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Originally published In Press as doi:10.1074/jbc.M402032200 on April 15, 2004

J. Biol. Chem., Vol. 279, Issue 25, 26266-26273, June 18, 2004
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ATP Augments von Willebrand Factor-dependent Shear-induced Platelet Aggregation through Ca2+-Calmodulin and Myosin Light Chain Kinase Activation*

Cécile Oury{ddagger}§, Elsie Sticker{ddagger}, Heidi Cornelissen{ddagger}, Rita De Vos¶, Jos Vermylen{ddagger}, and Marc F. Hoylaerts{ddagger}||

From the {ddagger}Center for Molecular and Vascular Biology and the Laboratory of Morphology and Molecular Pathology, University of Leuven, 3000 Leuven, Belgium

Received for publication, February 24, 2004 , and in revised form, April 14, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ib{alpha} and subsequent integrin {alpha}IIb{beta}3-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous L-{beta},{gamma}-methylene ATP, a stable P2X1 agonist, as well as by ADP, confirming that in addition to ADP (via P2Y1 and P2Y12), ATP (via P2X1) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X1 activation with L-{beta},{gamma}-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of {alpha}-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca2+ and VWF-glycoprotein Ib{alpha} interactions without involving {alpha}IIb{beta}3. Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X1 stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X1 with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X1 ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Blood platelets are constantly subjected to hemodynamic forces imposed by the blood flow, including fluid shear stress. High shear stress is generated at sites of arterial injury where laminar blood flow is forced through a stenosis (1, 2). Shear stress-triggered platelet activation and subsequent aggregation, termed SIPA1 (3), may thus contribute to the pathogenesis of vascular diseases. In addition, platelets from patients with acute myocardial infarction (4) or cerebral ischemia (5) display enhanced SIPA in vitro.

High shear stress is required for the interaction between von Willebrand factor (VWF) and platelet glycoprotein Ib{alpha} (GPIb{alpha}) (for review, see Ref. 6), but the effects of shear forces on GPIb{alpha} signaling are only just beginning to be defined. Downstream effectors that have been implicated in the VWF-dependent activation of {alpha}IIb{beta}3 include phosphatidylinositol 3-kinase (PI3K) (7), protein kinase C (PKC) (8), Syk, and Src, as well as co-associated immunoreceptor tyrosine-activated motif-containing transmembrane proteins and adaptor proteins (2). A recent study of platelet adhesion to dimeric VWF A1 domain, which recognizes only GPIb{alpha}, showed that GPIb{alpha} itself can signal to activate {alpha}IIb{beta}3 through sequential actions of Src kinases, Ca2+ oscillations, and PI3K/PKC (9). It has also been proposed that GPIb signals directly as a consequence of its association with structural cytoskeletal proteins (10), among which GPIb{alpha}-bound filamin A, filamentous actin cross-linked by {alpha}-actinin, vinculin, and talin would directly link the cytoplasmic domain of GPIb{alpha} with the {beta}3 tail of {alpha}IIb{beta}3 (11).

Studies examining SIPA have suggested that VWF primarily stimulates platelet activation through an indirect pathway linked to ADP release (12, 13). These studies have also shown that platelet activation initiated by VWF-GPIb{alpha} interaction requires a transmembrane Ca2+ influx independent of released ADP and VWF binding to {alpha}IIb{beta}3 (14, 15), which promotes dense granule secretion of ADP and activates integrin {alpha}IIb{beta}3 through engagement of the P2 receptors for ADP. A role for the platelet ADP receptors P2Y1 and P2Y12 (reviewed in Ref. 16) in SIPA has subsequently been confirmed (17, 18). Reséndiz et al. (19) recently reported that the selective P2Y12 antagonist AR-C69931MX inhibits the shear-induced aggregation of washed platelets and showed that P2Y12 mediates shear-induced PI3K activation, a process coupled to phosphorylation of PI3K-associated Syk tyrosine kinase.

In blood vessels, shear stress causes the release of high levels of nucleotides, including ATP and ADP, into the extracellular environment; these nucleotides mainly originate from endothelial cells (20) and platelet-dense granules (21). Although the shear-induced ATP release from endothelial cells probably involves vesicular exocytosis (22), the molecular mechanisms of such a release are largely unknown. In platelets, the time course of this ATP release appeared to parallel the association of myosin with the actin cytoskeleton, suggesting that these two processes are related (21). In addition, Ca2+ influx-dependent phosphorylation of myosin light chain, preceding the myosin-actin interactions, has been proposed to be an initial step during SIPA (21).

