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J. Biol. Chem., Vol. 277, Issue 17, 14738-14746, April 26, 2002

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RhoA Sustains Integrin _IIb3 Adhesion Contacts under High Shear^*

Simone M. Schoenwaelder, Sascha C. Hughan^§, Karen Boniface, Sujanie Fernando, Melissa Holdsworth, Philip E. Thompson, Hatem H. Salem, and Shaun P. Jackson

From the Department of Medicine, Australian Centre for Blood Diseases, Monash University, Box Hill Hospital, Arnold St., Box Hill, Victoria 3128, Australia

Received for publication, January 22, 2002, and in revised form, February 5, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The small GTPase RhoA modulates the adhesive nature of many cell types; however, despite high levels of expression in platelets, there is currently limited evidence for an important role for this small GTPase in regulating platelet adhesion processes. In this study, we have examined the role of RhoA in regulating the adhesive function of the major platelet integrin, _IIb3. Our studies demonstrate that activation of RhoA occurs as a general feature of platelet activation in response to soluble agonists (thrombin, ADP, U46619, collagen), immobilized matrices (von Willebrand factor (vWf), fibrinogen) and high shear stress. Blocking the ligand binding function of integrin _IIb3, by pretreating platelets with c7E3 Fab, demonstrated the existence of integrin _IIb3-dependent and -independent mechanisms regulating RhoA activation. Inhibition of RhoA (C3 exoenzyme) or its downstream effector Rho kinase (Y27632) had no effect on integrin _IIb3 activation induced by soluble agonists or adhesive substrates, however, both inhibitors reduced shear-dependent platelet adhesion on immobilized vWf and shear-induced platelet aggregation in suspension. Detailed analysis of the sequential adhesive steps required for stable platelet adhesion on a vWf matrix under shear conditions revealed that RhoA did not regulate platelet tethering to vWf or the initial formation of integrin _IIb3 adhesion contacts but played a major role in sustaining stable platelet-matrix interactions. These studies define a critical role for RhoA in regulating the stability of integrin _IIb3 adhesion contacts under conditions of high shear stress.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Platelet adhesion and aggregation at sites of vascular injury is essential for the arrest of bleeding and for subsequent vessel wall repair. The ability of platelets to adhere under conditions of rapid blood flow requires the synergistic contribution of multiple receptor-ligand interactions, foremost of which involves the interaction between von Willebrand factor (vWf)¹ and the two major platelet adhesion receptors, glycoprotein (GP) Ib/V/IX and integrin _IIb3. The vWf·GP Ib/V/IX interaction is characterized by a rapid association rate that enables efficient platelet tethering to the injured vessel wall, whereas subsequent integrin _IIb3 engagement of vWf is important for promoting platelet arrest (1). The vWf·GP Ib/V/IX interaction is indispensable for normal platelet function, because it slows platelet movement at the vessel wall thereby enabling receptors with slower intrinsic binding kinetics, i.e. integrins, to engage adhesive ligands. A similar dual-step adhesion mechanism, involving selectins and ₂ integrins, is employed by leukocytes to adhere to post-capillary venules at sites of inflammation (2-4).

A key requirement of the adhesion contacts formed by platelets and leukocytes is their ability to resist the detaching effects of rapidly flowing blood. In the case of platelets, the shear forces operating on adhesive bonds can be extremely high, particularly at sites of arterial narrowing where shear forces can exceed 380 dyne/cm² (10,000 s¹). The ability of the GP Ib/V/IX complex to maintain platelet adhesive interactions under such high shear conditions is due to the large number of bonds formed between vWf and GP Ib, the inherent biomechanical stability of the vWf·GP Ib interaction, and anchorage of the receptor complex to the membrane skeleton (1). The factors that regulate the stability of integrin _IIb3 adhesion contacts in a shear field remain less clearly defined. It is well established that integrin engagement of adhesive ligands leads to receptor clustering and the formation of firm anchorage points with the actin-based cytoskeleton (5), however, the importance of such post-ligand binding events for sustaining platelet adhesion in a shear field remains unclear.

The adhesive function of many integrins is regulated in part by RhoA, a member of the Ras homologous (Rho) family of small GTPases (6). RhoA activation has been extensively studied in a range of cultured cell and is critical for the formation of actin stress fibers and focal adhesions (7). Several RhoA effector proteins have been implicated in the formation of these structures, including p140mDia and Rho kinase (p160^ROCK, ROK). The effect of Rho kinase on focal adhesion formation appears to involve phosphorylation and inactivation of myosin light chain (MLC) phosphatase leading to increased MLC phosphorylation and enhanced myosin II contractility, resulting in tension on actin filaments and their subsequent alignment. Furthermore, RhoA-mediated activation of phosphatidylinositol-4-phosphate 5-kinase induces the formation of phosphatidylinositol (4,5)-bisphosphate, a widely acting phospholipid that promotes actin polymerization and cytoskeletal-membrane attachment (8, 9). The culmination of these and other RhoA-mediated signaling events leads to bundling of actin filaments into actin cables or thick stress fibers and the clustering of their associated integrin receptors into focal adhesions (7). These cytoskeletal changes induced by RhoA leads to strong cell attachment points with the extracellular matrix (8).

The platelet contains relatively high levels of RhoA, along with other RhoA effectors (10). However, despite this, very little is known about the mechanisms of RhoA activation or its contribution to integrin-mediated adhesive responses in platelets. Recent studies have demonstrated RhoA activation in response to thrombin (11) and the thromboxane mimetic, U46619 (12), with activation of RhoA by the latter agonist reported to occur independent of integrin _IIb3 (12). The role of RhoA in regulating the adhesive function of integrin _IIb3 remains controversial (10, 13-16). Although initial reports suggested a potentially important role in regulating integrin _IIb3 activation (10), more recent studies have not supported these conclusions (13, 14, 17). In general, these latter findings are in keeping with studies on cultured cells in which changes in the activation status of RhoA do not correlate with alterations in integrin affinity (18).

