the functional studies; only dye-positive platelets underwent GP IIb-IIIa activation and n-nanule secretion in response to GTPyS (Figs. 3E and 4 E ) . A cell-to-cell variability in the extent of agonist-induced activation has previously been descrihed for intact platelets (28). We observed considerahle heterogeneity in the activation of platelets permeahilized with C5h-9 (e.g. Figs. 3F and 4F), which mav

' K. Cichowski, L. F. Brass, and S. .J. Shattil. r~npuhlishetl ohser- vations.

18430 Intracellular Regulation of GP IIb-IIIa reflect this intrinsic variability of cell responsiveness com- bined with platelet-to-platelet variations in the extent of permeabilization of the plasma membrane. It therefore cannot be assumed that the averaged response of an entire set of cells subjected to a permeabilization protocol reflects the responses of the subset of cells that have been rendered permeable. This caveat may also hold true for other methods that have been used to permeabilize platelets, such as electropermeabilization or detergent permeabilization with saponin or digitonin. Therefore, flow cytometric analysis may find a more general use in cell permeabilization studies.

One of the consequences of permeabilizing cells is the rapid flux of Ca2' across the plasma membrane. Under experimental conditions in which the extracellular free Ca2+ concentration is 21 mM, permeabilization by C5b-9 causes Ca2+ influx, which stimulates the nonlytic activation of platelets. This results in secretion from storage granules and the formation of plasma membrane vesicles, which along with the remnant activated platelets can provide a catalytic surface for the phosphatidylserine-dependent enzyme reactions of the coag- ulation system (27, 30,40). Previous studies have shown that the remnant platelets do not spontaneously express binding sites for fibrinogen and do not aggregate. However, these platelets are able to bind fibrinogen and aggregate in response to ADP, indicating that their GP IIb-IIIa complexes remain functionally intact (17, 41). In the present study, we took advantage of these previous observations by designing a permeabilization protocol in which complement would create membrane pores without inducing cell activation or vesicu- lation while allowing agonist-induced activation of GP IIb- IIIa to take place. This goal was achieved by carrying out membrane C5b-9 assembly at an extracellular free Ca2+ concentration estimated at 1-10 p~ in the presence of 0.5 mM MgC12. These conditions are permissive for plasma membrane assembly of C5b-9 complexes, agonist-induced activation of GP IIb-IIIa, and fibrinogen binding (31, 42).

GP IIb-IIIa function was better preserved when C5b-9 was used as a permeabilization reagent instead of saponin, presumably because of differences in the membrane lesions created by these reagents. Kinetic and equilibrium measurements of the functional radius of the C5b-9 channel in human blood cell membranes provides evidence for heterogeneous pores with exclusion radii ranging from C0.4 to >10 nm, depending upon the number of C5b-9 complexes bound per cell and the stoichiometry of C9 within the C5b-9 complex (43-45). The markedly reduced susceptibility of human uersus non-primate blood cells to both cytolytic and pore-forming activities of the human C5b-9 proteins suggests that the exclusion radius of the complement pore formed in the platelet plasma membrane is attenuated by CD59, the complement-inhibitory protein that normally serves to protect these cells from activation and lysis by human serum complement (46). By contrast to the restricted pores formed by complement, the membrane lesions induced by saponin are governed solely by the affinity of this lipophilic molecule for membrane cholesterol and have been estimated at 0.1-1 pm (47). Not unexpectedly, therefore, C5b- %treated platelets do not leak lactate dehydrogenase, whereas saponin-treated platelets do (41, 48). It is possible that the maintenance of physiological responses in C5b-g-permeabilized platelets is due to better retention of key regulatory molecules within the cytoplasm and to less perturbation of the lipid-protein interactions essential to plasma membrane receptor function.

The observation that fibrinogen receptor expression in C5b- 9-permeabilized platelets could be stimulated with GTPyS is consistent with prior studies with saponin-permeabilized

platelets which suggested that a G protein is involved in the regulation of GP IIb-IIIa (14, 49). Indeed, many agonist receptors on platelets, including those for thrombin, epinephrine, and platelet-activating factor belong to a family of heterotrimeric G protein-coupled receptors containing seven transmembrane domains (19, 50, 51). Platelets have been shown to contain the G proteins G., Gil, Giz, Gi3, G,, and G, (52), but it remains to be determined which of these function to couple agonist receptors to effector pathways that activate GP IIb-IIIa. There is as yet no evidence that a heterotrimeric or small G protein interacts directly with GP IIb-IIIa.

