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Keywords:

  • 14-3-3ζ;
  • apoptosis;
  • GPIbα;
  • platelets;
  • von Willebrand factor

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Summary. Background: The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates platelet adhesion, and simultaneously triggers intracellular signaling cascades leading to platelet aggregation and thrombus formation. Some of the signaling events are similar to those occurring during apoptosis, however, it is still unclear whether platelet apoptosis is induced by the GPIbα–VWF interaction. Objectives: To investigate whether the GPIbα–VWF interaction induces platelet apoptosis and the role of 14-3-3ζ in apoptotic signaling. Methods: Apoptotic events were assessed in platelets or Chinese hamster ovary (CHO) cells expressing wild-type (1b9) or mutant GPIb–IX interacting with VWF by flow cytometry or western blotting. Results: Ristocetin-induced GPIbα–VWF interaction elicited apoptotic events in platelets, including phosphatidylserine exposure, elevations of Bax and Bak, gelsolin cleavage, and depolarization of mitochondrial inner transmembrane potential. Apoptotic events were also elicited in platelets exposed to pathologic shear stresses in the presence of VWF; however, the shear-induced apoptosis was eliminated by the anti-GPIbα antibody AK2. Furthermore, apoptotic events occurred in 1b9 cells stimulated with VWF and ristocetin, but were significantly diminished in two CHO cell lines expressing mutant GPIb–IX with GPIbα truncated at residue 551 or a serine-to-alanine mutation at the 14-3-3ζ-binding site in GPIbα. Conclusions: This study demonstrates that the GPIbα–VWF interaction induces apoptotic events in platelets, and that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα is essential for apoptotic signaling. This finding may suggest a novel mechanism for platelet clearance or some thrombocytopenic diseases.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

The interaction of glycoprotein (GP) Ibα with von Willebrand factor (VWF) initiates the adherence of platelets to sites of vascular injury [1], and simultaneously triggers intracellular signaling events such as elevation of cytoplasmic calcium [2] and activations of multiple protein kinase pathways [3], which result in the activation of the ligand-binding function of integrin αIIbβ3, leading to platelet activation and thrombus formation [2–4]. The intracellular signaling protein 14-3-3ζ and the membrane skeleton protein filamin A have been confirmed to interact with the intracellular domain of GPIbα and play important roles in the regulation of platelet function. Filamin A, which interacts with GPIbα at amino acids within the 557–579 sequence, attaches the GPIb–IX complex to the membrane skeleton [5]. Association of filamin A with the cytoplasmic domain of GPIbα is essential for VWF-induced platelet activation and GPIb–IX anchorage on the VWF surface under high-shear conditions [5]. The GPIbα binding sites for 14-3-3ζ have been mapped to the C-terminal region, and the association of 14-3-3ζ with the C-terminal domain of GPIbα is critical for the interaction between GPIb–IX and VWF [6]. The signaling events elicited by the GPIbα–VWF interaction, such as calcium mobilization and phosphatidylserine (PS) exposure [7,8], are similar to those occurring during apoptosis. In particular, the 14-3-3ζ-binding domain of GPIbα has been reported to be involved in the regulation of cell proliferation [9,10]. However, it is still unclear whether the GPIbα–VWF interaction induces platelet apoptosis.

Apoptosis (programmed cell death) is a common physiologic strategy for controlling the number of cells, as well as for the elimination of unwanted cells [11]. Platelet apoptosis induced by physiologic [12,13] or chemical [14–16] compounds, or platelet storage [17,18], has been reported to occur widely in vivo and in vitro. Generally, apoptotic events induced by weak platelet agonists include depolarization of the mitochondrial inner transmembrane potential (ΔΨm), increased Bcl-2 family protein expression, and caspase-3 activation [12,18,19]. In contrast, stimulation of platelets with strong agonists such as A23187 [16] or thrombin [12], or platelet storage under blood bank conditions [17,18], cause not only the above apoptotic events but also PS exposure. PS exposure occurs in both platelet activation and apoptosis; however, the signaling pathways leading to PS exposure in the two processes [20] are different, suggesting that platelet activation and apoptosis occur separately under physiologic conditions. Platelet apoptosis, which has been reported to be induced by the natural platelet agonist thrombin or pathologically high shear stress [21], involves the regulation of function and clearance of circulatory platelets. Thus, whether the GPIbα–VWF interaction causes platelet apoptosis has important pathophysiologic implications.

