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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:1466-1473.)
© 1995 American Heart Association, Inc.

Articles

Surface Recruitment but Not Activation of Integrin _IIbß₃ (GPIIb-IIIa) Requires a Functional Actin Cytoskeleton

John B. Addo; Paul F. Bray; Dmitriy Grigoryev; Nauder Faraday; Pascal J. Goldschmidt-Clermont

From the Divisions of Cardiology and Hematology, Department of Medicine, and Department of Cell Biology and Anatomy, Johns Hopkins University School of Medicine, Baltimore, Md.

Correspondence to Pascal J. Goldschmidt-Clermont, Bernard Laboratory, Ross 1023, Johns Hopkins University, 720 Rutland Ave, Baltimore, MD 21287.

	Abstract

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Abstract Binding of integrin _IIbß₃ (glycoprotein [GP] IIb-IIIa) to soluble fibrinogen requires that the receptor undergo a conformational change (receptor activation), which occurs rapidly in agonist-stimulated platelets. Agonist stimulation of platelets also results in _IIbß₃ recruitment from intracellular membranes (-granules and open canalicular system) to the platelet surface. Once activated and accessible, the receptor can engage, a process that corresponds to the binding of the receptor to its soluble fibrinogen ligand, leading to intracellular signaling reactions and centripetal migration of bound receptor molecules. Because these processes occur concurrently with a marked reorganization of the actin cytoskeleton, we investigated the role of actin in fibrinogen receptor activation and surface recruitment. We used a flow cytometric assay to directly quantitate the binding of _IIbß₃ to fluorescently labeled fibrinogen on the platelet surface. Cytochalasin D, which inhibits elongation of actin filaments, was used to prevent the actin response to platelet agonists. Despite its ability to inhibit the actin response and _IIbß₃ binding to the actin cytoskeleton, cytochalasin D did not alter the agonist-induced intramolecular changes resulting in increased affinity of _IIbß₃ for soluble fibrinogen and therefore did not inhibit ADP-induced aggregation. Thus, disruption of the actin network with cytochalasin D had no effect on the dissociation constant of the complex between activated _IIbß₃ and fibrinogen (K_d=0.26 to 0.28 µmol/L). However, cytochalasin D suppressed the recruitment of cryptic _IIbß₃ molecules to the platelet surface. While the physiological consequence of exposing additional _IIbß₃ molecules on the surface of platelets is unclear, it is tempting to speculate that this process plays an important role in consolidating intra-arterial platelet thrombi, despite the shear strain generated by the arterial blood flow.

Key Words: cytoskeleton • integrin • actin • fibrinogen receptor • platelets

	Introduction

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Integrin _IIbß₃ exists in four different states: resting, occupied (bound to fibrinogen), activated (agonist-induced conformational change), and engaged (activated and occupied).^{1 2 3 4} Activated _IIbß₃ corresponds to the conformation of the receptor whose affinity for fibrinogen in solution is markedly increased.² In addition, _IIbß₃ can be found in two different compartments relative to extracellular ligands (exposed to ligands, or cryptic, that is, not accessible to extracellular ligands).^{5 6} The switch from cryptic to exposed is referred to as recruitment or outward translocation of the receptor. Centripetal (inward) migration of receptor molecules also occurs upon fibrinogen binding.

Activation, engagement, and surface recruitment of _IIbß₃ occur concurrently with agonist-induced actin polymerization.^{7 8} However, not all aspects of the processing of the fibrinogen receptor require the actin response and associated cell shape changes. Activation of the fibrinogen receptor, which is necessary for aggregation, does not appear to require actin polymerization. Despite the fact that several laboratories (including ours⁹ ) have shown that key tyrosine kinase substrates, whose phosphorylation correlates with _IIbß₃ activation, correspond to proteins that have been linked to the organization of the actin superstructure, the actin cytoskeleton does not appear to be required for platelet aggregation ex vivo.¹⁰ In contrast to receptor activation for which actin polymerization is not necessary, the actin response is needed to mediate "outside-in" signals resulting from the binding of fibrinogen to its activated receptor (integrin _IIbß₃ engagement).^{1 3 4}

Aggregation assays do not measure fibrinogen receptor recruitment because the fraction of receptor molecules that are constitutively present on the plasma membrane of platelets appears to be sufficient to allow for platelet aggregation ex vivo. A recent study indicated that the cellular fraction of _IIbß₃ present in the membrane of -granules becomes physically bound to the actin cytoskeleton upon thrombin activation of platelets,¹¹ thus suggesting that the actin cytoskeleton may play an important role in _IIbß₃ recruitment. An intriguing finding of the report of Bertagnolli and Beckerle¹¹ is the fact that in contrast with previous studies,^{12 13} binding of internal or cryptic _IIbß₃ to the actin cytoskeleton occurred in activated but not aggregated platelets.