ATP is the physiological agonist at P2X1, but its role in platelet activation is only starting to be unraveled. In the aggregometer, the selective P2X1 agonists {alpha},{beta}-meATP and L-{beta},{gamma}-meATP evoke a transient Ca2+ influx accompanied by rapid and reversible platelet shape change and myosin light chain (MLC) phosphorylation, provided that measures are taken to avoid desensitization of P2X1 by ATP spontaneously released during platelet preparation (23–25). Recent studies, using P2X1 knock-out mice (26) or transgenic mice overexpressing this ion channel selectively in the megakaryocytic cell lineage (27), have reported a prominent role for P2X1 in platelet aggregation and thrombus formation in shear stress conditions. Notably, P2X1 overexpressing platelets displayed potent SIPA in conditions where wild-type platelets hardly aggregated (27). In the present study, we have investigated the role of the P2X1-mediated Ca2+ influx and the associated downstream signaling pathways in VWF-dependent shear-induced granule release and aggregation of washed human platelets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—ADP, L-{beta},{gamma}-meATP, the P2X1 antagonist MRS2159, the P2Y1 antagonist MRS2179, and the ectonucleotidase apyrase (grade I) were purchased from Sigma. AR-C69931MX was a gift from AstraZeneca R&D, Charnwood, UK. The P2X1 antagonist MRS2159 at the concentration used in this study was verified to selectively inhibit a P2X1-mediated platelet shape change without affecting platelet shape change and aggregation induced by ADP. The calmodulin inhibitor W-7, the MLC kinase inhibitor ML-7, the Rho kinase inhibitor Y-27632, and the PKC inhibitors GF109203X, Go6976, and Go6983 were purchased from Calbiochem. Human VWF was purified from plasma cryoprecipitate by gel filtration on a Sepharose 4B-CL column. Tirofiban (Aggrastat) was obtained from Merck Sharp and Dohme. The murine-neutralizing anti-VWF monoclonal antibody AJvW-2 was from Ajinomoto Co., Inc. (Kawasaki, Japan) (28), and the neutralizing monoclonal anti-GPIb{alpha} antibody G19H10 was raised in our laboratory. ADP and L-{beta},{gamma}-meATP were purified by high pressure liquid chromatography on an Adsorbosphere HS C18, 7 µm, 250 x 4.6-mm column (Alltech) as described by Oury et al. (24).

Preparation of Washed Human Platelets—Washed human platelets (2.5–3.5 x 105 platelets/µl) were prepared as described previously (29). Apyrase (0.5 units/ml) was added to the blood sample and platelet resuspension buffer when indicated. Platelet preparations were free of red blood cells, as validated via microscopy and blood cell counting (CellDyn 1300, Abbott, Abbott Park, IL).

Shear-induced Platelet Aggregation, ATP Secretion, and P-selectin Expression—Shear-induced aggregations of washed human platelets were performed in an annular ring-shaped viscometer generating laminar flow (Ravenfield viscometer; Heywood, Lancashire, UK) at 37 °Cin the presence of 2 mM CaCl2 and 10 µg/ml human soluble VWF unless otherwise indicated. A shear rate of 9000 s-1 was used, which corresponds to a shear stress of 80 dynes/cm2. The threshold shear rate causing platelet aggregation ranged between 3000 s-1 and 5000 s-1. At defined time points, platelet samples were collected and fixed in 1% paraformaldehyde; the percentage of platelet aggregation was calculated by comparing single platelet counts before and after shearing. Shear-induced P-selectin surface translocation on tirofiban-treated washed platelets was detected by flow cytometry with the phycoerythrin-conjugated anti-P-selectin CD62P antibody (BD Biosciences). For ATP secretion assays, the reactions were stopped with an ice-cold Hepes buffer (20 mM Hepes, pH 7.4, 150 mM NaCl, 5 mM EDTA). After immediate centrifugation at 4 °C, ATP was measured in the supernatants by the addition of a luciferase/luciferin reagent (Chrono-Lume, Kordia) in a microplate luminometer LB96V (Berthold Technologies, Vilvoorde, Belgium). At least five independent experiments were performed on platelets from different individuals. The data are represented as the mean ± S.D. Statistical analysis of the data was done using nonpaired Student's t tests.

Immunoblotting Analyses—Platelets (0.3-ml platelet suspensions) were lysed in SDS sample buffer (62.5 mM Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 50 mM dithiothreitol, 0.1% bromphenol blue). Sample aliquots were loaded on SDS-PAGE (12.5%) and subjected to Western blotting. Thr-18/Ser-19 MLC phosphorylation was detected with the anti-phospho-MLC polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) according to the instructions of the manufacturers.

Electron Microscopy—Washed platelets were immediately fixed overnight at 4 °C in 2.5% (w/v) glutaraldehyde, 0.1 M phosphate buffer, pH 7.2. After centrifugation at 800 x g for 10 min, a condensed pellet of platelets was formed. After fixation in 1% OsO4 (w/v), 0.1 M phosphate buffer, pH 7.2, and dehydration in a graded series of ethanol, the pellets were embedded in epoxy resin. Ultrathin sections were cut and stained with uranyl acetate and lead citrate before examination with a Zeiss EM 10 electron microscope (Oberkochen, Germany).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
VWF-GPIb{alpha} Interactions and Extracellular Ca2+ in SIPA—When suspensions of washed human platelets were subjected to uniform high shear stress (80 dynes/cm2 = 9000 s-1 shear rate) in the presence of soluble human VWF in an annular ring-shaped viscometer, aggregation rapidly occurred, reaching a plateau after 3 min (Fig. 1A). In contrast, a shear rate of 1000 s-1 did not cause platelet aggregation (Fig. 1A). The inclusion of the VWF A1 domain-blocking antibody AJvW-2, neutralizing anti-GPIb{alpha} antibody G19H10, or the {alpha}IIb{beta}3 antagonist tirofiban (Fig. 1B) confirmed the findings by others (30) that SIPA requires VWF interactions with GPIb{alpha} and the activation of {alpha}IIb{beta}3. We also confirmed that SIPA does not occur in the absence of added extracellular Ca2+ (14, 15) (Fig. 1B).



