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Keywords:

  • platelet;
  • lipid rafts;
  • G protein;
  • ADP;
  • P2Y12;
  • cholesterol depletion

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary.  ADP is important in propagating hemostasis upon its secretion from activated platelets in response to other agonists. Lipid rafts are microdomains within the plasma membrane that are rich in cholesterol and sphingolipids, and have been implicated in the stimulatory mechanisms of platelet agonists. We sought to determine the importance of lipid rafts in ADP-mediated platelet activation via the G protein-coupled P2Y1 and P2Y12 receptors using lipid raft disruption by cholesterol depletion with methyl-β-cyclodextrin. Stimulation of cholesterol-depleted platelets with ADP resulted in a reduction in the extent of aggregation but no difference in the extent of shape change or intracellular calcium release. Furthermore, repletion of cholesterol to previously depleted membranes restored ADP-mediated platelet aggregation. In addition, P2Y12-mediated inhibition of cAMP formation was significantly decreased upon cholesterol depletion from platelets. Stimulation of cholesterol-depleted platelets with agonists that depend upon Gαi activation for full activation displayed significant loss of aggregation and secretion, but showed restoration when simultaneously stimulated with the Gαz-coupled agonist epinephrine. Finally, Gαi preferentially localizes to lipid rafts as determined by sucrose density centrifugation. We conclude that Gαi signaling downstream of P2Y12 activation, but not Gαq or Gαz signaling downstream of P2Y1 or α2A activation, respectively, has a requirement for lipid rafts that is necessary for its function in ADP-mediated platelet activation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Platelet activation plays a central role in thrombosis and hemostasis [1]. When platelets are stimulated with agonists such as ADP, thromboxane A2 or thrombin, platelets aggregate, release their granule contents, and generate thromboxane A2. These agonists mediate their effects on platelets by utilizing G protein-coupled receptors on plasma membranes. Two receptors for ADP, the P2Y1 and P2Y12 receptors, stimulate the Gαq and Gαi protein signaling pathways, respectively, and their co-stimulation is needed for ADP-induced primary aggregation [2–5]. Activation of the Gαq pathway leads to phospholipase C stimulation, which leads to activation of PKC and an increase in intracellular calcium; on the other hand, activation of the Gαi pathway leads to inhibition of cyclic AMP formation, activation of the phosphoinositol-dependent protein kinase Akt [6,7] and potentiation of dense granule secretion [8].

The thromboxane A2 receptor (TP) also couples to Gαq but requires additional stimulation by ADP and epinephrine that are secreted from dense granules for activation of the Gαi and Gαz protein pathways, respectively, to initiate full aggregation [9,10]. Thrombin activates a family of protease-activated receptors (PARs) [11], two of which (PAR-1 and PAR-4) are present in human platelets and couple to Gαq [12]. While the signaling pathways downstream of both PARs rely upon dense granule release to initiate full platelet aggregation [13], PAR-4 has a lower affinity for thrombin and thus has a greater dependence upon secretion to initiate aggregation at low thrombin concentrations [14].

Lipid rafts, also called detergent-resistant membranes or glycolipid-enriched membranes, are microdomains within the plasma membrane that contain saturated phospholipids and glycosphingolipid conjugates of cholesterol that are highly ordered in comparison with the rest of the plasma membrane [15,16]. Lipid rafts are believed to serve as platforms for receptor-mediated signal transduction by selectively localizing certain proteins while excluding others, and have been isolated using density gradients by taking advantage of their relatively lower density and resistance to solubilization at low temperature by non-ionic detergents [15,17,18]. The presence of lipid rafts has been identified in several cell types including hematopoietic cells [19–21]. In particular, lipid rafts have been shown in platelets to be involved in the clustering of integrin proteins [22–25], as well as activation of phosphatidylinositol 3-kinase activation [26] which implied the presence of Gαi in lipid rafts. However, the extent to which lipid rafts are utilized by platelet signaling proteins is unknown.