For the present study, we tested the hypothesis that the actin cytoskeleton reorganization after agonist stimulation is required for the exposure of fibrinogen receptor molecules that are cryptic in resting platelets. We used CytoD to inhibit actin polymerization.¹⁴ We studied the changes in fibrinogen binding to the platelet surface induced by activation of the platelet purinergic receptor (P_2Y) with ADP by using a flow cytometric assay.¹⁵ The assay quantitates the binding of fluorescently labeled fibrinogen molecules to the surface of platelets.¹⁵

While we confirm that the actin polymerization in response to ADP is not necessary for the activation of _IIbß₃ and consequently for platelet aggregation in vitro, we demonstrate that a functional actin cytoskeleton is necessary for surface recruitment of the fibrinogen receptor. Our data are consistent with a mechanism whereby the actin network binds to a population of cryptic _IIbß₃ molecules and mediates the recruitment of these receptor molecules to the platelet surface.

	Methods

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Materials
CytoD, collagen (type I), pepstatin, antipain, chymostatin, leupeptin, phalloidin, and prostaglandin E₁ (PGE₁), were from Sigma Chemical Co. ADP and other reagents used for the aggregation assay were from Chrono-Log Corp. The 4-(2-aminoethyl) benzene-sulfonyl fluoride (AEBSF) protease inhibitor was from Calbiochem-Novabiochem Corp. Lyophilized human fibrinogen was from AB Kabi. FITC for labeling fibrinogen was from Pierce, and FITC calibration beads (Quantum Series) were from Flow Cytometry Standards Corp. The peptide Ser-Phe-Leu-Leu-Arg-Asn (TRAP) was from Bachem Bioscience Inc. Monoclonal antibody to PLC1 was from Upstate Biology. The anti–P-selectin (S12) and the anti-_IIb (Tab) were the generous gift of Rodger McEver (University of Oklahoma). Additional antibodies against _IIb (SZ-22), ß₃ (SZ-21), and _IIbß₃ (P2) were from Amac, Inc. The anti–pan-actin monoclonal antibody (clone C4) was from ICN Biomedicals, Inc. Horseradish peroxidase–labeled goat anti-rabbit and goat anti-mouse IgG and FITC-labeled goat anti-mouse antibody were obtained from Hyclone Laboratories. Protein concentrations were measured on an EL 312e Bio-Kinetics Reader (Bio-Tek Instruments, Inc). Platelet counts were obtained in a Coulter counter, model ZF (Coulter Electronics, Inc) with 70-µm window aperture.

Platelet Preparation and Processing
PRP was obtained by centrifugation of normal donor's blood at 180g for 15 minutes.⁹ For aggregation assay, platelets were suspended at a concentration of 3x10⁸ mL^-1. "Washed" platelets were obtained by centrifugation (750g for 10 minutes) in the presence of PGE₁ (1 µmol/L); the pellets were gently rinsed with Tyrode's solution (138 mmol/L NaCl, 2.9 mmol/L KCl, 12 mmol/L NaHCO₂, 0.36 mmol/L Na₂HPO₄, 5.5 mmol/L glucose, 1.8 mmol/L CaCl₂, 0.49 mmol/L MgCl₂, pH 7.4) and were eventually resuspended in Tyrode's solution without PGE₁ at 10⁷ cells/mL^-1 for flow cytometry assay and 10⁹ cells/mL^-1 for biochemical assays.⁹

Platelets were treated with CytoD (10 µmol/L) for 30 minutes to generate maximal inhibition of actin polymerization.¹⁴ CytoD was diluted in DMSO and was added to either PRP or washed platelets in such a way that the DMSO represents 0.1% of the total sample volume. Keeping the DMSO concentration low was necessary because DMSO by itself can inhibit platelet aggregation if it represents >1% of the total sample volume (data not shown).

Three concentrations of ADP were used to activate the platelet purinergic receptor: 1 µmol/L, 2 µmol/L, and 20 µmol/L (the latter concentration is saturating). For some experiments, TRAP (stock solution in deionized water, 5.4 mmol/L) was used as a strong agonist to provide maximal fibrinogen binding to platelets (final concentration, 54 µmol/L).