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  		FIG. 1.
Role of VWF and Ca2+ in shear-induced platelet aggregation and ATP release. A, time course of washed human platelet aggregation and ATP release induced at a shear rate of 9000 s-1 (black squares). The absence of platelet responses at a shear rate of 1000 s-1 is also shown (lower lines with open squares). B, effects of preincubation with the neutralizing anti-VWF monoclonal antibody AJvW-2 (20 µg/ml, 1 min), the neutralizing anti-GPIb{alpha} antibody G19H10 (20 µg/ml, 1 min), or the {alpha}IIb{beta}3 antagonist tirofiban (1 µg/ml, 1 min), as well as the absence of added extracellular Ca2+ on aggregation induced for 3 min at 9000 s-1. *, p < 0.0001; #, p < 0.0001 versus control; §, p < 0.0002.

 
Platelet aggregation was accompanied by gradually increasing shear stress-dependent ATP release (Fig. 1A). ATP release was initiated as early as 10 s after exposure to shear stress. When measured at the maximal aggregation time point, this release clearly relied on VWF-GPIb{alpha} signaling, as it was at least partly inhibited by AJvW-2 and G19H10 (Fig. 1B). Omitting the addition of extracellular Ca2+ also reduced the amount of released ATP, although only partly (Fig. 1B). The combined absence of VWF and extracellular Ca2+ almost abolished platelet degranulation (Fig. 1B). In contrast, {alpha}IIb{beta}3 antagonism had no effect on the degree of platelet degranulation (Fig. 1B). Therefore, both VWF-GPIb{alpha} signaling and Ca2+ contribute to platelet release under shear stress, a process that is independent of {alpha}IIb{beta}3 outside-in signals.

MLC Kinase (but Not PKCs) Controls the SIPA-induced Platelet Degranulation—Platelet granule release that is induced by most agonists requires an increase of intracellular Ca2+ and PKC activation (reviewed in Ref. 31). Whether or not PKC plays a role in SIPA is controversial (8, 32). In our experimental conditions, the nonselective PKC inhibitor GF109203X, which blocks both conventional ({alpha}, {beta}I, {beta}II, {gamma}) and novel ({delta}, {theta}, {eta}, {epsilon}) PKC isoforms, failed to inhibit either platelet aggregation or release produced by shear stress (Fig. 2A), although it prevented such responses to collagen (2 µg/ml) or to the PAR-1-activating peptide TRAP1–6 (1 µM) under stirring conditions (Fig. 2B). Similarly, the PKC {alpha}/{beta}-specific inhibitor Go6976 did not affect SIPA and associated platelet release (Fig. 2A), although shear stress-independent collagen-induced release and aggregation were fully blocked (Fig. 2B). In agreement with previous reports that PKC {alpha}/{beta} isoforms do not play a major role downstream of PAR-1 (33), Go6976 had a minimal inhibitory effect on TRAP1–6-induced granule release (Fig. 2B). Finally, to determine whether other PKC isoforms could be involved in platelet release accompanying SIPA, we used Go6983, which blocks the platelet-expressed atypical PKC {zeta} isoform in addition to PKC {alpha}, {beta}I, {beta}II, {gamma}, and {delta}. This inhibitor had no effect on shear-induced platelet release or aggregation, although it diminished TRAP1–6-induced ATP release by about 50% without affecting platelet aggregation (Fig. 2B). These results indicate that shear-induced platelet dense granule release and aggregation are dependent on signaling pathways distinct from those activated downstream of the collagen receptor GPVI or PAR-1. They also suggest that the known conventional, novel, and atypical PKC isoforms do not play a major role in platelet degranulation during SIPA.



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  		FIG. 2.
MLC kinase versus PKC in SIPA and platelet ATP release. A, aggregation of washed human platelets was induced for 3 min at a shear rate of 9000 s-1 following preincubation with the protein kinase C inhibitors GF109203X (+GF, 10 µM, 5 min), Go6976 (1 µM, 5 min), or Go6983 (1 µM, 5 min) or the MLC kinase antagonist ML-7 (10 µM, 10 min). The effect of these inhibitors on ATP secretion measured after 3 min of shearing is also shown. *, p < 0.05. B, aggregation of washed human platelets produced in an aggregometer by collagen (2 µg/ml) or TRAP1–6 (1 µM SFFLRN) in the absence (control) or presence (+) of the same inhibitors, as indicated. The corresponding concentrations (µM) of released ATP are shown in brackets.