The results of this study indicate that VWF binding to GPIbα elicits apoptotic events in human platelets, including ΔΨm reduction, gelsolin cleavage, elevations of Bax and Bak, and PS externalization. Elimination of the 14-3-3ζ-binding site in the C-terminal domain of GPIbα abolished the GPIbα–VWF interaction-induced platelet apoptosis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Reagents

The monoclonal antibodies SZ29, against VWF, SZ51, against P-selectin, and SZ2, against GPIbα, were from C. Ruan (Soochow University, Suzhou, China). Purified human VWF was kindly provided by X. Du (University of Illinois, Chicago, IL, USA). Ristocetin, the peptide RGDS and anti-human gelsolin antibody were purchased from Sigma (St Louis, MO, USA). Monoclonal antibodies against Bax, Bak, Bcl-2, and Bcl-XL, fluorescein isothiocyanate (FITC)-conjugated PAC-1, FITC-conjugated goat anti-mouse IgG and horseradish peroxidase-conjugated goat anti-mouse IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The monoclonal antibody AK2, against GPIbα, was from Research Diagnostics (Flanders, NJ, USA). The Annexin V–FITC Kit, the caspase-3 inhibitor z-DEVD-fmk and tetramethylrhodamine ethyl ester (TMRE) were from Bender Medsystem (Vienna, Austria). Prostaglandin E1 (PGE1), apyrase and calpain inhibitor 1 were purchased from Calbiochem (San Diego, CA, USA).

Cell lines expressing recombinant proteins

Chinese hamster ovary (CHO) cells expressing recombinant wild-type GPIb–IX (1b9), mutant GPIb–IX with a serine-to-alanine point mutation at Ser609 in GPIbα (S609A) [6] and mutant GPIb–IX with a truncated GPIbα cytoplasmic domain at residue 551 (Δ551) [22] had been established previously. Stable cell lines were selected by cell sorting, using the anti-GPIbα monoclonal antibody SZ2.

Preparation of washed platelets

Fresh blood from healthy volunteers was anticoagulated with a 1/7 volume of acid–citrate–dextrose (2.5% trisodium citrate, 2.0%d-glucose, and 1.5% citric acid). After centrifugation, isolated platelets were washed twice with CGS buffer (0.123 m NaCl, 0.033 m d-glucose, 0.013 m trisodium citrate, pH 6.5), resuspended in modified Tyrode’s buffer (2.5 mm Hepes, 150 mm NaCl, 2.5 mm KCl, 12 mm NaHCO3, 5.5 mm d-glucose, pH 7.4) to a final concentration of 3 × 108 mL−1, and supplemented with 1 mm CaCl2 and 1 mm MgCl2, and then incubated at room temperature (RT) for 1 h to allow them to recover to resting state, as described previously [6,22]. In some experiments, platelets were preincubated with 1 mm RGDS peptide at RT for 10 min (min) for evaluation of the effect of αIIbβ3.

Flow cytometry analysis of VWF binding to platelets

Flow cytometric analysis of VWF binding to platelets was performed as described previously [6,22]. Briefly, washed platelets (5 × 107 mL−1) were incubated with ristocetin (1.0 mg mL−1) in the presence or absence (control) of purified human VWF (35 μg mL−1) at 22 °C for 30 min and washed once. The platelets were further incubated with monoclonal antibody SZ29 and FITC-labeled goat anti-mouse IgG at 22 °C for 30 min, respectively, and then analyzed by flow cytometry.

Platelet surface staining and aggregation

Surface expression of P-selectin and αIIbβ3 activation in platelets were detected by flow cytometry using single-color analysis. For examination of P-selectin surface expression, washed platelets (5 × 107 mL−1) were incubated with VWF (35 μg mL−1) and ristocetin (1.0 mg mL−1) at 22 °C for 30 min, and then further incubated with SZ51 at RT for 30 min. Platelets were washed once, incubated with FITC-labeled anti-mouse IgG in the dark at RT for 30 min, and then analyzed by flow cytometry. For platelet inhibition studies, platelets were pretreated with PGE1 (100 μg mL−1) plus apyrase (0.5 U mL−1) at RT for 20 min, and then analyzed for P-selectin surface expression. As negative controls, platelets were incubated with mouse IgG and then incubated with FITC-labeled goat anti-mouse IgG. For evaluation of αIIbβ3 activation, washed platelets (3 × 108 mL−1) were incubated with VWF (35 μg mL−1) and ristocetin (1.0 mg mL−1) or control buffer at 22 °C for 30 min, added to a fluorescence-activated cell sorting tube containing FITC-labeled soluble PAC-1, and incubated at RT for 20 min in the dark. Platelets were fixed with 1% cold paraformaldehyde, further incubated at 4 °C in the dark for 30 min, and analyzed by flow cytometry.

Platelet aggregation was performed using a turbidometric platelet aggregometer (Xinpusen, Beijing, China). Briefly, washed platelets (3 × 108 mL−1) were added to glass aggregometer cuvettes in the presence of VWF (35 μg mL−1), and aggregation was then performed by addition of ristocetin (1.2 mg mL−1) at 37 °C under stirring (1000 r.p.m.) or non-stirring conditions.