Assay for Binding of _IIbß₃ to the Actin Cytoskeleton
Binding of _IIbß₃ to the actin cytoskeleton was measured in platelet lysates. Briefly, control or CytoD-treated (10 µmol/L for 30 minutes), resting or ADP-activated (20 µmol/L for 15 minutes, at room temperature, no stirring) platelets (10⁹ cells) were lysed in melting ice-cold buffer (L-buffer: 145 mmol/L NaCl, 0.1 mmol/L MgCl₂, 15 mmol/L HEPES, pH 7.0, 10 mmol/L EGTA, 1 mmol/L Na vanadate, 1.0% Triton X-100, and the protease inhibitors AEBSF [1 mmol/L], leupeptin [50 mg/L], chymostatin [25 mg/L], antipain [25 mg/L], and pepstatin [25 mg/L]). The extracts were centrifuged at 10 000g for 10 minutes at 4°C. The supernatants were transferred to Beckman polypropylene tubes (1 mL) and centrifuged at 100 000g for 30 minutes at 4°C. The 10 000g pellets and 100 000g pellets were resuspended in SDS sample nonreducing buffer (50 µL) and boiled. Aliquots (15 µL) for each one of the two pellets and the final supernatant were analyzed on SDS–polyacrylamide gel electrophoresis and Western blots^{16 17} developed with the antibodies SZ-21 and SZ-22 (1 µg/mL) and horseradish peroxidase–labeled goat anti-mouse IgGs (2 µg/mL), by use of the enhanced chemiluminescence technique (Amersham Corp).¹⁸ Densitometry was used for quantitation of the bands on chemiluminograms (Hewlett Packard Scanjet IIC).¹⁸

Quantitation of Filamentous Actin
To measure F-actin, phalloidin (5 µmol/L) was added to L-buffer to stabilize actin polymers present in the platelets at the time of lysis. Extracts were centrifuged at 100 000g for 30 minutes; pellets then were resuspended in SDS sample buffer and analyzed on Western blots.^{16 17} The concentration of F-actin within the various samples was determined by densitometric analysis of the single reactive band corresponding to actin on blots developed with the antiactin monoclonal antibody C4.¹⁸

Immunohistochemistry
Platelets (2.5x10⁶ cells in 120 µL) were loaded onto collagen-coated coverslips with and without pretreatment with CytoD.⁹ Activation was with 20 µmol/L ADP for 15 minutes at room temperature (no stirring). After 15 minutes on coverslips, the platelets were fixed with formaldehyde (3.7%) in phosphate-buffered saline (PBS). Fixed platelets were stained⁹ with the anti-ß₃ monoclonal antibody SZ-21 (2 µg/mL, 1 hour at 22°C) and an FITC-labeled goat anti-mouse secondary antibody (5 µg/mL, 1 hour at 22°C). The coverslips were mounted onto glass slides with use of Vectashield mounting medium (Vector Laboratories), and photomicrographs of representative fields were obtained by use of a Zeiss microscope and Kodak Ektachrome 400X film. Controls for specificity included omitting the first antibody or replacing the first antibody with the anti-PLC1, which is a cytoplasmic protein not expected to be detected on the surface of intact platelets. This assay also was used to rule out the presence of aggregates in experiments in which ADP-activated platelets were not stirred.

Platelet Aggregation Assay
Platelet aggregation in PRP was induced by adding ADP to stirred (10³ rpm) platelets at 37°C and recorded on a single-channel light densitometer (Chronolog).⁹ Aggregation also was performed on washed platelets resuspended in Tyrode's solution supplemented with fibrinogen (10 µmol/L).

Fluorescence-Activated Cell Sorting Assays
FITC-Fibrinogen Flow Cytometry Assay
This assay is based on the unique ability of activated _IIbß₃ to bind fibrinogen in solution on the surface of activated platelets. Labeling of fibrinogen with FITC, quantitation of fibrinogen in solution, and determination of the fluorescein to fibrinogen ratio were performed as reported previously.¹⁵ Each sample (500 µL in a plastic tube) contained washed platelets in Tyrode's solution (10⁷ cells/mL^-1) supplemented with FITC-fibrinogen (0.3 µmol/L) with or without cytochalasin (10 µmol/L). After 30-minute incubation, platelets were activated with ADP (various concentrations, see "Results") for 15 minutes unless otherwise indicated (in Tyrode's solution at room temperature). Samples were analyzed in a Fluorescence Activated Cell Sorter (FACScan, Becton Dickinson) that was calibrated daily for fluorescence with Cytofluor fluorescent beads.¹⁵ Data acquisition and processing from 10⁴ cells were carried out on a Hewlett Packard computer using FACSCAN Research Software (Version B). The median channel number was used as the measure of platelet fluorescence intensity,¹⁵ which was compared with a standard curve to calculate the number of FITC molecules.¹⁵ With this number and the known fluorescein to fibrinogen ratio, we were able to determine the number of FITC-fibrinogen molecules bound per platelet: FITC-fibrinogen per platelet=fluorescein equivalents per platelet/fluorescein equivalents per fibrinogen molecule.¹⁵