 
In addition to PKC, GPVI-mediated platelet dense granule release also depends on Ca2+-sensitive MLC kinase activation (25). Inhibiting this kinase with ML-7 abolished both the ATP release and platelet aggregation induced by collagen (2 µg/ml) (Fig. 2B). As shown in Fig. 2A, ML-7 reduced the shear-induced ATP release and platelet aggregation by about 30%, indicating a role for MLC kinase in SIPA and associated platelet degranulation.

Secreted ADP and ATP in VWF-dependent Shear-induced Platelet Release and Aggregation—Under stirring conditions in an aggregometer, MLC kinase is activated downstream of the P2X1-mediated Ca2+ influx, playing an essential role during cytoskeletal rearrangements evoked by selective agonists ({alpha},{beta}-meATP and L-{beta},{gamma}-meATP) of this ion channel (25). The activation of MLC kinase can also result from phospholipase C-dependent Ca2+ mobilization from internal stores downstream of the Gq protein-coupled P2Y1 receptor or of GPVI (34, 35). In agreement with earlier reports (17–19), the selective antagonists of P2Y1 (MRS2179, 10 µM) and P2Y12 (AR-C69931MX, 1 µM) inhibited SIPA by more than 50% (Fig. 3). In previous studies of platelet aggregation performed under stirring conditions in an aggregometer, a high concentration of the ATP/ADP scavenger apyrase always was required to demonstrate P2X1 function, as this ion channel is rapidly desensitized by ATP released during platelet preparation (23–25). In sharp contrast, the P2X1 antagonist MRS2159 (10 µM) reduced SIPA by about 40% even when apyrase was omitted (Fig. 3). Thus, the three platelet P2 receptors contribute to SIPA through secreted ADP and ATP. The effect of combined P2X1 and P2Y1 antagonism on SIPA was not significantly different from that of P2Y1 antagonism only (Fig. 3), suggesting that these receptors share common signaling pathways. Neither the antagonism of P2X1, P2Y1, nor their combination could inhibit the remaining SIPA observed in the presence of the P2Y12 antagonist (data not shown).



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  		FIG. 3.
P2X1, P2Y1, and P2Y12 in SIPA and ATP release. A, aggregation of washed platelets was induced for 3 min at a shear rate of 9000 s-1 following a 1-min preincubation with the selective P2 receptor antagonists for P2Y12 (AR-C69931MX,1 µM), P2Y1 (MRS2179, 10 µM), and P2X1 (MRS2159, 10 µM) or with combined MRS2179 + MRS2159. *, p < 0.001; #, p = 0.04 versus control. B, ATP secretion measured after 3 min of shearing in the presence or absence of the P2 receptor antagonists. *, p < 0.05; NS, not significant.

 
ATP secretion assays performed in the presence of the selective P2 receptor antagonists revealed a contribution of P2Y1, P2Y12, and P2X1 to VWF-dependent platelet degranulation (measured after 3 min) (Fig. 3). With the exception of P2X1 antagonism, the effects of P2Y1 and P2Y12 antagonism on secretion did not fully correlate with their ability to inhibit platelet aggregation (Fig. 3), which is in agreement with the interpretation that these receptors participate in other processes leading to SIPA besides secretion regulation.

P2X1 Enhances Degranulation and SIPA via Calmodulin-dependent MLC Kinase Activation—To further examine the contribution of individual nucleotides to SIPA, we have used platelets of a Hermansky-Pudlak patient lacking dense granules and secreting almost no ATP in response to supraoptimal doses of collagen (36). These platelets displayed severely impaired SIPA comparable with SIPA observed in the presence of P2Y1 or P2Y12 receptor antagonists (Fig. 4A). Interestingly, platelet aggregation could be largely restored by the addition of not only ADP but also of the selective P2X1 agonist L-{beta},{gamma}-meATP immediately prior to shearing, which supports the central roles for both ATP and ADP secreted during SIPA.



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  		FIG. 4.
P2X1 activation potentiates SIPA through CaM and MLC kinase. A, SIPA with platelets from a Hermansky-Pudlak patient (HPS) compared with platelets from healthy individuals (control). L-{beta},{gamma}-meATP (10 µM) or ADP (1 µM) was added to the Hermansky-Pudlak patient platelets prior to shearing, as indicated. B, aggregation of washed platelets and of apyrase (0.5 units/ml)-treated platelets was induced for 3 min at a shear rate of 9000 s-1 (control) in the presence or in the absence of the selective P2X1 agonist L-{beta},{gamma}-meATP (10 µM) following preincubation with the neutralizing anti-VWF monoclonal antibody AJvW-2 (20 µg/ml), the {alpha}IIb{beta}3 antagonist tirofiban (+tir, 1 µg/ml), the CaM antagonist W-7 (100 µM), the MLC kinase antagonist ML-7 (10 µM), or the P2Y antagonist AR-C69931MX, as indicated. 12$, p = 0.004 versus control; *, p = 0.001 versus apyrase - L-{beta},{gamma}-meATP; **, p < 0.005; #, p < 0.01 versus apyrase + L-{beta},{gamma}-meATP; ¶, p = 0.02 versus presence of W-7). The presence of apyrase and L-{beta},{gamma}-meATP are represented by solid black lines underscoring the graph.