PS externalization assay

Washed platelets (5 × 107 mL−1) were incubated with VWF (35 μg mL−1) and ristocetin (1.0 mg mL−1) at 22 °C for 30 min. Annexin V binding was performed according to the instructions with the Annexin V–FITC Kit. Briefly, annexin V binding buffer (10 mm Hepes, 10 mm NaOH, 140 mm NaCl, 2.5 mm CaCl2, pH 7.4) was mixed with treated platelets and annexin V–FITC at a ratio of 50 : 10 : 1. Samples were gently mixed by rocking, incubated at RT for 15 min in the dark, and then analyzed by flow cytometry.

ΔΨm measurement assay

Mitochondrial membrane potential was determined using the potential-sensitive dye TMRE. Briefly, washed platelets (5 × 107 mL−1) were incubated with VWF and ristocetin at 22 °C for 30 min, and TMRE was added to the platelet suspension to 100 nm final concentration. To evaluate the effect of platelet activation inhibitors, platelets were pretreated with PGE1 (100 μg mL−1) plus apyrase (0.5 U mL−1) at RT for 20 min. Then samples were further incubated in the dark at 37 °C for 20 min, and analyzed by flow cytometry. TMRE was excited using a 488-nm krypton–argon laser line, and its emissions were captured using filters at 625 nm.

Gelsolin cleavage and Bcl-2 family protein expression assay

Washed platelets (3 × 108 mL−1) were treated with VWF and ristocetin at 22 °C for 30 min, and lysed with an equal volume of lysis buffer containing 0.1 mm E64, 1 mm phenylmethylsulfonyl fluoride (PMSF) and 1/100 aprotinin on ice for 30 min, and whole lysate was resolved by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotted with anti-gelsolin antibody (1 : 2500). To study the effects of caspase, calpain, and platelet activation inhibitors, the platelets were preincubated with 50 μm z-DEVD-fmk, 50 μm calpain inhibitor 1, and PGE1 (100 μg mL−1) plus apyrase (0.5 U mL−1) for 20 min at RT, respectively. To evaluate Bcl-2 family protein expression, lysate was resolved by SDS-PAGE, and immunoblotted with anti-Bcl-2 (1 : 500), anti-Bcl-XL (1 : 500), anti-Bax (1 : 500), and anti-Bak (1 : 500).

Exposure of platelets to shear stress

Study of shear stress-induced GPIbα–VWF interaction was performed using a cone-and-plate viscometer, which consists of a stationary plate and rotating cone with an angle (= 1°). The cone rotated at a constant angular velocity, applying a uniform shear field. The cone and plate were silicon-coated before experiments. Shear stress was calculated with the following equation [23]: shear rate (s−1) = ω/tanα, where ω (s−1) is the cone rotation speed, and α is the cone–plate angle.

Two milliliters of washed platelets (3 × 108 mL−1) with or without VWF was placed between the cone and plate surfaces, and rotated at a shear rate of 7000 s−1 for 90 s; samples were then collected for platelet apoptosis assays. For study of the specific interaction between GPIbα and VWF, platelets were preincubated with monoclonal antibody AK2 (20 μg mL−1) for 5 min at 22 °C before exposure to shear stress.

Cell apoptosis assay

CHO cells expressing wild-type and mutant GPIb–IX were resuspended in modified Tyrode’s buffer as described previously [6,22]. The cells (2.5 × 106 mL−1) were incubated with VWF (35 μg mL−1) and ristocetin (1.0 mg mL−1) at 22 °C for 30 min, and then analyzed for PS exposure and ΔΨm reduction by flow cytometry. To evaluate gelsolin cleavage, the cells treated with VWF/ristocetin were lysed with an equal volume of lysis buffer containing 0.2 mm E64, 1 mm PMSF and 0.08 U mL−1 aprotinin on ice for 30 min. The lysates were centrifuged at 2000 × g for 5 min to remove the nuclei, and then resolved by SDS-PAGE and immunoblotted with anti-gelsolin antibody (1 : 2500).

Statistical analysis

Data are shown as means ± standard deviations. The statistical difference between groups was determined by paired Student’s t-test. A P-value of < 0.05 was considered to be significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

The GPIbα–VWF interaction induced by ristocetin elicits PS exposure and upregulation of Bax and Bak proteins in platelets

In order to investigate the specific effects of the GPIbα–VWF interaction on platelet apoptosis, platelets were isolated and washed to exclude possible interference from other serum proteins, and were then resuspended in modified Tyrode’s buffer. First, we tested VWF binding to washed platelets induced by ristocetin. As shown in Fig. 1A, most of the washed platelets were VWF-positive, indicating sufficient GPIbα–VWF interaction for platelet activation induced by ristocetin under the conditions used. The GPIbα–VWF interaction triggers intracellular signaling cascades leading to platelet activation, which is characterized by P-selectin surface expression and αIIbβ3 activation. Therefore, P-selectin surface expression and αIIbβ3 activation were assessed by flow cytometry, and the results indicated that the GPIbα–VWF interaction caused platelet activation (Fig. S1). Furthermore, the ristocetin-induced interaction of washed platelets with VWF resulted in a normal aggregation response under stirring conditions, but not non-stirring conditions (Fig. S1). In addition, washed platelets treated with ristocetin (or VWF) alone did not demonstrate any biochemical responses under either non-stirring or stirring conditions (data not shown). Thus, ristocetin-induced interaction of platelets with VWF under non-stirring conditions was employed for platelet apoptosis investigations.