Flow Cytometry Assay for _Iibß₃
Indirect immunofluorescence was used to detect _IIbß₃ molecules on the surface of resting and activated platelets. P2 is a monoclonal antibody that recognizes specifically an extracellular epitope of the _IIbß₃ complex, whereas Tab is a monoclonal antibody specific for an extracellular domain of the GPIIb subunit. Platelets were incubated with and without CytoD as described above for 30 minutes, activated with ADP for 15 minutes, and fixed by adding formaldehyde (3.7%) to the platelet suspension for 30 minutes. Fixed platelets were washed once in Tyrode's solution by spinning the cells at 1200 rpm in a Beckman GPR centrifuge equipped with a GH-3.7 rotor for 10 minutes and resuspended in 1 mL of Tyrode's solution. The primary antibody was added (1 µg/mL^-1) for 30 minutes, and the secondary antibody, FITC-labeled goat anti-mouse (5 µg mL^-1), then was added for another 30 minutes. Antibody binding was measured by FACScan analysis, with FITC beads used as standards. Controls for background included omitting the primary antibody or using a monoclonal against PLC1.

Flow Cytometry Assay for P-Selectin
A similar assay was used to quantitate the expression of P-selectin on the surface of platelets before and after agonist stimulation in the presence and the absence of CytoD. The antibody S12, a monoclonal antibody directed against the extracellular portion of P-selectin on the surface of activated platelets, was used as primary antibody. Controls included omitting the first antibody or using anti-PLC1 as primary antibody.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Inhibition by CytoD of _IIbß₃ Association With the Actin Cytoskeleton
We confirmed that agonist stimulation of platelets induced the fraction of filamentous actin to nearly double (Fig 1).^{7 8 19} Concurrently with the actin polymerization, a marked increase of the fraction of _IIbß₃ that cosedimented with the Triton X-100–insoluble 10⁴g pellet was observed: 12% of total _IIbß₃ in resting platelets versus 31% in cells activated with 20 µmol/L ADP. CytoD efficiently blocked both actin polymerization and _IIbß₃ association with the actin cytoskeleton.^{8 19}

View larger version (29K): [in this window] [in a new window]   Figure 1. CytoD prevents the association of IIbß3 with the actin cytoskeleton. Differential centrifugation of Triton X-100–permeabilized platelets allows the separation of actin cytoskeleton–associated proteins (104g pellet) from membrane-associated proteins (105g pellet) and cytosolic proteins (105g supernatant). Upon platelet activation with ADP, a fraction of IIbß3 became associated with the actin cytoskeleton. Such association could be efficiently blocked by preincubation of the platelets with CytoD. Platelet fractions were analyzed by SDS–polyacrylamide gel electrophoresis and Western blotting.16 17 18 Blots were developed by reaction with the two monoclonal antibodies SZ-21 and SZ-22 and by horseradish peroxidase–labeled goat anti-mouse IgGs, by the enhanced chemiluminescence technique. F-actin was quantitated in matching samples stabilized with phalloidin with use of the monoclonal antibody C4 for immunodetection on Western blots and densitometric analysis of the luminograms.18 Data are representative of three separate experiments.

CytoD Inhibits Platelet Morphological Changes
We also confirmed the ability of CytoD to inhibit coarse platelet morphological changes induced by agonists, collagen, and ADP.^{8 19 20} Platelets loaded on a collagen-coated surface displayed cellular extensions corresponding to lamellipodia and a few fillopodia (Fig 2). The addition of ADP led to further amplification of the same morphological changes. In the presence of CytoD, the platelets were able to adhere to the coverslip but did not display changes in cell shape detectable by light microscopy.

View larger version (59K): [in this window] [in a new window]   Figure 2. Inhibition of platelet remodeling by CytoD. Platelets loaded on collagen-coated coverslips underwent morphological changes that included the extension of filopodia and lamellipodia (left). Addition of ADP (20 µmol/L) potentiated the effect of collagen on platelet morphology. CytoD (10 µmol/L) inhibited the platelet morphological changes induced by collagen or collagen plus ADP that were detectable by light microscopy. Cells were stained for IIbß3 with use of the monoclonal antibody SZ-21.9 Bar corresponds to 2 µm. Panels correspond to representative areas of coverslips from two separate experiments.

Effect of Actin Cytoskeleton Disruption on Platelet Aggregation
Despite the ability of 10 µmol/L CytoD to suppress efficiently actin polymerization, _IIbß₃ association with the actin cytoskeleton, and coarse morphological changes resulting from agonist-induced actin cytoskeleton response, aggregation induced ex vivo by either 2 µmol/L or 20 µmol/L ADP was not decreased by CytoD pretreatment (Fig 3).¹⁰ If anything, aggregation induced by 2 µmol/L ADP was more extensive in the presence of CytoD than without the actin inhibitor. Because plasma proteins could have antagonized the inhibitory effect of CytoD on platelet aggregation, aggregation also was performed using washed platelets resuspended in Tyrode's solution containing purified fibrinogen (10 µmol/L). CytoD (10 µmol/L) did not detectably affect platelet aggregation of washed platelets resuspended in the presence of fibrinogen (data not shown). These data indicate that inhibition of the reorganization of the actin cytoskeleton does not appear to affect platelet aggregation under our standard assay, ex vivo conditions.