 
We pursued this aspect of SIPA using normal platelets but in the presence of the ectonucleotidase apyrase, which degrades secreted ATP and ADP. The addition of apyrase (0.5 units/ml) to the platelet resuspension buffer reduced the level of SIPA to 70% of the control (Fig. 4B). In this condition, the selective P2Y1 and P2X1 receptor antagonists MRS2179 and MRS2159 no longer inhibited the remaining SIPA (not shown), indicating that the reduction of aggregation by apyrase is due to the absent activation of P2Y1 and P2X1 signaling (Fig. 4A). In contrast, the P2Y12 antagonist still reduced the aggregation of apyrase-treated platelets from 26.3 ± 9.5% to 17.1 ± 3.2%, indicating the presence of sufficient remaining secreted ADP to activate this receptor.

The addition of the selective apyrase-resistant P2X1 agonist L-{beta},{gamma}-meATP to this set-up just prior to shearing fully restored the aggregation of apyrase-treated platelets (Fig. 4B). The resulting aggregation still depended on VWF and {alpha}IIb{beta}3 activation, as shown by its full inhibition by AJvW-2 and tirofiban (Fig. 4B). Therefore, this experimental approach enabled us to study the P2X1-driven signaling pathways in more detail during SIPA.

We found that the enhanced SIPA was the result of CaM and MLC kinase activation, as W-7 and ML-7 inhibited the L-{beta},{gamma}-meATP amplification (Fig. 4B). Notably, these inhibitors had no effect on the SIPA of apyrase-treated platelets without additional stimulation by L-{beta},{gamma}-meATP (data not shown), indicating that the activation of these pathways exclusively depends on P2X1 potentiation. The fact that W-7 inhibited the L-{beta},{gamma}-meATP-driven SIPA to a larger extent than ML-7 may indicate the existence of an additional CaM-dependent pathway activated downstream of P2X1. Thus, exogenous P2X1 activation selectively triggers CaM-dependent pathways contributing to SIPA. Moreover, in the presence of the P2Y12 receptor antagonist AR-C69931MX, the L-{beta},{gamma}-meATP-driven SIPA was reduced to the same level (16.2 ± 3.4%) as that achieved during inhibition of apyrase-treated platelets themselves, confirming a central role for ADP also in the L-{beta},{gamma}-meATP-driven SIPA.

P2X1 Activation Promotes Shear-induced Platelet Granule Centralization and Release—We investigated whether P2X1-mediated MLC kinase activation would be instrumental in platelet degranulation to explain the enhanced SIPA of Hermansky-Pudlak patient platelets or of apyrase-treated platelets in the presence of L-{beta},{gamma}-meATP. Flow cytometry analysis of P-selectin expression (as a marker of {alpha}-granule release) was done on the surface of apyrase-treated (0.5 units/ml) and tirofiban-treated platelets exposed to VWF and shear for 5 min in the presence or absence of L-{beta},{gamma}-meATP. Fig. 5 shows a significant increase of the percentage of platelets expressing P-selectin following their pretreatment with L-{beta},{gamma}-meATP. P-selectin expression was, in turn, reduced to the initial level in the presence of the MLC kinase inhibitor ML-7 (Fig. 5). Therefore, during SIPA, the P2X1-mediated MLC kinase activation can reinforce the shear-induced platelet degranulation.



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  		FIG. 5.
P2X1 activation enhances platelet degranulation. Upper panel, flow cytometry analyses of P-selectin surface expression on tirofiban- and apyrase (0.5 units/ml)-treated washed platelets exposed to VWF at a shear rate of 9000 s-1 for 5 min or not (control) in the presence or absence of L-{beta},{gamma}-meATP (10 µM) and the MLC kinase inhibitor ML-7 (10 µM) as indicated. #, p = 0.01 versus control; *, p = 0.02 versus absence of L-{beta},{gamma}-meATP; **, p = 0.04 versus presence of L-{beta},{gamma}-meATP. Lower panels (a–d), transmission electron microscopy of SIPA. Apyrase (0.5 unit/ml)-treated washed human platelets were subjected or not (a) to a shear rate of 1000 s-1 (b) or 9000 s-1 (c) in the absence or presence of L-{beta},{gamma}-meATP (10 µM) (d) for 3 min before being analyzed by electron microscopy. Scale bars = 1 µm.