In the current study, we have investigated the role of RhoA in regulating the adhesive function of integrin _IIb3. Our studies have demonstrated the existence of integrin _IIb3-dependent and -independent mechanisms regulating RhoA activation. Although RhoA activation appears to represent a general feature of platelet activation, our studies do not support an important role for RhoA in regulating the affinity status of integrin _IIb3. Rather, we demonstrate an important role for RhoA in regulating the stability of integrin _IIb3 adhesion contacts. The ability of RhoA to sustain integrin _IIb3·matrix interactions appears critical for efficient platelet adhesion in a shear field.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Antibodies-- The anti-₃ chimeric Fab fragment of the monoclonal antibody 7E3 (c7E3 Fab-abciximab) was from Eli-Lilly (Centocor, Leiden, The Netherlands). The RhoA monoclonal antibody was from Santa Cruz Biotechnology Inc. (Santa Cruz, CA), and the horseradish peroxidase-rabbit anti-mouse-IgG was from Jackson Laboratories. TRAP_(1-6) (SFLLRN), PAR4 agonist peptide (AYPGKF-NH₂), and RGDS peptide were prepared using standard solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl)-based procedures, on a Rainin PS3 automated synthesizer, according to the method Fields and Noble (19). All other reagents were from sources described previously (20-23).

Preparation of Native Human von Willebrand Factor-- Human von Willebrand factor (HvWf) was purified to homogeneity from plasma cryoprecipitate according to the method of Montgomery and Zimmerman (24).

Preparation of Asialo Human von Willebrand Factor-- Asialo vWf was prepared from vWf that had been purified from a human dried Factor VIII Fraction (CSL Ltd., Victoria, Australia). Briefly, reconstituted Factor VIII fraction was applied to a Sepharose CL-6B size-exclusion column, fractions were collected, and vWf was detected by SDS-PAGE and ristocetin cofactor activity. Fractions containing purified vWf were pooled and concentrated by centrifugation. Purified vWf (100 µg/ml) was dialyzed in 0.05 M sodium acetate, 150 mM NaCl, 8 mM CaCl₂, pH 6.0, at 4 °C, followed by incubation with 0.1 unit of 2-3,6,8-neuraminidase, Vibrio cholerae (Calbiochem-Novabiochem Corp.) for 3 h at 37^oC. De-sialylated vWf was dialyzed in 10 mM Tris-HCl, 150 mM NaCl, pH 7.4, and platelet aggregating activity was confirmed by addition of 20 µg/ml purified protein to citrated platelet-rich plasma.

Purification of Fibrinogen-- Human fibrinogen was purified from fresh frozen plasma, according to the method of Jakobsen and Koerulf (25).

Preparation of Washed Platelets and Red Blood Cells-- Whole blood (anticoagulated with acid-citrate-dextrose) was collected from healthy volunteers who had not received any anti-platelet medication in the preceding 2 weeks. Washed platelets were prepared as previously described (21) and resuspended in Tyrode's buffer (10 mM Hepes, 12 mM NaHCO₃, pH 7.4, 137 mM NaCl, 2.7 mM KCl, 5 mM glucose) containing 1 mM calcium, where indicated. Autologous red blood cells (RBCs) were obtained as described previously (23).

Preparation and Treatment of Platelets with C3 Exoenzyme-- A construct encoding recombinant C3 exoenzyme (a gift from Prof. Keith Burridge, University of North Carolina, Chapel Hill, NC) was expressed as a glutathione S-transferase (GST) fusion protein in Escherichia coli, followed by thrombin-mediated cleavage of the GST tag, as described by Dillon and Feig (26). Purified C3 exoenzyme was dialyzed against Tyrode's buffer overnight at 4 °C. Platelets in platelet washing buffer (pH 6.5) were incubated with purified C3 exoenzyme (100 µg/ml) in the presence of 200 units of hirudin for 4 h at room temperature. Using these conditions, we routinely achieved ~80% inhibition of RhoA (Fig. 3A), without significant loss of platelet reactivity (data not shown).

Preparation and Treatment of Platelets with Y27632-- The Rho kinase inhibitor Y27632 was prepared as described previously by Uehata and colleagues (63). Washed platelets resuspended in Tyrode's buffer were incubated with vehicle alone (Me₂SO) or Y27632 (20 µM) for 10 min at room temperature.

ADP-ribosylation of RhoA by C3 Exoenzyme-- ADP-ribosylation of RhoA in platelet extracts was analyzed using a modified method as described by Leng and colleagues, et al. (13). Briefly, platelets incubated with vehicle alone or the indicated concentrations of C3 exoenzyme, were lysed on ice by the addition of an equal volume of lysis buffer (20 mM Hepes, pH 7.4, 145 mM NaCl, 1.5% Triton X-100, 0.8% deoxycholate, 0.2% SDS, 3 mM EGTA, 2 mM MgCl₂, 50 µg/ml phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin). Immediately prior to lysis, an equivalent amount of C3 exoenzyme was added to control platelets. In vitro ADP-ribosylation was performed immediately at 30 °C for 60 min, by addition of 200 µM GTP, 10 µM NAD⁺, 10 µg/ml C3 exoenzyme, and 05-2.5 µCi/assay [³²P]NAD⁺. Reactions were terminated by the addition of sample buffer, boiled, and analyzed on SDS-PAGE, followed by autoradiography.

Assay for GTP-bound RhoA-- The level of GTP-bound RhoA in platelet lysates was measured as described previously (27, 28). Western blots were quantified by densitometry (GelPro software), and RhoA activation expressed as -fold increase over the level of active RhoA in resting platelets, after correcting for protein loading (histograms).