View larger version (14K): [in this window] [in a new window]   Figure 3. Tracings show effects of CytoD on platelet aggregation. Aggregation of platelets in PRP induced by 2 µmol/L (top) or 20 µmol/L (bottom) ADP was measured in vitro. Aggregation was studied in the absence of additive (control), in the presence of CytoD (10 µmol/L) in DMSO, or in the presence of DMSO alone. Tracings are representative of three independent experiments. Experiments performed with washed platelets in Tyrode's buffer supplemented with fibrinogen provided similar results (data not shown).

Actin Inhibition and Fibrinogen Binding to the Platelet Surface
Aggregometry measures the clustering of platelets. Such clustering depends on the intercellular bridging of multiple platelets, mediated by _IIbß₃ activation and binding of fibrinogen to its receptor on the surface of adjacent platelets. However, aggregation does not measure directly the binding of fibrinogen to _IIbß₃ because fibrinogen binding to its receptor can occur in the absence of aggregation. Therefore, we sought to achieve a more specific and sensitive measure of this ligand-receptor interaction. We used our FITC-fibrinogen flow cytometry assay¹⁵ to measure the effect of CytoD on the number of fibrinogen molecules capable of binding to resting and activated platelets.

CytoD reduced the number of FITC-fibrinogen binding sites on the surface of platelets activated with saturating concentrations of ADP (Fig 4). When platelets were stimulated with 20 µmol/L ADP, CytoD decreased the number of bound FITC-fibrinogen molecules per platelet by nearly 60%. In the presence of a submaximal ADP concentration (1 µmol/L), CytoD had no detectable effect on fibrinogen binding, supporting the concept that actin inhibition did not directly affect the molecular mechanism leading to _IIbß₃ activation but instead limited the extent of _IIbß₃ recruitment.

View larger version (33K): [in this window] [in a new window]   Figure 4. Bar graph shows effect of CytoD on FITC-fibrinogen binding to platelets. The number of activated IIbß3 molecules on the surface of platelets was quantitated by use of our flow cytometric FITC-fibrinogen binding assay (see "Methods"). Three conditions were tested: in the absence of additive (Control), in the presence of DMSO (DMSO), and in the presence of the same concentration of DMSO but with CytoD (CytoD). Two ADP concentrations were tested: 1 µmol/L ADP and 20 µmol/L ADP. Data correspond to the means of three separate experiments (±SEM).

Accordingly, when FITC-fibrinogen binding was measured as a function of the concentration of ADP, the main effect of CytoD was to reduce the level at which the binding plateaued (Fig 5a). Because ADP is not the strongest agonist for platelets, we used TRAP (54 µmol/L) to attempt to overcome the CytoD inhibitory effect on FITC-fibrinogen binding. While TRAP induced more FITC-fibrinogen binding to activated platelets than 20 µmol/L ADP, CytoD inhibition of fibrinogen-receptor recruitment was not overcome by activation of the thrombin receptor (Fig 5a).

View larger version (19K): [in this window] [in a new window]   Figure 5. a, Scatchard plot shows agonist concentration dependency of FITC-fibrinogen binding. ADP concentration dependency of FITC-fibrinogen binding to platelets in the absence (, control; , DMSO control) or in the presence of 10 µmol/L CytoD (). The initial increase in FITC-fibrinogen binding at low concentration of ADP was similar for CytoD-treated and control platelets; however, the binding level reached a plateau at lower ADP concentration in CytoD-treated cells. A saturating concentration of the TRAP Ser-Phe-Leu-Leu-Arg-Asn (54 µmol/L) was used to test the ability of a stronger agonist to overcome CytoD inhibition. On the right vertical axis TRAP-activated platelets (), in the presence of DMSO (), and in the presence of DMSO and CytoD (). Note that platelet activation with TRAP did result in slightly increased fibrinogen binding, but did not overcome CytoD inhibition. b, Effect of CytoD on Kd of IIbß3-FITC-fibrinogen complex. The effect of CytoD on FITC-fibrinogen binding to activated (20 µmol/L ADP) platelet surface was studied as a function of FITC-fibrinogen concentration. Bound and free FITC-fibrinogen were measured for each FITC-fibrinogen concentration by FACScan analysis in the presence and the absence of CytoD. The Kd for the IIbß3-FITC-fibrinogen complex was deduced from the slope of the best-fit line to the experimental data presented on Scatchard plots. Data points correspond to the means of two separate experiments: , control platelets; , cytoD-treated platelets. Intersection of the best fit-line with the x-axis confirms that CytoD reduces the binding sites for fibrinogen on the surface of ADP-activated (20 µmol/L) platelets by nearly 50%.