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Figure 1.  The glycoprotein Ibα–von Willebrand factor (VWF) interaction induced by ristocetin (Ris) elicits phosphatidylserine (PS) exposure and upregulation of Bax and Bak proteins in platelets. (A) Washed platelets in modified Tyrode’s buffer (5 × 107 mL−1) were incubated with ristocetin (1.0 mg mL−1) in the presence (VWF/Ris) or absence (control) of purified VWF (35 μg mL−1) at 22 °C for 30 min. The bound VWF was detected by anti-VWF antibody SZ29 and flow cytometry. (B) Washed platelets were treated with VWF/ristocetin or control buffer at 22 °C for 30 min. PS exposure was analyzed by fluorescein isothiocyanate (FITC)–annexin V and flow cytometry. The linear gate M2 denotes the position of annexin V-stained platelets. (C) Means ± standard deviations of the percentage of PS-positive platelets from three independent experiments are shown. **P < 0.01. (D) Washed platelets treated with VWF/ristocetin or control buffer were lysed, and the total lysate was resolved by 15% sodium dodecylsulfate polyacrylamide gel electrophoresis, and probed with anti-Bax, Bak, Bcl-2 and Bcl-XL antibodies. Actin levels demonstrate equal loading. Results are representative of three separate experiments with different donors.

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The interaction of GPIbα with VWF exposed at the injured vessel wall initiates platelet adhesion and simultaneously triggers intracellular signaling events such as elevation of intracellular calcium concentration [2]. It has been well established that the elevation of intracellular calcium concentration leads to the exposure of PS [24]. However, although it was reported that GPIb facilitated GPVI-mediated PS exposure under physiologic conditions of flow [7], up to now there has been no direct evidence that the GPIbα–VWF interaction elicits PS exposure. Thus, we evaluated the effect of the GPIbα–VWF interaction on PS exposure. As shown in Fig. 1B,C, incubation of platelets with VWF plus ristocetin, but not control buffer, induced a significant increase in PS exposure as measured by annexin V staining, whereas ristocetin or VWF alone failed to induce PS exposure (data not shown).

The consequences of platelet activation are associated with many specific biochemical and morphologic changes, some of which are similar to those occurring in apoptotic cells [7,8]. Although PS exposure is thought to be a typical event in both platelet activation and apoptosis, the signaling pathways leading to PS exposure are quite different between these two processes [20]. The proapoptotic proteins Bax and Bak, and the antiapoptotic proteins Bcl-2 and Bcl-XL, which regulate the mitochondrial pathway of apoptosis in nucleated cells, play essential roles in the regulation of platelet apoptosis [25]. To investigate whether PS exposure results from apoptosis induced by the VWF–GPIbα interaction, Bax, Bak, Bcl-2 and Bcl-XL protein levels were examined. Following VWF/ristocetin stimulation, the expression levels of Bax and Bak were significantly enhanced in platelets; however, no obvious variation was observed in Bcl-2 and Bcl-XL expression levels (Fig. 1D), suggesting that the GPIbα–VWF interaction triggers signaling cascades leading to platelet apoptosis.

The GPIbα–VWF interaction induced by ristocetin elicits gelsolin cleavage and ΔΨm depolarization in platelets

Bcl-2 family proteins, interacting with the mitochondrial outer membrane, regulate the depolarization of ΔΨm as well as the release of apoptogenic factors leading to activation of caspases such as executioner caspase-3 [25]. To further characterize the effects of the GPIbα–VWF interaction on the platelet apoptotic pathway, markers of the mitochondrial cell death pathway were evaluated. Gelsolin is a cytoskeletal regulatory protein that has been shown to be a specific substrate of activated caspase-3 [26]. Therefore, caspase-3 activation was measured by the cleavage of gelsolin. The 48-kDa cleaved fragment of gelsolin was present in platelets stimulated with VWF/ristocetin but not with control buffer (Fig. 2A). Although it is widely accepted that gelsolin is a caspase-3 specific substrate, there is also a report suggesting that gelsolin could be cleaved by calpain [16]. Therefore, calpain inhibitor was used to exclude the involvement of calpain in the current observation. The results showed that gelsolin cleavage was inhibited by the caspase-3 inhibitor z-DEVD-fmk but not by the cell-permeable calpain inhibitor 1 (Fig. 2B), indicating that gelsolin cleavage is mediated by caspase-3, but not by calpain. Thus, the GPIbα–VWF interaction causes caspase-3 activation in platelets.