 
The ability of P2X1 to potentiate platelet aggregation during SIPA was further investigated by electron microscopy of platelet aggregates (Fig. 5). Following 3 min of shearing, the apyrase (0.5 units/ml)-treated platelets formed small aggregates and displayed granule centralization and pseudopodia, but most of the granules were intact (Fig. 5c). On the contrary, in the presence of L-{beta},{gamma}-meATP, larger platelet aggregates were observed in which more platelets were degranulated; in the platelets still showing intact granules, their centralization appeared more advanced than in the absence of L-{beta},{gamma}-meATP, indicative of more pronounced cytoskeletal rearrangements (Fig. 5d). Platelets subject to low shear stress (1000 s-1 shear rate) did not show any morphological changes (Fig. 5b) as compared with resting platelets (Fig. 5a). Thus, P2X1-driven MLC kinase activation in SIPA contributes both to cytoskeletal rearrangements in platelets and to degranulation.

Shear-induced MLC Phosphorylation Depends on P2X1, P2Y1, and P2Y12—Activation of CaM-regulated MLC kinase and Rho kinase results in Ca2+-dependent and -independent phosphorylation of MLC (34), which is a key step in platelet activation. Shear stress caused phosphorylation of MLC (detected as early as 20 s and reaching its maximum after 3 min) (Fig. 6A), which was inhibited both by ML-7 and the Rho kinase inhibitor Y-27632 (Fig. 6B). Such phosphorylation was not affected by the {alpha}IIb{beta}3 antagonist tirofiban (Fig. 6B), indicating that it occurred independently of {alpha}IIb{beta}3 outside-in signals. In agreement with a role for Rho kinase in SIPA, platelet aggregation was reduced from 43.9 ± 9.5% to 23.6 ± 8.7% by adding Y-27632 (5 µM) during SIPA (37).



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  		FIG. 6.
Shear-induced MLC phosphorylation depends exclusively on secreted ATP and ADP. A, Western blotting of MLC phosphorylation (P-MLC) at a shear rate of 9000 s-1. The time course of MLC phosphorylation in the absence of apyrase is shown. B, MLC-phosphorylation produced after 3 min of shear following preincubation with the selective P2 receptor antagonists (as described in the legend for Fig. 3), ML-7 (in Fig. 2), Y-27632 (5 µM), or tirofiban (in Fig. 1B), as indicated. C, same experiment as in A performed on platelets treated with 0.5 units/ml apyrase in the absence or presence of L-{beta},{gamma}-meATP (10 µM). D, platelets were treated with 5 units/ml apyrase, and shear was applied for 3 min in the presence of 10 or 20 µg/ml VWF, as indicated.

 
We found that antagonizing P2X1 (MRS2159), P2Y1 (MRS2179), or P2Y12 (AR-C69931MX) inhibited phosphorylation of MLC to a variable degree (Fig. 6B). Phosphorylation was inhibited potently by P2X1 neutralization. Correspondingly, the addition of 0.5 units/ml apyrase (i.e. abolishing platelet activation via P2Y1 and P2X1) almost abolished MLC phosphorylation (Fig. 6C); moreover, shearing for 20 s resulted in a phosphorylation state below that of resting platelets (Fig. 6C). The addition of 5 units/ml apyrase abrogated the overall response (Fig. 6D), which could not be restored by doubling the concentration of exogenous VWF (20 µg/ml) (Fig. 6D), even though this led to an ~20% increase of aggregation (not shown). Thus, VWF does not trigger MLC phosphorylation without involving secreted nucleotides.

Fig. 6C also shows that, in the presence of 0.5 units/ml apyrase, the addition of L-{beta},{gamma}-meATP prior to shear potently enhanced MLC phosphorylation induced after 20 and 60 s; such L-{beta},{gamma}-meATP-elicited phosphorylation was quickly reversible as it was no longer detected after 3 min. Therefore, the ability of L-{beta},{gamma}-meATP to cause MLC phosphorylation coincides with its ability to potentiate the shear-induced aggregation of apyrase-treated platelets.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we describe a role for the P2X1-mediated CaM and MLC kinase activation in VWF-dependent SIPA through secreted ATP. This pathway contributes to the extracellular Ca2+-dependent platelet degranulation induced by shear stress and acts in conjunction with VWF-GPIb{alpha} signaling. As schematically represented in Fig. 7, platelet activation initiated by the VWF-GPIb{alpha} interaction required a transmembrane Ca2+ influx independent of both ADP and VWF binding to {alpha}IIb{beta}3 (14, 15). Our observation that the P2X1 receptor antagonist MRS2159 only partly inhibits platelet degranulation and aggregation (to a maximum of 60%), whereas omitting extracellular Ca2+ or neutralizing VWF abolishes it, suggests that the VWF/GPIb-associated transmembrane Ca2+ influx only partly relies on P2X1-mediated Ca2+ influx.