To conclude that the reduced binding of fibrinogen to the surface of maximally activated platelets with ADP (or TRAP) induced by CytoD was due to reduced receptor recruitment, we had to exclude two alternative mechanisms: (1) that a lower affinity (increased dissociation constant [K_d]) of fibrinogen for its receptor was induced by CytoD or (2) that the presence of CytoD resulted in marked slowing of the activation rate of the receptor.

We measured the dissociation constant (K_d) for the binding of FITC-fibrinogen to the surface of activated platelets in the presence and the absence of CytoD pretreatment (Scatchard plot, Fig 5b). While CytoD reduced by half the number of binding sites for FITC-fibrinogen (Fig 5b, x-intersect), we could not detect any change in the K_d of the complex between _IIbß₃ and FITC-fibrinogen resulting from actin disruption (0.28 versus 0.26 µmol/L). Therefore, it is unlikely that the effect of CytoD on fibrinogen binding was mediated by a change in receptor affinity.

We next tested the possibility that CytoD slowed the rate of _IIbß₃ activation. FITC-fibrinogen binding to the platelet surface was tested at three time points (15, 30, and 45 minutes), either in the absence of agonist or in the presence of 1 µmol/L or 20 µmol/L ADP. If CytoD acted to slow the _IIbß₃ activation rate, we reasoned that the amount of fibrinogen binding to CytoD-treated platelets should approach that of the controls over the time course of this experiment. However, there was no significant change in FITC-fibrinogen binding beyond 15 minutes of incubation with or without ADP or CytoD treatments, indicating that actin inhibition did not merely retard activation of the fibrinogen receptor (data not shown).

Effect of Actin Inhibition on _IIbß₃ Detection on the Surface of Platelets
To further support that CytoD impaired fibrinogen receptor recruitment to the platelet surface, where it becomes accessible to extracellular ligands, we measured the effect of CytoD on _IIbß₃ expression on the surface of platelets, using antibodies specific for this integrin. It was previously reported that the number of _IIbß₃ molecules detectable on the surface of activated platelets increases twofold to threefold upon platelet activation, although this increase varied with the type of agonist used.⁶

With 20 µmol/L ADP, the number of _IIbß₃ molecules expressed on the platelet surface nearly doubled. However, no detectable increase in _IIbß₃ expression was observed when cells were pretreated with CytoD (Fig 6). Similar results were obtained independent of the type of anti-_IIbß₃ antibody used in these experiments (anti-_IIbß₃ complex versus anti-_IIb). The extent of CytoD-inhibition of _IIbß₃ recruitment to the cell surface was sufficient to account for the reduction in FITC-fibrinogen binding to activated platelets induced by CytoD (compare Fig 4 and Fig 6).

View larger version (60K): [in this window] [in a new window]   Figure 6. Bar graphs show detection of IIbß3 on platelet surface with use of monoclonal antibodies. Platelets, either treated with CytoD in DMSO or with DMSO alone, were fixed in the resting state or after 15-minute incubation with ADP (20 µmol/L). Indirect immunofluorescence using monoclonal antibodies against IIbß3 as primary antibodies was quantitated by FACScan analysis. Antibody P2 is directed toward a complex-specific epitope in the extracellular domain of IIbß3, whereas Tab recognizes specifically the extracellular domain of GPIIb. The data, expressed in percent of maximum signal obtained with 20 µmol/L ADP in the absence of CytoD, correspond to the mean of three separate experiments (±SEM). Background immunofluorescence was measured by omitting the first antibody or by using a monoclonal antibody against PLC1 as primary antibody (data not shown). No effect of CytoD or ADP could be detected on the fluorescent signal corresponding to either of the two controls.

Effect of Actin Inhibition on -Granule Secretion
These data suggest that an intact actin superstructure is necessary for the recruitment to the platelet surface of _IIbß₃ in response to ADP. The intracellular sites from which this cryptic pool of _IIbß₃ arises could consist of -granules and the OCS.⁵ However, ADP is generally considered a weak platelet agonist that causes shape change and aggregation but weak granule secretion.^{21 22} Therefore, the membrane of the OCS represented the most likely source of recruited _IIbß₃ molecules. Nevertheless, to rule out a contribution of the -granule pool to the observed increase in cytoskeleton-mediated _IIbß₃ surface expression, we measured the effect of CytoD on -granule secretion. We used the expression of P-selectin on the surface of activated platelets as a marker for -granule translocation to and fusion with the plasma membrane. As reported,^{21 22} platelets activated with ADP demonstrated only a relatively small increase in cell surface expression of P-selectin compared with cells stimulated with thrombin (Fig 7). Treatment with CytoD did not block P-selectin expression, and, if anything, P-selectin expression triggered by ADP was increased in the presence of CytoD. These experiments indicate that the inhibitory effect of CytoD on _IIbß₃ surface expression is unlikely to involve -granule secretion in our system.