What second messenger pathways within the platelet are involved in activation of GP IIb-IIIa? Based on studies with intact and permeabilized platelets, it would appear that cer- tain agonists, such as thrombin and thromboxane A2, are capable of initiating GP IIb-IIIa activation through a pathway that involves G protein-mediated stimulation of phospholi- pase C and activation of protein kinase C. In this case, GP IIb-IIIa activation is presumably the result of phosphorylation of an as yet unidentified protein substrate on serine/threonine residues. Consistent with this, we observed fibrinogen receptor expression when C5b-9-permeabilized platelets were stimulated with an agonist peptide for the thrombin receptor and with PMA, a direct activator of protein kinase C. These effects were inhibited by RFARKGALRQKNV, a peptide inhibitor of protein kinase C, which gained selective entry into the permeabilized cells. Since this peptide inhibits the enzyme's active site, it is likely to be more selective than other inhibitors of the enzyme (21).

Despite evidence pointing to involvement of protein kinase C in GP IIb-IIIa activation, studies of GP IIIa phosphorylation using 32Pi indicate that it is unlikely that GP IIb-IIIa activation can be explained by direct phosphorylation of this integrin by a serine/threonine kinase (53). In addition, cyclic AMP functions as a negative modulator of GP IIb-IIIa, presumably by stimulating another serine/threonine kinase, protein kinase A. Although a number of substrates for this latter enzyme have been identified in platelets, GP IIb-IIIa has not been shown to be one of them (reviewed in Ref. 54). This suggests an alternative model whereby the cytoplasmic domain of GP IIb and/or IIIa might interact with one or more regulatory molecules that are themselves substrates for protein kinase C and protein kinase A. However, even this mechanism would not easily explain how some platelet agonists, such as epinephrine and ADP, can induce fibrinogen receptor expression under conditions in which activation of protein kinase C is not readily detected (55, 56). Thus, more than one signaling pathway may couple agonist receptors to GP IIb-IIIa. In this context, we found that tyrphostin-23, a selective inhibitor of protein tyrosine kinases, partially inhibited FITC-PAC1 binding to permeabilized platelets in response to GTPyS and SFLLRN. In preliminary studies, this tyrphostin has also been shown to inhibit ADP-induced PAC1 binding and GTPyS-induced protein tyrosine phosphorylation in platelets.' Platelets contain five members of the Src family of protein tyrosine kinases (Src, Yes, Fyn, Lyn, and Hck) which could be affected by this inhibitor (57). The relative contributions of platelet protein tyrosine kinases, serine/threonine kinases, and protein phosphatases to the process of GP IIb-IIIa activation remain to be determined.

Recent studies have begun to examine the interaction of purified GP IIb-IIIa with potential regulatory molecules. For example, phosphatidic acid has been shown to induce fibrinogen binding to purified GP IIb-IIIa (58). In addition, a 21- kDa GTP-binding protein from platelets appears to co-purify with GP IIb-IIIa (59). However, no signaling molecule has yet


been shown to directly interact with and regulate the function of GP IIb-IIIa in platelets or in platelet membranes. Of note in this regard, short synthetic peptides derived from specific domains of several cell signaling proteins have been used successfully to study the function and molecular interactions of these proteins (60-65). In the future, it may be possible to identify molecules that directly interact with and regulate the receptor function of GP 1%-IIIa by inserting relevant peptides into CSb-9-permeabilized platelets.

Acknowledgments-The technical assistance of Elizabeth Smith in the purification of the complement proteins is gratefully acknowl- edged. We also thank Drs. John Lambris and David Manning, Uni- versity of Pennsylvania, for providing peptides and Mark Ginsberg and Rodger McEver for providing monoclonal antibodies.

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regulation of glycoprotein iib-iiia receptor function ...fitc-pac1 binding to csb-9-permeabilized...

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