Aggregation Studies-- Washed platelets were stimulated with the indicated concentrations of agonist, in the presence of calcium (1 mM) and fibrinogen (500 µg/ml). Aggregation initiated by thrombin was performed in the absence of fibrinogen. All aggregations were initiated by stirring the suspensions at 950 rpm for 10 min at 37 °C in a four-channel automated platelet analyzer (Kyoto Daiichi, Japan). The extent of platelet aggregation was defined as the percentage change in optical density as measured by the automated platelet analyzer.

Static Adhesion and Spreading Assays-- Static adhesion assays were performed using a modified method of Yap et al. (23). Briefly, glass coverslips were coated with 10 µg/ml HvWf for 2 h at room temperature, followed by blocking with 10% heat-inactivated human serum for 60 min at room temperature. Platelets were incubated with immobilized matrices for between 30-60 min, unless otherwise indicated, and visualized using phase contrast microscopy. Images were captured, and the surface area of adherent platelets was determined using MCID software (Imaging Research Inc.). The number of C3 exoenzyme-treated adherent platelets and surface area of C3 exoenzyme-treated adherent platelets was expressed as a percentage of vehicle-treated control platelets.

In Vitro Flow-based Adhesion Assays-- Flow assays were performed using glass microcapillary tubes (Microslides, Vitro Dynamics Inc.) coated with 100 µg/ml HvWf, according to the method described by Yap et al. (23). Microcapillary tubes were blocked with 10% heat-inactivated human serum prior to use. Washed platelets reconstituted with RBCs (50% hematocrit) were perfused across coated microcapillary tubes at shear rates of 600 and 1800 s¹. Platelet tethering and stationary adhesion was visualized in real time using differential interference contrast microscopy and the first 2 min of flow video recorded for off-line analysis. In all studies, off-line analyses of platelet tethering and stationary adhesion were performed as described previously (23). Briefly, any cell forming an adhesion contact with immobilized HvWf for greater than 40 ms was scored as a tethered cell. Stationary adhesion was arbitrarily defined as cells not moving more than a single cell diameter over a 10-s period. The duration of stationary platelet adhesion contacts was determined on individual platelets that had formed stationary interactions with immobilized vWf for a period of 1 s. Individual cells were analyzed every second for a period up to 30 s, and stationary adhesion was arbitrarily defined as cells not moving more than a single cell diameter over a 1-s period. For analysis of platelet spreading, adherent platelets were washed for 15 min with Tyrode's buffer (1800 s¹), visualized using phase contrast microscopy, and video recorded for off-line analysis. Platelet spreading was analyzed as described for the static adhesion assays.

Shear-induced Platelet Aggregation Studies-- Control or Y27632- or C3 exoenzyme-treated platelets, resuspended in Tyrode's buffer, pH 7.5, and containing 1 mM CaCl₂, were subjected to shear (5000 s¹) in the presence of 20 µg/ml HvWf for 5 min, using a cone-and-plate viscometer, as described previously (29). Platelets were visualized using inverse phase contrast microscopy (×5 objective), and images were captured using a charge-coupled device camera. Five random fields were captured from each experimental sample, and quantification of single platelets was performed using MCID software.

Immunofluorescence Microscopy-- PAC1 immunofluorescence was performed under static and flow conditions, as described previously (23). Platelets were imaged via confocal microscopy, using ×100 oil immersion, and quantitation was performed using Leica software.

Statistical Analysis-- Significant differences were detected using an unpaired t test with one-way analysis of variance, using Prism software (GraphPad Software for Science, San Diego, CA).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Integrin _IIb3-dependent and Independent Pathways Regulating RhoA Activation-- Platelets respond to a multitude of activating stimuli in vivo, including soluble agonists generated at the site of vascular injury, adhesive substrates present within the damaged vessel wall, and hemodynamic forces generated by the flow of blood. In an attempt to gain further insight into the functional role of RhoA in platelets, we initially examined whether RhoA activation was restricted to a subset of platelet-activating stimuli or occurred as a general feature of platelet activation. Initially, platelets were stimulated in suspension with a diverse range of soluble agonists, including potent agonists such as thrombin, collagen, and the thromboxane A₂ mimetic, U46619, or weaker agonists such as ADP. To monitor RhoA activation directly, we utilized an assay that selectively isolates GTP-bound active RhoA from cell lysates (27) by precipitation with the RhoA binding domain of the downstream effector Rhotekin (GST-RBD), as described under "Experimental Procedures." Consistent with previous reports (11, 12), RhoA activation was induced by thrombin (1 unit/ml) and U46619 (500 nM) (Fig. 1, left). In addition, RhoA activation was also induced by collagen (10 µg/ml) and ADP (25 µM), but with the latter agonist the rate and extent of RhoA activation was substantially less than with the other agonists (Fig. 1A, and data not shown).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   RhoA activation represents a general feature of platelet activation and is regulated by integrin IIb3. A and B, washed platelets resuspended in Tyrode's buffer supplemented with calcium (1 mM) were incubated with buffer alone (Resting), thrombin (1 unit/ml, 1 min), collagen (10 µg/ml, 1-2 min), U46619 (0.5 µM, 1-2 min), or ADP (25 µM, 5-10 min), in the presence (+) or absence () of c7E3 Fab (20 µg/ml). In part (C), platelets were stimulated with 1.0 µM U46619 for 90 s in the presence of control buffer, c7E3 Fab (20 µg/ml), or RGDS (1 mM) for 10 min prior to stimulation. Following stimulation, platelets were lysed, and RhoA activity was measured as described under "Experimental Procedures." Immunoblots demonstrating active RhoA (RhoA GTP) are from one experiment representative of three. Histograms represent the means ± S.E. of three separate experiments (n = 3), the results of which are presented as the -fold increase over levels obtained in resting platelets (= 1.0).