In this paper, we show that the ability of Gαi to potentiate ADP-mediated platelet aggregation is highly dependent upon its localization to lipid rafts. Also, the activation of ADP-mediated Gαq and epinephrine-mediated Gαz pathways do not require their presence in lipid rafts.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Reagents

The PAR-4 activating peptide agonist AYPGKF was synthesized by Invitrogen Research Genetics (Huntsville, AL, USA). The thromboxane A2 analog 15(S)-hydroxy-9,11-epoxymethanoprosta-5Z,13E-dienoic acid (U46619) was purchased from Biomol (Plymouth Meeting, PA, USA). SC-57101 was obtained from Searle Research and Development (Skokie, IL, USA). Fura-2 was purchased from Molecular Probes (Eugene, OR, USA). [3H]5-hydroxytryptamine was purchased from Perkin Elmer Life Sciences (Boston, MA, USA). Mouse anti-Gαi2 antibody was purchased from Lab Vision, Inc. (Fremont, CA, USA). Mouse anti-Gαq and anti-flotillin-2 antibodies were purchased from BD Pharmingen (San Diego, CA, USA). Mouse anti-Gαz antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were purchased from Sigma Chemical Co. (St Louis, MO, USA).

Platelet isolation

Whole human blood was drawn into one-sixth final volume of acid–citrate–dextrose (ACD – 85 mm sodium citrate, 70 mm citric acid, 110 mm glucose) from informed, healthy volunteers at the Sol Sherry Thrombosis Research Center of Temple University. Human platelets were treated with aspirin while suspended in platelet-rich plasma (PRP), then isolated and resuspended in Tyrode's buffer as previously described [27].

Depletion/repletion of platelet cholesterol

For cholesterol depletion, a one-tenth final volume of 50 mm methyl-β-cyclodextrin (MβCD) in 1:5 ACD:phosphate-buffered saline was added to PRP to achieve a concentration of 5 mm (ACD:PBS alone was added as a mock control) and was incubated at 37 °C for 15 min. Platelets were then isolated and resuspended as described above. For cholesterol repletion, platelets were treated as described above but were resuspended in Tyrode's buffer containing 5 mm MβCD and 200 μg mL−1 cholesterol, and were incubated at room temperature for 30 min.

Measurement of platelet aggregation and secretion

Agonist-induced platelet aggregation was analyzed using a Chrono-Log model 440-VS aggregometer (Havertown, PA, USA) with sample volumes of 0.5 mL in a cuvette holder thermostatted at 37 °C and set at constant stirring. Aggregometer output was recorded using a Kipp & Zonen type BD 12E flatbed chart recorder (SCI-TEC, Saskatoon, SK, Canada) set at 0.2 mm s−1. Dense granule secretion was performed concurrently with aggregation assays as previously described [28].

Intracellular calcium release

For platelet measurements, PRP was incubated with 2 μm fura-2 or an equal amount of DMSO (vehicle) and incubated simultaneously with acetylsalicylic acid. Platelets were then isolated and washed as described above. The integrin αIIbβ3 antagonist SC-57101 (10 μm) was added prior to each assay to prevent agonist-mediated platelet aggregation from interfering with fluorescence measurement. Changes in fluorescence were measured using an Aminco-Bowman Series 2 luminescence spectrometer with a water-jacketed cuvette holder thermostatted at 37 °C and set at constant stirring. Sample volumes of 0.5 mL were analyzed with an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Fluorescence measurements were converted to calcium concentrations using the equation reported by Grynkiewicz et al. [29] where Fmin and Fmax were determined with each respective platelet preparation.

Measurement of cAMP

PRP was incubated with 2 μCi mL−1 of [3H]adenine and aspirin (1 mm) for 1 h at 37 °C. Platelets were isolated as described above. Reactions were stopped by the addition of 1 m HCl and 4000 dpm [14C]cyclic AMP as a recovery standard. Cyclic AMP was determined as detailed earlier and expressed as a percentage of total [3H]adenine nucleotides.

Isolation and Western blotting of lipid raft fractions

Sucrose density gradient centrifugation was performed to isolate the cholesterol-rich lipid rafts from other cellular lipids. Platelet samples (250 μL of 3 × 1010 mL−1) were solubilized with an equal volume of ice-cold 2× lysis buffer (final concentrations 50 mm Tris, pH 7.5, 150 mm NaCl, 10 mm EGTA, 1% Triton X-100, 10 μg mL−1 leupeptin, 10 μg mL−1 antipain) and placed on ice for 15 min. Samples were then mixed with an equal volume of ice-cold 80% sucrose (final volume 1 mL at 40% sucrose) and placed at the bottom of polyallomer bell-top Quick-Seal centrifuge tubes (Beckman Instruments, Fullerton, CA, USA). Successive volumes of 30% (1.5 mL) and 5% (0.75 mL) sucrose were consecutively layered upon the solubilized platelet sample, and the tubes were centrifuged at 100 000 g at 4 °C for 18 h. Gradient fractions (0.32 mL) were sequentially removed from the top of the gradient and analyzed by Western blotting as previously described [7].