View larger version (47K): [in this window] [in a new window]   Figure 7. Bar graph shows effect of CytoD on P-selectin expression. P-selectin was detected by indirect immunofluorescence by use of FACScan analysis. Platelets were treated with CytoD and DMSO or DMSO alone and fixed after incubation with or without ADP (20 µmol/L) for 15 minutes. Data represent the mean of two separate experiments (±SD). Fluorescence data were converted to number of P-selectin molecules per platelet, assuming a ratio of 4 to 1 between FITC and the secondary antibody (information provided by the manufacturer). Controls included assays performed in the absence of primary antibody or using anti-PLC1 as primary antibody (data not shown).

Because the increased P-selectin expression in the presence of CytoD could have resulted from nonspecific alteration of platelet membrane, we tested the integrity of our CytoD-treated platelets by using a monoclonal antibody directed toward an intracellular antigen, PLC1, which is not expressed on the surface of platelets. The fluorescent signal corresponding to PLC1 did not exceed background level and was not affected by CytoD treatment or ADP activation (data not shown).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
We conclude that CytoD inhibition of FITC-fibrinogen binding to the surface of activated platelets results from impairment of actin polymerization and _IIbß₃ binding to the actin cytoskeleton. As a result, the externalization of the OCS fails, a process that is necessary to the unmasking of cryptic _IIbß₃ in response to ADP.

The physiological maintenance of normal hemostasis and the pathological development of an intracoronary thrombus require platelet aggregation and formation of a platelet plug at the site of vessel injury. Several rapid events occur upon platelet stimulation by an agonist, including a remarkable change in platelet shape that requires polymerization of actin and fibrinogen binding to the _IIbß₃ receptor. The present study provides evidence linking these two events and indicates that as much as half of the total amount of platelet _IIbß₃ normally present on the surface of maximally activated platelets was lacking in the presence of CytoD. Our data are consistent with receptor molecules being sequestered within the membranes of the OCS in CytoD-treated cells. In this cryptic site, _IIbß₃ is not accessible to fibrinogen or to antibodies used for our study⁵ and requires a functional actin cytoskeleton in order to be translocated to the platelet surface.

Previous studies on spread platelets using colloidal gold-labeled fibrinogen and electron microscopy have shown that the centripetal movement of _IIbß₃ triggered by ligand binding is not blocked by cytochalasin B.²³ Although this result appears to be sensitive to experimental conditions,²⁴ it suggests that, unlike for surface recruitment, bound receptor molecules undergoing centripetal migration can use the residual actin cytoskeleton of cytochalasin-treated platelets or a microtubular system instead of the actin cytoskeleton. Actually, bound receptor translocation to the open canalicular system at the center of spread platelets might even be enhanced by the effect of cytochalasin B on the platelet actin cytoskeleton.²³ Previous studies have indicated that the open canalicular system functions as "a two-way street"²⁵ for fibrinogen receptor molecules in such a way that _IIbß₃ molecules migrate both inward and outward (OCS externalization), but only the externalization of the OCS is blocked by cytochalasins. Inhibition of _IIbß₃ outward migration accompanying OCS externalization is supported by our results showing reduced binding of FITC-fibrinogen and anti-_IIbß₃ antibodies to the surface of CytoD-treated platelets. Thus, the apparent increase in receptor migration to the cell center²³ may result in part from inhibition of further OCS externalization.

Externalization of _IIbß₃ appears to require binding to the actin cytoskeleton, although the precise conditions leading to binding remain controversial. We confirmed the finding of Bertagnolli and Beckerle¹¹ that association of _IIbß₃ with the Triton X-100–insoluble pellet can be observed in unstirred, unaggregated, agonist-stimulated platelets. However, in contrast with the latter study, in which the source of cryptic _IIbß₃ appeared to correspond to -granules, our data suggest that the source of cryptic receptor molecules corresponds to the OCS. This conclusion is based on the inability of CytoD to reduce the limited -granule secretion induced by ADP at concentrations that blocked _IIbß₃ recruitment. It is possible that the discrepancy between our data and those provided by Bertagnolli and Beckerle¹¹ results from the fact that they have used the stronger secretion agonist thrombin in their experiments.