In cultured cells, the activation status of RhoA is regulated by soluble growth factors and integrin adhesion receptors (18, 30). However, to date, there is no evidence that platelet integrins are involved in regulating RhoA. To examine the potential involvement of the major platelet integrin _IIb3 in this process, platelets were pretreated with the integrin _IIb3-blocking antibody c7E3 Fab, prior to the performance of platelet aggregation studies. In thrombin-stimulated platelets, c7E3 Fab-pretreatment resulted in a reduction of ~70% in RhoA activation (Fig. 1B), whereas with U46619, collagen, and ADP, integrin _IIb3 inhibition resulted in >90% inhibition of RhoA activation. Our findings with respect to U46619 are in direct contrast to those recently reported by Gratacap and colleagues (12) who demonstrated that integrin _IIb3 blockade with RGDS peptides had no inhibitory effect on RhoA activation. To investigate potential explanations for this difference, we compared the effects of RGDS peptide and c7E3 Fab on the activation of RhoA induced by U46619 under identical experimental conditions employed by Gratacap et al. (12). Consistent with previous findings (12), we did not detect any inhibitory effect of RGDS peptide on RhoA activation induced by U46619, however, c7E3 Fab dramatically inhibited RhoA activation under identical experimental conditions (Fig. 1C). Findings similar to c7E3 Fab were observed with another potent integrin _IIb3 antagonist, aggrastat (data not shown). These findings suggest that RGDS peptide may not completely block ligand binding to integrin _IIb3, a finding consistent with previous reports (31).

To examine whether RhoA activation represents a general feature of platelet activation, changes in RhoA activity were assessed following platelet adhesion to immobilized substrates and in platelets exposed to high shear stress. As demonstrated in Fig. 2, adhesion and spreading of platelets on either a fibrinogen (100 µg/ml) or human vWf (10 µg/ml) matrix was associated with significant RhoA activation, similar to the levels observed with soluble agonist stimulation. Pretreating platelets with c7E3 Fab prior to adhesion to vWf inhibited RhoA activation by >95% (Fig. 2B). To examine the effects of high fluid shear stress on RhoA activation, shear-induced platelet activation (SIPA) studies were performed using a cone-and-plate viscometer, as described under "Experimental Procedures." In control studies, we demonstrated that high shear rates (typically 3000 s¹) were required to induce aggregation of washed platelets independent of the addition of exogenous stimuli. Furthermore, aggregation under these experimental conditions was dependent on vWf binding to GPIb and integrin _IIb3, because it was completely inhibited by pretreating platelets with blocking antibodies against either receptor (data not shown). As demonstrated in Fig. 2A, exposure of platelets in suspension to high shear (5000 s¹) in the presence of purified soluble human vWf, induced platelet aggregation and robust RhoA activation. In contrast, exposure of platelets to relatively low levels of shear (<1000 s¹) failed to induce RhoA activation (data not shown), consistent with the requirement for a threshold level of shear to induce platelet activation independent of other exogenous stimuli. Similar to the findings on immobilized vWf, blocking ligand binding to integrin _IIb3 with c7E3 Fab abolished RhoA activation (Fig. 2B). Taken together, these studies suggest that RhoA activation represents a general feature of platelet activation. Furthermore, they demonstrate a major role for integrin _IIb3 in regulating the activation state of RhoA.

View larger version (28K): [in this window] [in a new window]   Fig. 2.   RhoA activation induced by platelet adhesion or high shear. A and B, washed platelets resuspended in Tyrode's buffer supplemented with calcium (1 mM) were incubated with immobilized vWf (10 µg/ml) or fibrinogen (100 µg/ml) for 30 min, or sheared (5000 s1) in the presence of 20 µg/ml human vWf for 5 min (HvWf+Shear) using the cone-and-plate viscometer, in the presence (+) or absence () of c7E3 Fab (B). Activated platelets were lysed and RhoA activity measured. Immunoblots demonstrating active RhoA (RhoA GTP) are taken from one experiment representative of three. Histograms represent the means ± S.E. of three separate experiments (n = 3), the results of which are presented as the -fold increase over levels obtained in resting platelets (= 1.0).