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Figure 2.  The glycoprotein Ibα–von Willebrand factor (VWF) interaction induced by ristocetin elicits gelsolin cleavage and mitochondrial inner transmembrane potential (ΔΨm) depolarization in platelets. (A) Washed platelets treated with VWF/ristocetin (Ris) or control buffer were lysed. Total lysate was resolved by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis and probed with anti-gelsolin antibody. (B) Washed platelets were preincubated with the caspase-3 inhibitor z-DEVD-fmk (50 μm) or calpain inhibitor 1 (50 μm) at room temperature for 20 min, treated with VWF/ristocetin, and lysed. Total platelet lysate was separated and probed for gelsolin. (C, D) After treatment of platelets with VWF/ristocetin or control buffer, ΔΨm depolarization was determined with tetramethylrhodamine ethyl ester dye and flow cytometry. Representative flow cytometric histograms are shown (C). (D) Depolarization was quantified as the geometric (Geo) mean channel fluorescence (left), and as the percentage of depolarized cells (right). Means ± standard deviations from three independent experiments are shown. *P < 0.05, **P < 0.01.

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To further confirm mitochondria-dependent platelet apoptosis induced by the GPIbα–VWF interaction, depolarization of ΔΨm was measured with the cell-permeable lipophilic cationic dye TMRE, which accumulates in the mitochondrial matrix, driven by ΔΨm [27]. Figure 2C,D shows that treatment of platelets with VWF/ristocetin resulted in ΔΨm depolarization, as monitored by decreased fluorescence of TMRE-stained platelets, and an increase in the number of depolarized cells. In addition, ΔΨm reduction, gelsolin cleavage, elevations of Bax and Bak and PS externalization were not inhibited by the αIIbβ3-specific inhibitor RGDS peptide (data not shown). Taken together, these data indicate that the interaction of GPIbα with VWF induced by ristocetin elicits platelet apoptosis.

The GPIbα–VWF interaction-induced apoptotic events are independent of platelet activation

Several lines of evidence indicate that platelet activation and apoptosis are two different groups of platelet responses that may be induced by different mechanisms and require different levels of stimuli [28,29]. The GPIbα–VWF interaction occurs commonly under pathophysiologic conditions to induce platelet activation. Thus, in order to investigate whether the GPIbα–VWF interaction-induced apoptosis is a result of platelet activation, platelets were preincubated with the activation inhibitors PGE1 and apyrase. As shown in Fig. 3A, PGE1 and apyrase abolished the GPIbα–VWF interaction-induced platelet activation as measured by P-selectin surface expression. However, the platelet activation inhibitors had no effect on ΔΨm reduction and gelsolin cleavage in platelets treated with VWF/ristocetin (Fig. 3B,C). These data indicate that the GPIbα–VWF interaction-induced apoptotic events are independent of platelet activation.

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Figure 3.  The apoptotic events induced by the glycoprotein Ibα–von Willebrand factor (VWF) interaction are independent of platelet activation. Washed platelets were pretreated with or without prostaglandin E1 (PGE1) (100 μg mL−1) plus apyrase (0.5 U mL−1) at room temperature (RT) for 20 min, and then incubated with ristocetin (Ris) (1.0 mg mL−1) and VWF (35 μg mL−1) or control buffer at 22 °C for 30 min. (A) The platelets were further incubated with the anti-human P-selectin antibody SZ51 at RT for 30 min. After being washed once, platelets were incubated with fluorescein isothiocyanate-labeled anti-mouse IgG in the dark at RT for 30 min, and then analyzed by flow cytometry. Representative flow cytometric histograms are shown. (B) Tetramethylrhodamine ethyl ester dye (100 nm) was added to the treated platelet suspension, further incubated in the dark at 37 °C for 20 min, and analyzed by flow cytometry. (C) The platelets were lysed, the total lysate was resolved by 10% sodium dodecylsulfate polyacrylamide gel electrophoresis, and probed with anti-gelsolin antibody. Note that PGE1 and apyrase inhibited P-selectin surface expression, but did not affect ΔΨm reduction and gelsolin cleavage. Results are representative of three separate experiments.