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  		FIG. 7.
Schematic representation of the central role of P2 receptor signaling in SIPA. In addition to the shear-induced interaction of VWF with GPIb{alpha}, a role for all three P2 receptors is depicted in SIPA. Extracellular Ca2+-dependent GPIb{alpha} signaling triggers cytoskeletal rearrangements and {alpha}IIb{beta}3 activation but also early degranulation leading to subsequent activation of P2 receptors via released ADP and ATP. Downstream P2 receptor pathways include P2Y-mediated Ca2+ mobilization and P2X1-mediated Ca2+ influx, as well as P2Y12-dependent PI3K and Akt activation and RhoA/Rho kinase activation. The three P2 receptors are needed to cause MLC phosphorylation through CaM/MLC kinase and/or RhoA/Rho kinase pathways, leading to shear-associated cytoskeletal reorganization, facilitating platelet aggregation and enhancing platelet secretion. The fast ATP-activated Ca2+ influx through P2X1 triggers rapid CaM activation, which is critical for a process requiring a prompt platelet-activating signal. eCa2+ and iCa2+, extracellular and intracellular Ca2+, respectively.

 
In contrast to the effects on aggregation, the removal of VWF or antagonizing its binding to GPIb{alpha} only partially reduced the shear-induced platelet ATP release unless extracellular Ca2+ was omitted at the same time. The antagonist of {alpha}IIb{beta}3, tirofiban, had no effect on shear-induced platelet release, excluding contributions by VWF-{alpha}IIb{beta}3 outside-in signaling to secretion regulation. Antagonizing P2X1 with MRS2159 diminished the ATP release, which was also seen with the antagonism of P2Y1 and P2Y12, indicating a role for all of these receptors in the amplification of the VWF/GPIb{alpha}-triggered platelet degranulation (Fig. 7).

The ATP-gated P2X1 ion channel only operates in the presence of physiological extracellular Ca2+ concentrations (23, 24). Ca2+ is a key element in granule secretion (reviewed in Ref. 31). The proteins that mediate the effects of Ca2+ in secretion fall into two categories: the EF hand proteins and the Ca2+/phospholipid-binding proteins. EF hand proteins found in platelets include calmodulin and calcyclin. Calmodulin binds to platelet {alpha}-granules. Ca2+/calmodulin-dependent phosphorylation of MLC kinase mediates secretion via activation of MLC with subsequent contraction of the actomyosin; therefore, the concurrent centralization of granules is thought to facilitate granule secretion (38). Calmodulin may also bind specifically to the vesicle-associated membrane protein (39), contributing to granule release by directly affecting the exocytotic core complex.

Here, we found that shear-induced MLC phosphorylation involves both Ca2+-dependent MLC kinase and Ca2+-independent Rho kinase signaling, as it was inhibited by the MLC kinase inhibitor ML-7 as well as by the Rho kinase inhibitor Y-27632. The phosphorylation of MLC was abolished by the ATP/ADP scavenger apyrase and was potently reduced by the selective antagonism of P2X1, P2Y1, or P2Y12, indicating a central role for secreted ATP and ADP in this event.

Compatible with a prominent role for the P2X1 ion channel under high shear stress (26, 27), apyrase was not needed to demonstrate P2X1 function during SIPA. On the other hand, apyrase could be used under these conditions to control P2X1 function via the addition of stable selective P2X1 agonists and to drive platelet activation through P2X1-dependent pathways of signaling. The ability of the selective P2X1 agonist L-{beta},{gamma}-meATP to restore the aggregation of apyrase-treated platelets coincided with its potency in causing early phosphorylation of MLC and increased platelet P-selectin expression, a marker of {alpha}-granule release. The potentiating effect of L-{beta},{gamma}-meATP on SIPA was prevented by inhibitors of CaM (W-7) and of MLC kinase (ML-7). These findings were substantiated by electron microscopy analyses of the platelet aggregates, depicting more pronounced granule centralization as well as degranulation in the presence of L-{beta},{gamma}-meATP. Our observation that exogenous P2X1 activation can restore the severely defective SIPA of platelets lacking dense granules (Hermansky-Pudlak patient) corroborates a role for P2X1-mediated cytoskeletal rearrangements in facilitating {alpha}-granule release.

Thus, our findings suggest that P2X1 participates in the extracellular Ca2+-dependent shear-induced platelet granule release through CaM and MLC kinase-triggered MLC phosphorylation and concurrent cytoskeletal rearrangements, facilitating SIPA and leading to granule centralization as an onset to degranulation (Fig. 7). Because P2Y1 antagonism also inhibited shear-induced MLC phosphorylation (although weakly), a role for Ca2+ mobilized from internal stores in MLC kinase activation is possible (34, 35). Rapid P2X1 and slower P2Y1 signals (activated by co-released ATP and ADP, respectively) converge to a common effector (MLC kinase) contributing to cytoskeleton remodeling and platelet release (Fig. 7). Accordingly, combined P2X1 and P2Y1 antagonism leads to the same inhibition of SIPA at 3 min as P2Y1 antagonism only.

The study by Schoenwaelder et al. (37) defines an important role for RhoA and Rho kinase in SIPA but not in platelet release. The inhibition of shear-induced MLC phosphorylation by selective P2Y1 or P2Y12 antagonism suggests that ADP activates Rho kinase through Gq (40) and Gi{alpha}2 (41) also under shear stress (Fig. 7). Further investigations are required to determine the function of these pathways during SIPA. The failure of Rho kinase inhibitors to affect platelet release (Ref. 37 and the present study) may be explained by the existence of two pools of myosin present in different cell compartments (42) and emphasizes the role of MLC kinase activation in nucleotide-mediated granule release. In agreement with findings by Reséndez et al. (19), we further found that P2Y12 mediated PI3K activation during SIPA. The resulting Akt (protein kinase B) phosphorylation was inhibited by P2Y12 neutralization. PI3K inhibition partially inhibited SIPA but had no effect on ATP release (not shown); i.e. P2Y12-recruited PI3K activates platelets via a separate pathway (Fig. 7) potentially by regulating the function of {alpha}IIb{beta}3 via Akt and the integrin-linked kinase ILK (43).

Platelet granule release induced by most platelet agonists depends on PKC activation (reviewed in Ref. 31). However, none of the PKC inhibitors used in this study affected the shear-induced platelet release or aggregation. The nonselective PKC inhibitor GF109203X (which blocks both diacylglycerol- and Ca2+-sensitive conventional PKC isoforms and the diacyl-glycerol-sensitive Ca2+-insensitive novel isoforms), the more potent PKC {alpha}/{beta}-specific inhibitor Go6976, and Go6983 (which inhibits the atypical {zeta} isoform in addition to {alpha}, {beta}, {gamma}, and {delta}) were all without effect, even though they inhibited platelet release in response to collagen or TRAP1–6. This finding is consistent with a previous study in which the nonselective protein kinase inhibitor staurosporine failed to inhibit SIPA (32). This is in contrast to the study by Kroll et al. (8) showing inhibition of the full aggregation response by the staurosporine analogue Ro 31-7549. The effect of this inhibitor on platelet release was not reported in their study. Ro 31-7549, similar to Ro 31-8220 and GF109203X, belongs to a category of nonselective PKC inhibitors showing similar potency in inhibiting collagen-, thromboxane A2 analogue-, and thrombin-induced platelet release and aggregation (29, 33, 44). These authors show that SIPA is associated with the phosphorylation of pleckstrin, a PKC substrate that occurs in the absence of diacylglycerol or hydrolysis of phosphatidylinositol 4,5-bisphosphate in a Ca2+-dependent manner. It has to be noticed that EGTA, a chelator of extracellular Ca2+, had minimal effect on such phosphorylation, excluding a role for transmembrane Ca2+ influx. They conclude that a diacylglycerol-independent pathway of PKC activation contributes to SIPA. Thus, although involvement of an unknown PKC isoform in shear-induced platelet granule release cannot be ruled out, our data clearly distinguished this process from the GPVI- and PAR-1-mediated granule release (33). Finally, the study by Nakai et al. (21) describes the shear-induced cytoskeleton translocation of actin, actin-binding protein, and myosin as a rapid and transient movement of MLC kinase wherein the amount of cytoskeletal PKC did not change, unlike what is seen with thrombin. This observation further suggests distinct roles of PKC (if any) and MLC kinase during platelet responses to shear stress. The transient association of MLC kinase with the cytoskeleton may coincide with the quickly reversible MLC phosphorylation produced via P2X1 activation.

In conclusion, P2X1 contributes to SIPA through its ability to rapidly elicit Ca2+ influx-triggered signals leading to prompt CaM-dependent signaling, platelet activation, and platelet granule release. Further studies are required to elucidate the mechanisms of VWF-GPIb{alpha}-dependent platelet granule release. It also remains to be determined whether the presently identified P2X1/CaM/MLC kinase pathway plays a role during platelet activation processes accompanying platelet recruitment and adhesion on immobilized VWF and, most importantly, during thrombosis.


   FOOTNOTES
 
* This work was supported by Fonds voor Wetenschappelijk Onderzoek-Vlaanderen (FWO) Project G.0227.03 and by Geconcerteerde Onderzoeksactie/2004/09. 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. Back

§ Recipient of a postdoctoral research mandate from the FWO. Back

|| To whom correspondence should be addressed: University of Leuven, Center for Molecular and Vascular Biology, Herestraat 49, 3000 Leuven, Belgium. Tel.: 32-16-346145; Fax: 32-16-345990; E-mail: marc.hoylaerts{at}med.kuleuven.ac.be.

1 The abbreviations used are: SIPA, shear-induced platelet aggregation; meATP, methylene ATP; CaM, calmodulin; MLC, myosin light chain; PI3K, phosphatidylinositol 3-kinase; PKC, protein kinase C; VWF, von Willebrand factor; GP, glycoprotein; PAR, protease-activated receptor; TRAP1–6, thrombin receptor activating peptide SFFLRN. Back


   ACKNOWLEDGMENTS
 
We gratefully acknowledge critical comments by Dr. J. W. M. Heemskerk, Maastricht, The Netherlands.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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