Effect of Inhibiting RhoA on Platelet Adhesion and Aggregation-- The role of RhoA in regulating platelet aggregation remains controversial. Several studies have demonstrated inhibition of platelet aggregation in the presence of the specific RhoA inhibitor, C3 exoenzyme (10, 16), or the Rho kinase inhibitor Y27632 (15), whereas others have failed to show any inhibitory effect (13). To investigate the role of RhoA in regulating platelet aggregation, platelets were pretreated with C3 exoenzyme. Incubation of platelets with increasing concentrations of C3 exoenzyme (0-100 µg/ml) demonstrated a dose-dependent ADP-ribosylation of RhoA, with ~80% inhibition of this small GTPase following incubation with 100 µg/ml C3 exoenzyme for 4 h at room temperature (Fig. 3A). In all subsequent experiments, high concentrations (100 µg/ml) of C3 exoenzyme were used to inhibit RhoA. As demonstrated in Fig. 3B, pretreating platelets with C3 exoenzyme had no inhibitory effect on platelet aggregation induced by thrombin or the PAR1 specific agonist, TRAP. Furthermore, the same concentration of C3 exoenzyme had no effect on the ability of these agonists to induce binding of the activation-specific integrin _IIb3 antibody, PAC1 (Fig. 3C). Similar findings were observed using the Rho kinase inhibitor Y27632 (20 µM) (Fig. 3B and data not shown). Moreover, neither C3 exoenzyme nor Y27632 had any inhibitory effect on platelet aggregation induced by collagen, U46619, or ADP (Fig. 3B), demonstrating that RhoA is unlikely to play a major role in regulating integrin _IIb3 activation induced by soluble agonists.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Examination of the role of RhoA and Rho kinase in regulating platelet aggregation and integrin IIb3 activation. Platelets were pretreated with the indicated concentrations of C3 exoenzyme or Y27632, using conditions indicated under "Experimental Procedures." A, inhibition of RhoA by C3 exoenzyme. The amount of RhoA ADP-ribosylated by C3 exoenzyme was assessed by performing an ADP-ribosylation assay in the presence of [32P]NAD+, as described under "Experimental Procedures." The autoradiograph is taken from one experiment representative of three. The histogram depicts the means ± S.E. from three separate experiments (n = 3). These results demonstrate that, although 100% of RhoA was available for in vitro ribosylation following the incubation of platelets with vehicle alone, this amount was reduced in the samples preincubated with C3 exoenzyme, indicating that a significant proportion of RhoA in these platelets had become ADP-ribosylated during the initial incubation period. B, effect of C3 exoenzyme or Y27632 on platelet aggregation. Aggregation was initiated by the addition of either TRAP (1 µM), thrombin (1 unit/ml), collagen (10 µg/ml), ADP (25 µM), or U46619 (0.5 µM), to washed pretreated platelets, and monitored in a four-channel automated platelet analyzer. These results represent the means ± S.E. from four separate experiments. C, effect of C3 on integrin IIb3 activation in suspension-activated platelets. Washed platelets resuspended in Tyrode's buffer were incubated with buffer alone, TRAP (1 µM), or thrombin (1 unit/ml), in the presence of PAC-1 (1 µg/ml), in the absence of stirring. PAC-1 binding was quantified by FACS analysis, as described under "Experimental Procedures." These results represent the means ± S.E. of three separate experiments.

We next examined the consequences of inhibiting RhoA activity on platelet adhesion, shape change, and spreading on immobilized fibrinogen or HvWf. Previous studies have demonstrated a potentially important role for RhoA in regulating platelet shape change in response to soluble agonist stimulation (11, 32-34). As demonstrated in Fig. 4 (A and B), inhibiting RhoA or RhoA kinase had no effect on platelet adhesion, shape change, and spreading on immobilized vWf or on the ability of these cells to bind PAC-1 (Fig. 4C). Similar findings were apparent on a fibrinogen matrix (data not shown). Platelet adhesion and spreading on these matrices is associated with dramatic reorganization of the cytoskeleton, leading to the formation of two distinct actin-based structures, filopodial bundles and broad lamellipodial networks (35, 36). Although Cdc42 and Rac are well known to regulate filopodial and lamellipodial formation, respectively, some degree of RhoA activity is also required for these cytoskeletal changes in a number of cell types (37). To investigate the effect of inhibiting RhoA on actin cytoskeletal remodeling, C3 exoenzyme- or Y27632-treated platelets were fixed following adhesion to immobilized vWf and F-actin stained with tetramethyl rhodamine isothiocyanate-conjugated phalloidin. In contrast to the well-documented inhibitory effects of RhoA/Rho kinase inhibitors on cytoskeletal reorganization of cultured motile cells, both C3 exoenzyme- and Y27632-treated platelets exhibited a similar pattern of F-actin staining to control platelets (data not shown). Collectively, these studies do not support a major role for the RhoA signaling pathway in regulating integrin _IIb3 activation and cytoskeletal reorganization in spreading platelets.

View larger version (107K): [in this window] [in a new window]   Fig. 4.   Examination of a role for RhoA and Rho kinase in platelet adhesion, spreading, and integrin IIb3 activation under static conditions. Washed platelets pretreated with vehicle alone (Control) or C3 exoenzyme (100 µg/ml, C3), were applied to coverslips coated with vWf (10 µg/ml), as described under "Experimental Procedures." C, in some experiments, adhesion was carried out in the presence of PAC-1 antibody (1 µg/ml). Adhesion (A) and spreading (B) were examined using differential interference contrast microscopy (A, ×100 oil objective; B, ×100 oil objective with 4× zoom). C, PAC-1 binding was quantified using confocal microscopy (×100 oil objective, with 2× zoom). Images are taken from one experiment representative of three. Histograms represent the means ± S.E. from three separate experiments (n = 3).

Effect of RhoA Inhibitors on Shear-induced Platelet Aggregation-- To investigate the effects of RhoA signaling inhibitors on shear-induced platelet aggregation (SIPA), Y27632 or C3 exoenzyme-treated platelets were sheared for 5 min in a coneand-plate viscometer. Inhibiting RhoA or Rho kinase had a major effect on SIPA. As demonstrated in Fig. 5, both Y27632 and C3 exoenzyme prevented the formation of large platelet aggregates with the majority of cells remaining as single platelets or small aggregates (<10 cells). SIPA is a complex process initiated by shear-induced binding of soluble vWf to the GPIb/V/IX, leading to ADP release and the activation of integrin _IIb3 (38). In an attempt to gain further insight into the mechanism by which RhoA regulates SIPA, we investigated the effects of the RhoA inhibitors on platelet aggregation induced by asialo-vWf. This latter platelet agonist utilizes a similar multistep adhesion process as SIPA, except that it does not require high shear or artificial modulators to initiate platelet activation, because removal of the sialic acid residues from vWf results in spontaneous vWf binding to GP Ib. Similar to shear-induced platelet aggregation, As-vWf binding to GP Ib induces platelet aggregation in an ADP- and integrin _IIb3-dependent manner (39, 40). To investigate the role of RhoA in regulating As-vWf-induced platelet aggregation, C3 exoenzyme-treated platelets were resuspended in platelet-poor plasma, and aggregation induced by addition of As-vWf, as described under "Experimental Procedures." As demonstrated in Fig. 5B, C3-treated platelets were able to aggregate at a similar rate and extent as control platelets stimulated with As-vWf, with no disaggregation observed after 10 min of constant stirring. These findings suggest that the effects of the RhoA inhibitors are primarily manifested under high shear conditions.