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The shear-induced GPIbα–VWF interaction elicits platelet apoptotic events

In vivo, the interaction of GPIbα with VWF at the injured vessel wall initiates platelet tethering and subsequent adhesion under flow conditions [1,4]. Particularly under pathologically high shear stress, the shear-induced GPIbα–VWF interaction results in platelet aggregation and thrombus formation [1,4,30]. Therefore, we examined whether the shear-induced GPIbα–VWF interaction triggers platelet apoptosis, to explore the pathophysiologic implications of the GPIbα–VWF interaction. Washed platelets were exposed to pathologically high shear stress in the presence of VWF, using a cone–plate viscometer. As occurred with the ristocetin-induced GPIbα–VWF interaction, the shear-induced VWF–platelet interaction also elicited PS exposure (Fig. 4A,B) and upregulation of the proapoptotic proteins Bax and Bak (Fig. 4C). To further characterize the specific effect of the GPIbα–VWF interaction on platelet apoptotic events under conditions of high shear stress, the monoclonal antibody AK2, which specifically blocks the interaction of GPIbα with VWF, was preincubated with platelets, or platelets were sheared without VWF. The presence of AK2, but not of the control IgG (not shown), or the absence of VWF, completely abolished shear-induced platelet apoptotic events.

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Figure 4.  The shear-induced glycoprotein (GP)Ibα–VWF interaction elicits phosphatidylserine (PS) exposure and upregulation of Bax and Bak proteins in platelets. (A) Washed platelets were sheared by a cone-and-plane viscometer at 7000 s−1 for 90 s in the presence or absence of VWF or anti-GPIbα antibody AK2, and then analyzed for PS exposure by flow cytometry. Representative flow cytometric histograms are shown. (B) Means ± standard deviations of the percentage of PS-positive platelets from three independent experiments are shown. **P < 0.01. (C) Sheared platelets in the presence or absence of VWF or AK2 were lysed, separated, and probed for Bax, Bak, Bcl-2, and Bcl-XL. The figure is representative of three independent experiments.

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To further confirm shear-induced platelet apoptotic events, gelsolin cleavage and ΔΨm depolarization were examined in platelets treated with high shear stress. Similar to the ristocetin-induced GPIbα–VWF interaction, gelsolin cleavage and ΔΨm depolarization occurred in platelets under flow conditions (Fig. 5). Furthermore, AK2 or VWF absence completely abolished shear-induced gelsolin cleavage and ΔΨm depolarization in platelets. The extents of apoptotic responses were compared between platelets treated with VWF/ristocetin and those treated with VWF/shear stress. We found that the extent of apoptotic responses was lower in VWF/ristocetin-treated platelets (data not shown). Taken together, these data demonstrate that the shear-induced GPIbα–VWF interaction elicits platelet apoptosis.

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Figure 5.  The shear-induced glycoprotein Ibα–von Willebrand factor (VWF) interaction elicits gelsolin cleavage and mitochondrial inner transmembrane potential (ΔΨm) depolarization in platelets. (A) Washed platelets were exposed to 7000 s−1 shear stress for 90 s in the presence or absence of VWF or AK2. Platelets were lysed, separated, and probed for gelsolin. (B) Sheared platelets were analyzed for ΔΨm depolarization by flow cytometry, using tetramethylrhodamine ethyl ester. Representative flow cytometric histograms are shown. M2, platelet mitochondrial matrix; M1, depolarized platelets. (C) Depolarization was quantified as the geometric (Geo) mean channel fluorescence (left), and as the percentage of depolarized cells (right). Means ± standard deviations of three experiments are presented. **P < 0.01.

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The GPIbα–VWF interaction induces apoptotic events in CHO cells stably expressing wild-type GPIb–IX

To definitively establish that the apoptotic events were specifically elicited by the GPIbα–VWF interaction, to exclude the possible involvement of other platelet receptors in the apoptotic process, and to investigate whether the GPIbα–VWF interaction induces apoptosis in eukaryotic cells, apoptotic events were induced by the GPIbα–VWF interaction in CHO cells stably expressing wild-type GPIb–IX (1b9). Similar to what was observed in platelets, incubation of 1b9 cells with VWF/ristocetin but not ristocetin alone caused PS exposure, cleavage of gelsolin, and ΔΨm depolarization (Fig. 6). These data provide evidence indicating that the GPIbα–VWF interaction is specific and strong enough to induce cell apoptosis.

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Figure 6.  The glycoprotein (GP)Ibα–von Willebrand factor (VWF) interaction induces apoptotic markers in Chinese hamster ovary (CHO) cells expressing wild-type GPIb–IX. CHO cells expressing wild-type GPIb–IX (1b9) were incubated with or without (control) VWF (35 μg mL−1) in the presence of ristocetin (Ris) (1.0 mg mL−1) at 22 °C for 30 min. Phosphatidylserine (PS) exposure, mitochondrial inner transmembrane potential (ΔΨm) depolarization and gelsolin cleavage were detected by flow cytometry or western blotting as described for platelets. (A) Representative flow cytometric dot plots of three experiments are shown for PS exposure. Means ± standard deviations from three independent experiments are presented. **P < 0.01. (B) Western blotting analysis of gelsolin proteolysis in 1b9 cells stimulated with VWF/ristocetin or ristocetin (control). Data representative of three separate experiments are shown. (C) Representative flow cytometric histograms of three separate experiments are shown for ΔΨm reduction. Means ± standard deviations from three experiments are presented. **P < 0.01. (D) 1b9 cells were incubated with SZ2 and then with fluorescein isothiocyanate-labeled goat anti-mouse IgG to detect GPIb–IX surface expression.