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Gαi-specific platelet intracellular signaling mechanisms are associated with lipid rafts

We first set out to determine whether lipid rafts played a role in ADP-mediated platelet activation by investigating the effect of membrane cholesterol depletion on platelet aggregation. Upon stimulation with ADP, aggregation was significantly reduced after cholesterol depletion with MβCD when compared with the mock-treated control (Fig. 1). In an effort to replete platelet membranes with cholesterol and to account for any effects due to MβCD alone, platelets were also treated with MβCD into which cholesterol was dissolved. ADP-mediated aggregation of non-depleted platelets were unaffected by the combined MβCD-cholesterol treatment, whereas aggregation was restored to control levels by the repletion of cholesterol to previously depleted platelets (Fig. 1).

image

Figure 1. Effect of cholesterol depletion on ADP-mediated platelet aggregation. Treatment of platelets with either 5 mm MβCD or its vehicle, or treatment with MβCD plus 200 μg mL−1 cholesterol, prior to the addition of 10 μm ADP is described in ‘Materials and methods.’ Results are representative of three separate experiments.

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We next proceeded to examine platelet responses that were specifically downstream of either ADP receptor in order to determine whether one or both of the receptors was affected by the disruption of lipid rafts. ADP signaling downstream of the Gαq-linked P2Y1 receptor was first examined by observing platelet shape change and calcium mobilization, both of which are directly downstream of Gαq activation [30,31]. Upon depletion of cholesterol by MβCD treatment, stimulation with ADP showed no difference in platelet shape change or calcium mobilization compared with the mock control (Fig. 2).

image

Figure 2. ADP-mediated Gαq signaling is unaffected by lipid raft disruption. Treatment of platelets with either MβCD (5 mm, +) or its vehicle (−) prior to addition of 10 μm ADP is described in ‘Materials and methods.’ (A) ADP-mediated platelet shape change. (B) ADP-mediated intracellular calcium release. Both experiments were performed in the presence of the fibrinogen receptor antagonist SC-57101A (10 μm) and are representative of at least three separate experiments.

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One of the specific markers of Gαi activation is a decrease in the level of stimulated cyclic AMP formation resulting from the inhibition of adenylate cyclase [32]. Figure 3 shows that in reference to the ability of ADP to inhibit forskolin-mediated cAMP production in untreated platelets, cholesterol depletion by MβCD prevents this inhibition of cAMP production.

image

Figure 3. Lipid raft disruption abolishes ADP-mediated inhibition of cyclic AMP formation. Data are expressed as mean ± SD of the percentage of total tritiated adenine nucleotides present and are representative of at least three separate experiments.

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Secretion-dependent platelet aggregation is affected by cholesterol depletion

We have shown previously that Gαi activation can potentiate the secretion of dense granule components [8]. Some platelet agonists, e.g. thromboxane A2 and low concentrations of the PAR-4 activating peptide AYPGKF, are dependent upon secreted ADP for potentiation and full onset of the aggregation response; if ADP-mediated signaling was associated with lipid rafts, then cholesterol depletion and the resulting lipid raft disruption should have an effect on aggregation and secretion mediated by these agonists. Platelet aggregation mediated by either the thromboxane analog U46619, or low concentrations of thrombin (0.5 nm) or the PAR-4 agonist peptide AYPGKF, is abrogated by cholesterol depletion (Fig. 4A, top and middle panels). Similar to the pattern of aggregation, dense granule secretion is likewise affected by cholesterol depletion (Fig. 4B). In contrast, aggregation and secretion mediated by a high concentration of thrombin (10 nm) is insensitive to cholesterol depletion.

image

Figure 4. Effect of lipid raft disruption upon Gαi- and Gαz-dependent agonist-mediated aggregation and secretion. Epinephrine (epi, 10 μm) or its vehicle was added simultaneously with an agonist to initiate aggregation (A) and dense granule secretion (B). Results were normalized to that induced by 10 nm thrombin and are expressed as mean ± SD of three separate experiments.

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Gαz-specific platelet intracellular signaling mechanisms are not associated with lipid rafts

The α2A receptor agonist epinephrine, which activates the Gαi subclass member Gαz, also stabilizes and potentiates agonist-mediated platelet aggregation and has been used previously to substitute for Gαi activation in the absence of P2Y12 stimulation [9,28]. When epinephrine was added to cholesterol-depleted platelets simultaneously with ADP, U46619 or low concentrations of thrombin or AYPGKF, the extent of platelet aggregation was restored to control levels (Fig. 4A, bottom panel). Platelet secretion was likewise affected by cholesterol depletion and was correspondingly restored by supplementation with epinephrine (Fig. 4B).