View larger version (81K): [in this window] [in a new window]   Fig. 5.   Examination of a role for RhoA and Rho kinase in shear-induced platelet aggregation (SIPA). A, washed platelets were preincubated with C3 exoenzyme (100 µg/ml) or Y27632 (20 µM). Pretreated platelets were sheared at 5000 s1 for 5 min using a cone-and-plate viscometer. Single platelets and aggregates were imaged using phase-contrast microscopy (×5 objective). Images are taken from one experiment representative of three. SIPA was quantified by capturing five random fields from each experiment. The number of single platelets in each field was quantified offline using computer-assisted analysis (MCID software). Results represent the means ± S.E. of three independent experiments. Statistical analysis was performed using a t test comparing control versus C3 exoenzyme- or Y27632-treated platelets (*, p < 0.05; **, p < 0.01). B, effect of RhoA on asialo-vWf-mediated aggregation. Washed platelets preincubated with either vehicle alone or C3 exoenzyme (100 µg/ml) were stirred in the presence of asialo-vWf (20 µg/ml), and aggregation was monitored in a four-channel automated platelet analyzer. The aggregation tracing is taken from one experiment representative of five. Tabulated results represent the means ± S.E. of five separate experiments (n = 5).

Effects of RhoA Inhibition on Shear-dependent Platelet Adhesion on vWf-- To gain further insight into the mechanism by which RhoA regulates platelet adhesion under shear conditions, we examined the effect of C3 exoenzyme and Y27632 on platelet adhesion to immobilized vWf using an in vitro flow-based platelet adhesion assay (23). A major advantage of this assay system is that it allows detailed real-time analysis of each step of the platelet adhesion process, providing information on changes in dynamics of the vWf·GPIb and vWf·integrin _IIb3 adhesion contacts. In these studies, washed platelets were preincubated with either vehicle, C3 exoenzyme or Y27632, then reconstituted with RBCs prior to perfusion through vWf-coated microcapillary tubes at 600 or 1800 s¹, as described under "Experimental Procedures." Analysis of the dynamics of individual platelet·matrix contacts demonstrated that the majority of platelets were able to tether normally to HvWf, regardless of pretreatment by C3 exoenzyme (Fig. 6A, line graph) or Y27632 (Fig. 6B, line graph). In contrast, the number of platelets forming sustained stationary adhesion contacts with the vWf matrix was significantly reduced by C3 exoenzyme at 600 s¹ (Fig. 6A) and 1800 s¹ (data not shown). Similar findings were observed with Y27632 (Fig. 6B, and data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Examination of a role for RhoA and Rho kinase in regulating platelet tethering or stationary adhesion under flow conditions. Washed platelets were incubated with vehicle alone (Control), C3 exoenzyme (100 µg/ml) (A), or Y27632 (20 µM) (B). Pretreated platelets were reconstituted with RBCs and perfused over immobilized vWf (100 µg/ml) at 600 (A) or 1800 s1 (B). A and B, platelet tethering (line graphs) and stationary adhesion (histograms) was assessed as described under "Experimental Procedures." Results represent the means ± S.E. from three independent experiments. Statistical analysis was performed using a t test comparing control versus C3- or Y27632-treated platelets (*, p < 0.05; **, p < 0.01; ***, p < 0.001).

To investigate whether the decrease in the ability of C3 exoenzyme-treated platelets to form stable adhesion contacts was due to a decrease in integrin _IIb3 activation, we compared the binding of PAC-1 to control or C3 exoenzyme- or Y27632-treated platelets following adhesion to HvWf at 1800 s¹. These studies demonstrated no difference in PAC-1 binding to platelets treated with either inhibitor (data not shown). However, a potential limitation of these studies was the possibility that we were biasing our results by only selecting a subset of cells for analysis, i.e. those cells that had activated sufficient levels of integrin _IIb3 to form stable adhesion contacts. To overcome this technical limitation, flow-based adhesion studies were performed on a human vWf matrix in the presence of ristocetin, or alternatively, on a purified bovine vWf matrix. Ristocetin promotes stationary platelet adhesion in a shear field independent of integrin _IIb3, due to the ability of ristocetin to increase the affinity of the GPIb·vWf interaction. Similarly, bovine vWf has a high affinity for human GPIb, allowing sustained stationary platelet adhesion independent of integrin _IIb3 (data not shown). In contrast to human vWf alone, inhibiting RhoA or Rho kinase had no effect on the number of platelets forming stationary adhesion contacts on either bovine vWf or human vWf in the presence of ristocetin (data not shown), consistent with the lack of effect of RhoA signaling on the adhesive function of GPIb. Furthermore, neither C3 exoenzyme nor Y27632 had any inhibitory effect on the level of PAC-1 binding to these cells (Fig. 7). These studies do not support an important role for RhoA in regulating the affinity status of integrin _IIb3 under shear conditions.

View larger version (72K): [in this window] [in a new window]   Fig. 7.   Examination of a role for RhoA in regulating integrin IIb3 activation under flow conditions. Washed platelets were incubated with vehicle alone (Control) or C3 exoenzyme (100 µg/ml). Pretreated cells were reconstituted with RBCs prior to perfusion through vWf-coated microcapillary tubes (100 µg/ml) (1800 s1). Platelets were incubated with PAC-1 antibody prior to fixation and staining with a fluorescein isothiocyanate-conjugated secondary antibody. PAC-1 binding was imaged using a confocal microscope (×100 oil objective 2× zoom). Images were taken from one experiment representative of three. The histogram represents the means ± S.E. from three separate experiments.