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The cytoplasmic 14-3-3ζ-binding site of GPIbα is essential for GPIbα–VWF interaction-induced cell apoptosis

14-3-3ζ, which associates with the cytoplasmic tail of GPIbα, is an intracellular signaling molecule that is present in all eukaryotic cells [31]. The cytoplasmic tail of GPIbα is involved in the regulation of megakaryocyte proliferation and ploidy [10]. G1 arrest induced by recombinant GpIbα in heterologous cells requires signaling through the 14-3-3ζ-binding domain of GPIbα [9]. In particular, 14-3-3 proteins were reported to be involved in the regulation of apoptosis [29]. Therefore, the role of 14-3-3ζ in GPIbα–VWF interaction-induced cell apoptosis was investigated using cell lines expressing GPIb–IX. In addition to the wild-type 1b9 cells, two GPIbα mutant cells were used in the investigation. In the Δ551 mutant, the C-terminal residues 551–610 of GPIbα are eliminated, so that it lacks the binding sites for both filamin A and 14-3-3ζ [22]. In the S609A mutant, Ser609 is replaced with alanine, eliminating the 14-3-3ζ-binding site in the cytoplasmic tail [6]. The cells were allowed to bind soluble VWF in the presence of ristocetin, and were then analyzed for GPIbα–VWF interaction-induced apoptotic markers. As shown in Fig. 7, PS exposure, gelsolin cleavage and ΔΨm reduction were observed in 1b9 cells, but were significantly reduced in both Δ551 and S609A cells. Thus, these data indicate that elimination of the cytoplasmic 14-3-3ζ-binding site of GPIbα abolishes GPIbα–VWF interaction-induced cell apoptosis.

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Figure 7.  The glycoprotein (GP)Ibα–von Willebrand factor (VWF) interaction-induced apoptotic events are diminished in Δ551 and S609A cells. 1b9, Δ551 (lacking C-terminal residues 551–610) or S609A (bearing a Ser609 to alanine mutation of GPIbα) cells were incubated with or without VWF in the presence of ristocetin at 22 °C for 30 min. Phosphatidylserine (PS) exposure (A), mitochondrial inner transmembrane potential (ΔΨm) depolarization (B) and gelsolin cleavage (C) were detected by flow cytometry or western blotting as described for platelets. (A, B) Quantitative data for PS exposure (A) and ΔΨm depolarization (B) are shown as means ± standard deviations from three separate experiments. **P < 0.01. (C) Representative western blotting analysis of gelsolin proteolysis of three separate experiments is shown. (D) The cells were assessed for GPIb–IX surface levels by SZ2 binding.

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

The GPIbα–VWF interaction initiates platelet adhesion and thrombus formation. The results presented here demonstrate that the GPIbα–VWF interaction also induces apoptotic events in human platelets, including PS externalization, ΔΨm reduction, gelsolin cleavage, and upregulation of Bax and Bak. Elimination of the 14-3-3ζ-binding site in the cytoplasmic domain of GPIbα abolishes the GPIbα–VWF interaction-induced platelet apoptosis.

Apoptosis occurs widely in platelets stimulated with physiologic or chemical compounds or subjected to shear stress, or in stored platelets [12–16]. However, except for the well-known function of initiating platelet adhesion, the role of the GPIbα–VWF interaction in platelet apoptosis is still unclear. In order to explore the specific effects of the GPIbα–VWF interaction on platelet apoptosis, washed platelets were employed for the apoptotic investigations. First, the results indicate that the ristocetin-induced GPIbα–VWF interaction elicits PS exposure. PS exposure is present in both platelet activation and apoptosis processes, so the following investigations were performed to find direct evidence for apoptosis. There are two distinct pathways, the tumor necrosis factor family death receptor pathway, and the mitochondrial pathway [17], leading to apoptosis in nucleated cells. Up to now, most of the platelet apoptotic events have been considered to arise from the mitochondrial pathway, regulated by proapoptotic (Bax and Bak) and antiapoptotic (Bcl-2 and Bcl-XL) proteins of the Bcl-2 family. Therefore, the expression levels of Bcl-2 family proteins were examined, and we found that expressions of Bax and Bak were significantly enhanced in platelets stimulated with VWF/ristocetin, suggesting that the GPIbα–VWF interaction triggers signaling cascades leading to platelet apoptosis. Next, two typical apoptotic events, caspase-3 activation and depolarization of ΔΨm, were examined, and the results apparently indicate that apoptotic events were elicited after VWF/ristocetin stimulation. In order to investigate the relationship between platelet activation and apoptosis, the GPIbα–VWF interaction-induced apoptotic events were examined in the presence of the platelet activation inhibitors PGE1 and apyrase, and the data indicated that platelet apoptosis occurred independently of platelet activation. In addition, no difference in GPIbα–VWF interaction induced-apoptotic events was observed between platelets treated with and without RGDS, excluding the possible role of αIIbβ3 in the current observations. Furthermore, apoptotic events were induced in CHO cells expressing GPIb–IX in the presence of VWF and ristocetin, indicating that the GPIbα–VWF interaction not only specifically induces apoptotic events in platelets, but could also elicit apoptotic events in eukaryotic cell.