Platelet activation results in a shift of actin subunits from the monomeric pool to the filamentous pool. The mechanism leading to actin polymerization is not fully characterized but must involve the generation of an excess of actin monomers over the critical concentration of the filament ends.^{7 26} This could be achieved by lowering the critical concentration of actin through the uncapping of filament barbed ends and/or the generation of new barbed ends by severing or nucleating filaments.⁸ Lowering the actin critical concentration also could result from increasing the concentration of free, polymerization-competent, ATP-bound actin subunits or a combination thereof.^{26 27} Actin polymerization is necessary to allow the substantial changes in cell shape that occur upon platelet activation, and it is known that addition of CytoD to platelets prevents shape changes after addition of agonists.^{7 8 19 20}

Integrins participate in protein complexes whose stability is regulated by the small GTP-binding protein Rho^{28 29} and represent the membrane anchorage site for actin filaments.³⁰ While integrins are usually not believed to bind directly to actin filaments, Bertagnolli and Beckerle¹¹ have reported that in thrombin-activated platelets, a subpopulation of _IIbß₃ (22% of total cellular _IIbß₃) establishes a strong, direct interaction with the actin cytoskeleton. The molecular link that seals this complex is not yet characterized, and it is not clear to which extent this direct interaction is regulated by small GTP-binding proteins of the Rho family.

Like the ß₂ integrins, _IIbß₃ must be activated in order to bind to its ligand in solution.^{2 31} The mechanism of activation is divalent cation dependent and presumably involves a conformational change in the _IIbß₃ molecule.² Our data do not support an active role of actin in the intramolecular isomerization of _IIbß₃ because platelet aggregation was not inhibited by CytoD treatment, and FITC-fibrinogen binding to platelet surface in response to 1 µmol/L ADP was not affected by CytoD. However, a competent actin cytoskeleton was required for the recruitment of cryptic _IIbß₃ in response to ADP and more specifically, the _IIbß₃ subpopulation, which is stored in the membrane of the OCS.

Recent evidence suggested that the cross-linking of two _IIbß₃ molecules by fibrinogen on the surface of a platelet generates intracellular signals and in particular the activation of a tyrosine kinase pathway.^{1 4} Thus, it is possible that a mechanism of positive reinforcement exists whereby agonist activation of _IIbß₃ leads to the stimulation of tyrosine kinases within target platelets. Tyrosine kinase pathways represent key regulators of the actin cytoskeleton^{32 33} and may be controlling the actin reorganization after agonist activation of platelets. Actin reorganization leading to shape change and extroversion on the OCS provides additional _IIbß₃ molecules. New binding sites for fibrinogen may in turn induce increased outside-in signals and therefore generate a positive feedback mechanism for this process.¹

Several important questions are raised by our study and are presently being investigated in our laboratory. (1) What is the role for platelet adhesion and aggregation in vivo of the subpopulation of _IIbß₃ (50% of total) that requires an intact actin response for expression on the surface of activated platelets? Aggregation ex vivo does not require the participation of this cryptic fraction of _IIbß₃ molecules. However, one may speculate that aggregation ex vivo might require these additional binding sites for fibrinogen to consolidate a forming thrombus, particularly in arterial vessels in which shear forces are stringent. (2) Our data indicate that the actin network does not contribute to the initial intramolecular changes that convert _IIbß₃ into a receptor capable of binding soluble fibrinogen (K_d, 0.26 µM). Thus, if interaction with actin is not needed, what are the "activating" factors involved in this process? (3) The actin cytoskeleton is necessary to generate outside-in signals in response to fibrinogen binding to its receptor. In turn, does tyrosine phosphorylation in response to fibrinogen cross-linking of _IIbß₃ promote the reorganization of the actin cytoskeleton leading to the translocation of cryptic _IIbß₃ molecules to the platelet surface? While occupancy of the _IIbß₃ receptor by the peptide Arg-Gly-Asp-Ser does not alter the actin reorganization in activated platelets,³⁴ it is possible that intracellular _IIbß₃ receptor cross-linking by fibrinogen, which induces a tyrosine kinase response,^{1 3 4} modulates the structure of the actin network.

	Selected Abbreviations and Acronyms

 

CytoD = cytochalasin D DMSO = dimethyl sulfoxide FITC = fluorescein isothiocyanate OCS = open canalicular system PLC1 = phospholipase C1 PRP = platelet-rich plasma SDS = sodium dodecyl sulfate TRAP = thrombin receptor tethered peptide

	Acknowledgments

 
P.J.G-C. has been a Syntex Scholar since 1992. Support for this study was also provided by the American Heart Association, Maryland Affiliate, and the Bernard Foundation.

Received July 25, 1994; accepted June 22, 1995.

	References

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