To investigate the possibility that RhoA modulates firm platelet adhesion by influencing the stability of the integrin _IIb3·vWf interaction, we examined the duration of stationary platelet adhesion contacts with the human vWf matrix in control or C3 exoenzyme- or Y27632-treated platelets. In initial control studies, we demonstrated that blocking the ligand binding function of integrin _IIb3 by pretreating platelets with the integrin _IIb3 antagonist, c7E3 Fab, reduced stationary adhesion contact formation beyond 1 s down to 10-20% of cells. The ability of a small percentage of platelet to remain stationary longer than 1 s under these experimental conditions was due to the formation of thin membrane tethers (41), rather than incomplete blockade of integrin _IIb3. In contrast, ~60-65% of control, C3 exoenzyme-, and Y27632-treated platelets tethering to the vWf surface formed stationary adhesion contacts for at least 1 s. The lack of inhibitory effect of C3 exoenzyme or Y27632 on initial adhesion contact formation is consistent with the ability of integrin _IIb3 to become activated and engage vWf under these experimental conditions. Analysis of the proportion of platelets maintaining stationary adhesion contacts over time revealed a significant difference between control, C3 exoenzyme-, and Y27632-treated platelets. As demonstrated in Fig. 8, in control platelets there was a time-dependent reduction in the percentage of platelets remaining stationary from 1 to 10 s with ~25% sustaining firm adhesion contacts for at least 10 s. In contrast, with both C3 exoenzyme- and Y27632-treated platelets, <10% of cells sustained stable adhesion contacts with the vWf matrix for up to 10 s. These results demonstrate an important role for RhoA in maintaining the strength of integrin _IIb3 adhesion contacts with vWf under shear conditions.

View larger version (28K): [in this window] [in a new window]   Fig. 8.   A role for RhoA and Rho kinase in maintaining the strength of integrin IIb3 adhesion contacts with vWf under flow conditions. Washed platelets were incubated with vehicle alone (Control), C3 exoenzyme (100 µg/ml) (A) or Y27632 (20 µM) (B). Pretreated platelets were reconstituted with RBCs and perfused over immobilized vWf (100 µg/ml) at 600 (A) or 1800 s1 (B). Real-time tethering and stationary adhesion was captured by video microscopy (×63W objective), for offline analysis. Stationary adhesion of individual platelets at each time point was assessed as described under "Experimental Procedures." The results represent the means ± S.E. from three separate experiments. Statistical analysis was performed using a t test comparing control versus C3 exoenzyme- or Y27632-treated platelets at each time point (*, p < 0.05). Inset, results are expressed as percentage of control for each time point and highlight the decreased stability of C3 exoenzyme- and Y27632-treated platelets, when compared with controls.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Platelet thrombus formation is critically dependent upon the adhesive and signaling function of integrin _IIb3. Despite considerable progress in defining the mechanisms initiating integrin _IIb3 activation and adhesion (14, 23, 42-48), there remains limited information on the factors involved in sustaining integrin _IIb3 adhesion contacts, especially under pathophysiologically relevant shear conditions. The studies in this manuscript demonstrate an important role for the small GTPase RhoA in this process. Our studies demonstrate that RhoA activation occurs as a general feature of platelet activation, and, with the majority of platelet stimuli examined, integrin _IIb3 outside-in signaling appears to play a major role in regulating RhoA activity. Analysis of the effects of C3 exoenzyme or Y27632 on platelet function, over a broad range of experimental conditions, does not support an important role for RhoA or Rho kinase in regulating the affinity status of integrin _IIb3. Rather, our studies suggest a potentially important role for RhoA signaling in stabilizing integrin _IIb3 adhesion contacts, thereby promoting efficient platelet adhesion and aggregation in a shear field.

Our studies have demonstrated the existence of integrin _IIb3-dependent and -independent signaling pathways linked to RhoA activation. The relative importance of these two pathways for RhoA activation appears to be agonist-specific. For example, in thrombin-stimulated platelets, a substantial proportion of RhoA activation occurs independent of ligand binding to integrin _IIb3, whereas, with ADP, collagen, vWf, and U46619, integrin _IIb3 signaling appears to play the predominant role in promoting RhoA activation, at least under the experimental conditions employed in this study. Our findings with regards to U46619 appear to contradict a recent study by Gratacap et al. (12), who demonstrated that RhoA activation by U46619 occurred independent of ligand binding to integrin _IIb3. However, as demonstrated in the current study, and from other previous reports (31), the exclusion of integrin _IIb3 from platelet signaling events must be interpreted with caution when based on studies utilizing RGDS peptides in isolation. This technical issue aside, it is quite possible, and likely, that U46619 and other agonists utilize integrin _IIb3-independent mechanisms to promote RhoA activation. For example, several previous studies have demonstrated a potentially important role for RhoA in the regulation of integrin _IIb3-independent functional responses, such as platelet shape change (11, 32, 34, 49). Consistent with this, thrombin induces rapid RhoA/Rho kinase activation (11),² the timing of which mirrors MLC phosphorylation and platelet shape change (11). Furthermore, there is evidence supporting a direct role for G-protein-coupled receptors in the regulation of RhoA activity independent of integrins (50-53). Although the precise mechanisms regulating RhoA activity in platelets remain to be fully elucidated, the existence of integrin _IIb3-dependent and -independent pathways in this process provides for distinct spatial and temporal roles for RhoA in the modulation of platelet activation and adhesion responses.