Interestingly, however, the extent of apoptotic events was lower in CHO cells than in platelets. It has been reported previously that almost all wild-type GPIb–IX molecules expressed on CHO cells are in a resting form, so the level of VWF binding to 1b9 cell is low [32]. In contrast, more than 30% of GPIb–IX molecules expressed on the platelet surface are in an active form in vitro, so almost all platelets are VWF-positive in the VWF-binding experiments [32]. Therefore, the lower levels of apoptotic events in CHO cells appear to result from the lower extent of the GPIbα–VWF interaction.

Pathologic shear stresses have been reported to induce not only platelet activation but also apoptosis [21], whereas the mechanism of shear-induced platelet apoptosis remains unclear. To further investigate whether the GPIbα–VWF interaction causes platelet apoptosis under conditions of high shear flow, apoptotic events were examined in platelets subjected to shear stress in the presence of VWF. Consistent with the results obtained with ristocetin/VWF, apoptotic events occurred in platelets in response to the shear-induced GPIbα–VWF interaction, indicating that the GPIbα–VWF interaction could induce platelet apoptosis under pathophysiologic flow conditions. In addition, the GPIbα–VWF interaction-induced apoptotic events occurred independently of platelet activation, further suggesting the physiologic importance of the GPIbα–VWF interaction in the normal circulation.

14-3-3 has been reported to inhibit apoptosis by regulating mitogen-activated protein kinase activation or inactivating the proapoptotic protein BAD [31]. The cytoplasmic 14-3-3ζ-binding site of GPIbα is involved in the regulation of megakaryocyte proliferation and ploidy [10]. In particular, Feng et al. [9] reported that GPIbα expressed in CHO cells caused growth arrest in the G1 phase of the cell cycle, and that this effect was reversed by the GPIbα mutation eliminating the cytoplasmic 14-3-3ζ-binding site; however, the mechanism is still unclear. In the current investigations, GPIbα–VWF interaction-induced apoptotic events were diminished in Δ551 cells, indicating that the intracellular signal elicited by the GPIbα–VWF interaction is essential for apoptosis. As known, filamin A and 14-3-3ζ both interact with the cytoplasmic domain of GPIbα. Therefore, the S609A mutant, which has all of the capabilities of the cytoplasmic domain of GPIbα except for 14-3-3ζ binding [6], was selected to further specify the role of 14-3-3ζ in the apoptotic process. The results indicate that elimination of the cytoplasmic 14-3-3ζ-binding site of GPIbα abolishes the GPIbα–VWF interaction-induced cell apoptosis. Taken together, these data indicate that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα is essential for GPIbα–VWF interaction-induced cell apoptosis.

In summary, the data demonstrate that the GPIbα–VWF interaction induces apoptotic events in human platelets, and that the association of 14-3-3ζ with the cytoplasmic domain of GPIbα is essential for apoptosis signaling. The finding may represent a novel mechanism for platelet clearance and dysfunction in vivo or in vitro, and also for some thrombocytopenic diseases such as thrombotic thrombocytopenic purpura. The reagents that block the 14-3-3ζ–GPIbα interaction might have the potential to be developed into new classes of antiapoptosis reagents for platelet storage or antithrombocytopenia agents.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

We thank X. Du for providing reagents, and G. Liu for drawing figures. This work was supported by grants from National Natural Science Foundation of China (NSFC 30770795), Program for New Century Excellent Talents in University (NCET-06-0167), and A Foundation for the Author of National Excellent Doctoral Dissertation of China (FANEDD 200560). S. Li is a recipient of the Innovation Foundation of BUAA for PhD Graduates.

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  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure of Conflict of Interests
  9. References
  10. Supporting Information

Figure S1. Interaction of washed platelets with von Willebrand factor (VWF) induced by ristocetin results in platelet activation and platelet aggregation under stirring conditions.

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Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.