Platelet Gαi2 associates with lipid rafts while Gαq and Gαz do not

Finally, to address the implication raised by the data that Gαi was associated with lipid rafts, lysed platelets were fractionated by sucrose density centrifugation in order to isolate the lower density lipid raft membranes from other higher density membranes. Western blotting of the gradient fractions with an antibody against flotillin-2, a protein known to associate with lipid rafts [19], indicate that the upper fractions contained primarily low-density membranes (Fig. 5A). Western blotting of the same material for Gαi2 showed that Gαi was also localized to the upper gradient fractions containing low-density membranes (Fig. 5B). Furthermore, disruption of lipid rafts by cholesterol depletion caused a redistribution of flotillin-2 and Gαi to the heavier gradient fractions, and cholesterol repletion enabled the two proteins to relocalize to the lighter fractions. Consistent with the inability of Gαq and Gαz to be affected by cholesterol depletion, the localization of both G proteins remained unchanged regardless of treatment (Fig. 5C,D).

image

Figure 5. Gαi partitions to lipid rafts. Platelets were treated with MβCD or its vehicle prior to solubilization and isolation of lipid rafts by sucrose density gradient centrifugation as described in ‘Materials and methods.’ Bands denote the positions of (A) flotillin-2, (B) Gαi2, (C) Gαq, and (D) Gαz as determined by Western blotting of fractions obtained from the gradients.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

The importance of Gαi in platelet activation is underscored by the success of platelet-inhibiting drugs such as clopidogrel that block signaling via P2Y12, which couples to Gαi2. Also, the identification of a large number of signaling molecules that localize to lipid rafts (including heterotrimeric G proteins) attests to the importance of these membrane microdomains as mediators of intracellular signaling [33]. Indeed, localization of Gαi to lipid rafts has been previously reported in other cells [34] and has been suggested in platelets [26,35]. However, this is the first paper to conclusively report on the presence of platelet Gαi in lipid rafts.

Other platelet membrane proteins have been shown to localize to lipid rafts, including the GPIb-IX-V complex [22,23] and GPVI [24,36]. However, the aforementioned proteins are receptors that migrate to lipid rafts upon agonist stimulation while Gαi is a membrane-associated G protein that appears to be resident in lipid rafts regardless of the platelet activation state (data not shown). The fibrinogen receptor integrin αIIbβ3 does not appear to partition to lipid rafts [37], and therefore the decrease in agonist-mediated platelet aggregation that is seen upon cholesterol depletion is not reflective of a disruption in integrin αIIbβ3 function [38].

Based on the facts that ADP-mediated platelet activation requires coactivation of both Gαq- and Gαi-coupled pathways [10] and Gαi localizes to lipid rafts, one might expect that Gαq would also be associated with lipid rafts as is Gαi. However, it appears that the P2Y1-coupled Gαq does not localize to lipid rafts as observed by the failure of cholesterol depletion to inhibit ADP-induced platelet responses mediated by Gαq and the absence of significant Gαq protein from platelet low-density membranes. Likewise, Gαz and/or the α2A receptor do not appear to localize to lipid rafts since Gαz appears primarily in high-density platelet membranes, and aggregation and dense granule secretion in cholesterol-depleted platelets are restored when supplementally stimulated with the α2A receptor agonist epinephrine.

In conclusion, the presence of the P2Y12-coupled Gαi protein in lipid rafts appears to be necessary for its ability to activate platelets. It is unknown whether Gαi maintains its association with P2Y12 during cholesterol depletion or sucrose density centrifugation; therefore, it is unclear whether Gαi partitions to lipid rafts solely on its own merit or by virtue of association with P2Y12. Furthermore, while Gαq and Gαz activation are necessary for the onset and potentiation of platelet activation, respectively, their presence in lipid rafts is not. Determination of the association of P2Y12 and Gαi in lipid rafts, as well any implication of the difference in membrane localization between Gαq and Gαi, is currently under investigation.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We wish to thank Dr Florin Tuluc for his technical advice and Ms Patricia Quinter for her helpful discussions.

This work was supported by grants HL60683 and HL64943 from the National Institutes of Health (S.P.K.).

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  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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