Our findings that RhoA activity in platelets is regulated by both soluble agonists and integrin _IIb3 is consistent with a large volume of evidence in cultured cells that RhoA activation is modulated through a complex interplay between soluble stimuli and integrins (27, 54-58). The upstream signaling elements involved in RhoA activation remain a complex and relatively undefined area. RhoA, like all Rho family members, is activated by guanine-nucleotide exchange factors (GEFs) (59). Exchange factor activity can be regulated by a variety of signaling events, depending upon the nature of the stimulus. Many of these signaling events remain to be determined, however, some progress has been made into the mechanism of RhoA GEF activation mediated by G-protein-coupled receptors. Studies in mammalian cells have demonstrated that G_12/13 is capable of RhoA activation, via the direct binding of G_12/13 to p115 Rho GEF (50-52), leading to an increase in its exchange factor activity (51). Whether this same signaling pathway exists in platelets remains to be determined; however, studies by Klages et al. (32) have demonstrated that selective G_12/13 activation is sufficient to induce Rho/Rho kinase-mediated phosphorylation of MLC. Activation of RhoA by integrins remains poorly understood (60), although there is evidence for a potentially important role for tyrosine kinases such as Src and Syk in this process (61, 62).

Our findings do not suggest a major role for RhoA in regulating cytoskeletal changes necessary for platelet shape change and spreading on adhesive substrates, a finding consistent with previous studies by Leng et al. (13). This may not be surprising, given that the cytoskeletal changes associated with these events, such as formation of actin bundles in filopodia and actin networks in lamellipodia, have been attributed to the activation of other Rho family members, such as Cdc42 and Rac, respectively. However, in other cells types abolishing RhoA signaling has been demonstrated to inhibit cell spreading (37). For example, under experimental conditions leading to complete inhibition of RhoA, cell adhesion contact formation with the matrix is significantly weakened, thereby undermining the ability of cells to efficiently spread. In addition, changes in RhoA activity have been demonstrated to indirectly influence the activation status of other Rho family members (8). In platelets, the lack of effect of C3 exoenzyme on cytoskeletal reorganization and platelet spreading may be due to incomplete inhibition of RhoA, or alternatively, inhibiting RhoA in platelets may not significantly influence the activation state of other Rho family members. Future studies will be required to address this issue.

A major finding from the present study is the requirement for RhoA signaling for maintaining integrin _IIb3 adhesion contacts in a shear field. Our studies do not support an important role for RhoA in regulating integrin _IIb3 activation, a finding consistent with observations in many other cell types. The precise mechanism by which RhoA sustains integrin _IIb3 adhesion contacts remains to be established, however, based on findings in other cells, it is likely that RhoA plays an important role in regulating integrin _IIb3 avidity. In cultured cells, activation of RhoA results in the development of large integrin clusters that are anchored to actin stress fibers, forming focal adhesion contacts (FAs). The ability of RhoA to induce focal contact assembly has been extensively investigated and involves downstream targets, including Rho kinase. In its activated form, this serine/threonine kinase catalyzes the phosphorylation of myosin phosphatase, down-regulating its activity and elevating MLC phosphorylation, thereby promoting contractility, integrin clustering, and FA formation (7). Although platelets do not contain typical FAs such as those observed in fibroblasts, they do contain "focal adhesion-like" complexes, which share many of the components of true FAs. In support of a role for RhoA in regulating these structures, Leng and colleagues (13) have previously reported that C3 exoenzyme treatment of platelets reduces the number of vinculin-containing adhesion complexes in spread platelets.

Finally, our studies provide new insight into the importance of integrin _IIb3 outside-in signaling for the process of shear-induced platelet aggregation. More specifically, we have demonstrated that integrin _IIb3-dependent RhoA activation following platelet adhesion to vWf is essential for stabilizing integrin _IIb3 adhesion contacts. These findings have potentially important implications for the regulation of platelet adhesive behavior in vivo. For example, pathological thrombi typically form at sites of arterial narrowing where shear forces may be as high as 380 dyne/cm² (10,000 s¹). The demonstration that Rho signaling has a relatively selective role in regulating integrin _IIb3 adhesiveness under shear conditions raises the interesting possibility that Rho inhibitors may reduce platelet thrombus formation in vivo. The ability to inhibit integrin _IIb3 adhesiveness, without potentially undermining the normal signaling mechanisms regulating integrin _IIb3 activation, would represent a novel approach for regulating platelet adhesiveness in vivo.

	ACKNOWLEDGEMENT

We acknowledge Dr. Robert Andrews (Baker Institute for Cardiovascular Research, Melbourne, Australia), for assistance with the preparation of As-vWf.

	FOOTNOTES

^* This work was supported in part by grants from the National Health and Medical Research Council (NHMRC) of Australia and the National Heart Foundation of Australia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Recipient of an NHMRC R. D. Wright Fellowship and a Monash University (Australia) Logan Fellowship. To whom correspondence should be addressed: Dept. of Medicine, Monash University, 5th Level, Clive Ward Bldg., Box Hill Hospital, Arnold Street, Box Hill, Victoria 3128, Australia. Tel.: 61-3-9895-0350; Fax: 61-3-9895-0332; E-mail: Simone.Schoenwaelder@med.monash.edu.au.

^§ Recipient of the Australian Post-Graduate Research Award.

Published, JBC Papers in Press, February 5, 2002, DOI 10.1074/jbc.M200661200

² S. M. Schoenwaelder and K. Boniface, unpublished observations.

	ABBREVIATIONS

The abbreviations used are: vWf, von Willebrand factor; GP, glycoprotein; MLC, myosin light chain; HvWf, human vWf; RBC, red blood cells; GST, glutathione S-transferase; GEF, guanine-nucleotide exchange factor; SIPA, shear-induced platelet activation; As-vWf, asialo-vWf; FA, focal adhesion; RGDS, arginine-glycine-aspartic acid-serine; TRAP, thrombin receptor agonist peptide.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES