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From the W.K. Warren Medical Research Institute, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City.
Correspondence to George L. Dale, PhD, Department of Medicine; BSEB-330, OU Health Sciences Center, 941 Stanton Young Blvd, Oklahoma City, OK 73104. E-mail george-dale@ouhsc.edu
Abstract
Objective— Coated-platelets are a subset of cells observed during costimulation of platelets with collagen and thrombin. Important characteristics of coated-platelets include retention of multiple -granule proteins and expression of phosphatidylserine on the cell surface. The mitochondrial permeability transition pore (MPTP) is a key step in apoptosis and is suggested to be involved in some forms of platelet activation. The objective of this study was to examine the role of MPTP in the synthesis of coated-platelets.
Methods and Results— Flow cytometric analysis of coated-platelet production was used to examine the impact of pharmacological effectors of MPTP formation. Cyclosporin A, coenzyme Q, and bongkrekic acid all inhibit MPTP formation as well as production of coated-platelets. Phenylarsine oxide and diamide, both potentiators of MPTP formation, stimulate coated-platelet synthesis. Atractyloside, another inducer of MPTP formation, does not affect the percentage of coated-platelets synthesized; however, it does increase the level of phosphatidylserine exposed on the surface of coated-platelets.
Conclusions— These findings indicate that MPTP formation is an integral event in the synthesis of coated-platelets. Although the exact function of the MPTP remains to be determined, these data support a growing body of evidence that apoptosis-associated events are vital components of the platelet activation process.
Formation of coated-platelets involves a complex set of activation events initiated by dual agonist activation. The mitochondrial permeability transition pore (MPTP) is a key intermediate in apoptosis and has been suggested to impact platelet activation. This report demonstrates that MPTP formation is essential to production of coated-platelets.
Key Words: coated-platelet ? mitochondrial permeability transition pore ? coenzyme Q ? phosphatidylserine ? cyclosporin A ? phenylarsine oxide ? diamide
Introduction
Coated-platelets are a subset of platelets resulting from dual agonist activation with collagen and thrombin.1–4 These unusual cells, formerly known as COAT platelets (collagen and thrombin-activated platelets, see below), are characterized by high-affinity retention on the platelet surface of several -granule proteins,2 expression of surface phosphatidylserine (PS),1 and high prothrombinase activity.1 -Granule proteins bound to coated-platelets are derivatized with serotonin,2 and binding sites on the cell surface for serotonin-derivatized proteins are provided by fibrinogen and thrombospondin.3 The putative structure of the coated-platelet surface includes an intertwined network of -granule proteins, each retained on the cell surface through multivalent interactions with membrane receptors and neighboring proteins.4 Although considerable progress has been made in molecular characterization of coated-platelets, their physiological significance remains largely speculative.
Agonist(s) other than collagen plus thrombin can also produce a subpopulation of platelets with many, if not all, the characteristics of coated-platelets; included among these agonists are thrombin plus Fc receptor engagement,5 high-dose thrombin,6 and immobilized collagen.7,8 As mentioned, coated-platelets were referred to previously as COAT platelets, an acronym for the collagen and thrombin agonists used in their formation.1 However, the finding that additional agonist(s) can also produce a similar subpopulation of cells indicates this nomenclature is too limited. With the newly proposed designation, "coated" refers to the coating of adhesive and procoagulant proteins on the cell surface, which serves as the hallmark of these platelets.4 This terminology focuses on the final cell product rather than the agonists used in its formation.
Several reports have identified apoptotic processes involved in the synthesis,9,10 activation,11–13 and in vitro storage14,15 of platelets. For example, during platelet stimulation with tumor necrosis factor, activation of caspase-3 and caspase-9 has been observed,12 suggesting that these key components of apoptosis may have a function in platelet physiology. Additional studies have focused on apoptotic parameters associated with prolonged in vitro storage of platelets.14,15 Among these storage-induced defects, changes in mitochondrial integrity have been observed.15 Specifically, mitochondrial integrity, as measured by organelle potential (m), is lost during storage, and this loss of m is similar to events associated with the apoptotic cascade, which results in mitochondrial swelling and cytochrome C loss.16 The mechanism for loss of m during apoptosis involves the mitochondrial permeability transition pore (MPTP), a 2.5-nm pore composed of several components, including adenine nucleotide translocase, voltage-dependent ion channel, peripheral benzodiazepine receptor, and other proteins.17,18 Although the precise mechanism for production of MPTP, as well as its exact composition, is still the subject of considerable debate, it is clear that its generation is critical to the apoptotic process.
This study resulted from the unexpected observation that cyclosporin A, an inhibitor of MPTP formation, also inhibited production of coated-platelets. As a result, a systematic examination of MPTP formation and its effect on coated-platelet synthesis was undertaken.
Materials and Methods
Reagents
Human fibrinogen, BSA, bovine thrombin, goat-anti-human fibrinogen, Tween-20, N-hydroxy succinimidyl biotin, Sepharose CL-2B, cyclosporin A, coenzyme Q0, atractyloside, phenylarsine oxide (PAO), JC-1, and diamide (1,1'azobis[N,N-dimethylformamide]) were purchased from Sigma. Bongkrekic acid was from Calbiochem. Calcein-acetoxy methyl ester (calcein-AM) and MitoTracker green were obtained from Molecular Probes. Additional reagents have been described previously.1,2 Biotinylated fibrinogen (B-Fbg) was synthesized by reacting 125 μg/mL N-hydroxysuccinimidyl biotin with 500 μg/mL human fibrinogen in 75 mmol/L NaCl, 25 mmol/L borate, pH 8.5, for 45 minutes at 37°C. B-Fbg was dialyzed at room temperature (RT) against 25 mmol/L Na3 citrate, 150 mmol/L NaCl, filtered through a sterile 0.2-μm filter, and stored at 4°C.
Buffers
Acid citrate dextrose was composed of 38.1 mmol/L citric acid, 74.8 mmol/L Na3 citrate, and 136 mmol/L glucose. Buffered saline-glucose-citrate (BSGC) was composed of 129 mmol/L NaCl, 13.6 mmol/L Na3 citrate, 11.1 mmol/L glucose, 1.6 mmol/L KH2PO4, and 8.6 mmol/L NaH2PO4, pH 7.3. PBS was composed of 150 mmol/L NaCl, and 10 mmol/L NaH2PO4, pH 7.4. And HEPES/saline was composed of 150 mmol/L NaCl and 10 mmol/L HEPES, pH 7.5.
Platelet Isolation and Activation
Informed consent was obtained in accordance with local institution review board guidelines. Platelets were purified by gel filtration as detailed previously1 and normalized to a concentration of 4x104 platelets/μL in BSGC. All platelet activation reactions were in 100-μL total volume containing 1 mg/mL BSA, 2 mmol/L CaCl2, 1 mmol/L MgCl2, 140 mmol/L NaCl, and 10 mmol/L HEPES, pH 7.5 (reaction mix), plus 4x105 platelets and agonist (500 ng/mL convulxin and 0.5 U/mL thrombin) for 10 minutes at 37°C. Single agonist activations were performed similarly. For the standard coated-platelet assay, 5 μg/mL B-Fbg was included in the assay mixture, and the reaction was terminated with 200 μL 1.5% (wt/vol) formalin in HEPES/saline. After fixation for 20 minutes at RT, 3.5 mL of PBS containing 1 mg/mL BSA (PBS/BSA) was added, and the sample was centrifuged at 1500g for 15 minutes. The pellet was resuspended in 200 μL PBS/BSA with 5 μg/mL phycoerythrin-streptavidin. After 25 minutes at RT, platelets were washed and resuspended for flow cytometry. In some experiments, fluorescein isothiocyanate (FITC)–antifibrinogen was used instead of B-Fbg to detect coated-platelets; the assay with FITC-antifibrinogen has been described.2
For calcein-loss assay, calcein-AM was dissolved at 2 mmol/L in dimethyl sulfoxide (DMSO), and aliquots were frozen at –80°C. Platelets were incubated with 2 μmol/L calcein-AM for 10 minutes at RT before dilution to 4x106/ mL with assay mix and activation as detailed above. After 10 minutes at 37°C, the sample was diluted with HEPES/saline and left at RT for 20 minutes before assay; the sample was not formalin fixed.
For JC-1, the dye was stored at 1 mg/mL in DMSO. An initial 1:10 dilution was made in DMSO. Platelets were initially activated with agonist(s) for 10 minutes at 37°C and then incubated with 2 μg/mL JC-1 (final) for 20 minutes at RT. Platelets were analyzed immediately by flow cytometry, without fixation or washing, using parameters reported previously.15 For experiments with MitoTracker green, platelets were preincubated for 10 minutes at RT with 200 nmol/L dye and then activated as detailed for JC-1. PS exposure was quantitated with FITC-annexin binding.1 P-Selectin analysis and FITC-PAC-1 binding have been described previously.2
Effector Studies
For studies with cyclosporin A, coenzyme Q and atractyloside, platelets were preincubated with the effector for 5 minutes at RT before activation with convulxin plus thrombin. For experiments with PAO and diamide, these effectors were added to platelets simultaneously with convulxin plus thrombin. For experiments with bongkrekic acid, platelets at 4x107/mL in BSGC were incubated with 10 to 50 μmol/L bongkrekic acid (10 mmol/L stock in DMSO) for 20 minutes at RT; platelets were then diluted 1:10 into assay mix and activated as before. Whenever relevant, a vehicle control was included in each assay set.
Results
Coated Platelets Lose Mitochondrial Potential
JC-1 is a fluorescent dye sensitive to mitochondria potential (m), and quiescent platelets labeled with JC-1 demonstrate an intense FL2 fluorescence indicative of an intact mitochondrial potential (Figure 1A). Reaction of platelets with calcium ionophore A23187 (Figure 1B) results in platelet activation and a near total collapse of m, as indicated by the decrease in FL2 and an increase in FL1 (region R1), as reported previously.15 In contrast, activation of platelets with convulxin alone (Figure 1C) does not significantly impact m, whereas activation with thrombin alone results in a small subpopulation of platelets with decreased mitochondrial potential. However, activation with thrombin plus convulxin results in 2 populations of platelets (Figure 1E), including a subpopulation of cells (42.4% of total) with altered JC-1 fluorescence (region R1). Similar to the results in Figure 1B, FL2 decreased and FL1 increased for the platelets in region R1 of Figure 1E. Two-color flow cytometry indicates that the cells in region R1 of Figure 1E with low m also bound fibrinogen (data not shown), demonstrating they are coated-platelets.2 When the experiments shown in Figure 1 were repeated with MitoTracker Green, a mitochondrial dye that is not sensitive to m, no changes in fluorescence occurred and no second population of platelets was observed during agonist activation (data not shown).
Figure 1. Loss of mitochondrial potential by coated-platelets. Gel-filtered platelets were treated with a variety of agonists and then incubated with 2 μg/mL JC-1. Control (Con) platelets are shown in A; the high FL2 fluorescence indicates an intact mitochondrial m. Activation of platelets with 1 μmol/L ionophore A23187 (B) resulted in a near-total collapse of the m, as indicated by the decrease in FL2 and increase in FL1; 98.3% of platelets in region R1. Activation with convulxin (Cvx) alone (C) did not significantly affect JC-1 fluorescence, whereas activation with thrombin (Thr) alone (D) resulted in 8% of platelets with collapsed m (region R1). However, platelet activation with convulxin plus thrombin (C/T; E) generated a large population of cells (42.4%) with decreased FL2 and increased FL1. For F, 4 μmol/L cyclosporin A (Cys A) was included during activation with convulxin plus thrombin; no decrease in FL2 was observed.
Loss of mitochondrial m is commonly observed during apoptosis, and under those circumstances, JC-1 fluorescence changes reflect depolarization of mitochondria by MPTP.15 One standard inhibitor of MPTP formation is cyclosporin A, which blocks a peptidyl prolyl cis/trans isomerase required for MPTP formation.18 Therefore, we tested the ability of cyclosporin A to attenuate the loss of m observed during coated-platelet formation. Figure 1F demonstrates that 4 μmol/L cyclosporin A prevents mitochondrial depolarization during platelet stimulation with thrombin plus convulxin. As a result of this observation, the following experiments were designed to define the impact of inhibitors and activators of MPTP formation during coated-platelet synthesis.
Inhibition of Coated-Platelet Formation by MPTP Inhibitors
The results in Figure 1F suggest that coated-platelet synthesis might also be impacted by inhibitors of MPTP formation. To test this possibility, 3 separate markers of coated-platelets were used: fibrinogen retention,2 exposure of PS,1 and release of calcein.19 This third assay results from the observation that coated-platelets are permeable to calcein and several other low-molecular weight fluorescent probes that are normally retained within platelets.19 The mechanism by which coated-platelets release calcein is unclear, but it is the focus of ongoing investigations. Figure 2 indicates the impact of 1 μmol/L cyclosporin A on these markers of coated-platelet formation, and in each instance, coated-platelet production was inhibited. The concentration dependence of cyclosporin A inhibition of Fbg retention on coated-platelets is shown in Figure 3A; the IC50 for cyclosporin was 0.31 μmol/L.
Figure 2. Inhibition of coated-platelet synthesis by cyclosporin A (Cys-A). Three separate probes of coated-platelet formation were used: fibrinogen (Fbg) retention2 (top row; A through C), calcein loss19 (middle row; D through F), and annexin binding1 (bottom row; G through I). For the center column, the impact of platelet activation with convulxin plus thrombin (C/T) is demonstrated for Fbg retention (B), calcein loss (E), and annexin binding (H). In each case, the designated region indicates coated-platelets. The right column depicts activation of platelets with convulxin plus thrombin in the presence of 1 μmol/L cyclosporin A. Note that cyclosporin A inhibits retention of Fbg (C), loss of calcein (F), and binding of annexin (I).
Figure 3. Dose-dependent inhibition of coated-platelets (Plts) by cyclosporin A (Cys A), coenzyme Q0, and bongkrekic acid. Coated-platelet synthesis was monitored by Fbg retention after stimulation with convulxin plus thrombin (Thr) in the presence of graded doses of cyclosporin A (A), coenzyme Q0 (B) or bongkrekic acid (C). Data are expressed as percent of control coated-platelet level (mean ±1 SE; n=4 for A; n=5 for B and C). Also in C, the impact of bongkrekic acid on the subpopulation of cells with mitochondrial depolarization (hatched bars) was monitored simultaneously with inhibition of coated-platelet formation (filled bars). In D, the impact of 2 μmol/L cyclosporin A and 5 μmol/L coenzyme Q0 on P-selectin expression by thrombin-stimulated platelets is demonstrated (filled bars). Data are expressed as fluorescence of bound FITC-S12 (mean±1 SD; n=3). Also in D, the impact of 2 μmol/L cyclosporin A and 5 μmol/L coenzyme Q0 on glycoprotein IIb/IIIa activation after thrombin stimulation of platelets was tested (hatched bars). Data are expressed as fluorescence of bound FITC-PAC-120 (mean±1 SD; n=3).
Coenzyme Q0 is another inhibitor of MPTP formation,17 and it is also able to inhibit coated-platelet synthesis regardless of whether Fbg retention, PS exposure, or calcein loss is measured (data not shown). The dose-dependent inhibition by coenzyme Q0 of Fbg retention on coated-platelets is shown in Figure 3B; the IC50 for coenzyme Q0 was 1.3 μmol/L.
Bongkrekic acid is considered one of the most specific inhibitors of MPTP formation,17 and its impact on coated-platelet production in shown in Figure 3C. There is a dose-dependent decrease in coated-platelet production with increasing levels of bongkrekic acid. In addition, Figure 3C also demonstrates the impact of bongkrekic acid on m loss on convulxin plus thrombin stimulation (as shown in Figure 1E). Generation of a subpopulation of platelets with m loss was inhibited to the same extent by bongkrekic acid as was the production of coated-platelets (Figure 3C).
Cyclosporin A and coenzyme Q0 were also tested for their impact on more classical markers of platelet activation, P-selectin expression, and glycoprotein IIb/IIIa activation. As shown in Figure 3D, neither inhibitor had a significant impact on P-selectin expression by thrombin-stimulated platelets. Cyclosporin A also had no impact on thrombin-induced activation of glycoprotein IIb/IIIa, as measured by PAC-1 binding20 (Figure 3D). Coenzyme Q0 at 5 μmol/L inhibited PAC-1 binding significantly (34% decrease; Figure 3D) but less than it attenuated coated-platelet formation (76% decrease; Figure 3B).
Potentiation of Coated-Platelet Formation by MPTP Activators
PAO, a known MPTP activator,17 was tested for its effect on coated-platelet formation. PAO stimulated formation of coated-platelets by convulxin plus thrombin (Figure 4A) with 50 μmol/L PAO, resulting in an 80% increase in the percentage of coated-platelets formed. This increase was independent of which coated-platelet marker was used. In these experiments, PAO was added simultaneously with thrombin and convulxin. However, additional studies found that PAO could be added up to 60 seconds after platelet activation with thrombin plus convulxin, and a similar degree of PAO-dependent stimulation was still observed (data not shown).
Figure 4. PAO and diamide potentiation of coated-platelet formation. Platelets (Plts) were activated with convulxin plus thrombin in the presence of graded concentrations of PAO (A) or diamide (B). Fbg retention was used to identify coated-platelets. Data are expressed as percentage of coated-platelet level formed without PAO or diamide (mean±1 SE; n=5 for A; n=4 for B).
Diamide is also known to activate MPTP,17 and its impact on coated-platelet formation by thrombin plus convulxin (Figure 4B) was very similar to that observed with PAO. However, it should be noted that neither diamide nor PAO alone is capable of producing coated-platelets (data not shown).
Atractyloside is another MPTP activator,17 but 500 μmol/L atractyloside had no effect on the percentage of coated-platelets produced by thrombin plus convulxin, as measured by Fbg retention, PS exposure, or calcein release (data not shown). However, atractyloside did increase the level of FITC-annexin binding to coated-platelets generated by thrombin plus convulxin. Figure 5 demonstrates the dose response for atractyloside, potentiating the apparent level of PS expressed on coated-platelets without affecting the actual percentage of annexin-positive cells.
Figure 5. Atractyloside potentiates annexin binding to coated-platelets. Platelets were activated with thrombin plus convulxin in the presence of increasing concentrations of atractyloside. Binding of FITC-annexin was monitored, and data are expressed as bound fluorescence (mean±1 SD; n=3). The percentage of annexin-positive platelets did not change with atractyloside, although the level of bound FITC-annexin did increase.
Discussion
In this report, we observed that platelet stimulation with convulxin plus thrombin resulted in loss of mitochondrial potential in a subpopulation of cells corresponding to coated-platelets.2 In addition, cyclosporin A not only prevented loss of mitochondrial potential on platelet activation, but it also inhibited synthesis of coated-platelets. These observations lead to a systematic study of MPTP activators and inhibitors and their impact on coated-platelet formation. The data summarized in the Table indicate that 6 MPTP inhibitors/activators had an effect on at least 2 separate markers of coated-platelet production (PS exposure and calcein loss), and 5 of 6 inhibitors/activators affected all 3 markers of coated-platelet production examined. In each case, the consequence of an effector on coated-platelet production was the same as on MPTP formation (ie, all inhibitors of MPTP formation inhibited coated-platelets, and all activators of MPTP activated coated-platelet production). We are acutely aware that any inhibitor or activator can have additional effects unknown or unintended, especially when dealing with an intact cell; as a result, extreme caution is necessary in interpreting such pharmacological manipulations. However, the cumulative effect of the data in the Table strongly supports an essential role for MPTP formation in coated-platelet synthesis considering that all 6 effectors of MPTP had similar effects on coated-platelet markers.
Summary of the Effect of MPTP Inhibitors/Activators on Coated-Platelet Formation
The function of MPTP in coated-platelet formation is unclear. Before these observations, MPTP has been examined exclusively for its role in apoptotic and necrotic pathways. In apoptosis, MPTP transiently opens, resulting in mitochondrial swelling and subsequent leakage of mitochondrial contents (eg, cytochrome C) into the cytoplasm18; these released mitochondrial components are critical for propagation of the apoptotic signal (ie, caspase-3 activation). In necrosis, MPTP also opens, although it remains open, thereby preventing mitochondria from synthesizing ATP. In the absence of ATP, cell death takes the more chaotic path of necrosis rather than the orderly process of apoptosis.17 Although apoptotic-associated events have been reported to be involved in platelet synthesis and activation, these previous studies have focused primarily on caspase activation.9,12 However, with platelet storage in vitro, loss of mitochondrial potential has been reported, although its contribution to the platelet storage defect is unclear.14,15
The activation pathways and biosynthetic mechanisms responsible for synthesis of coated-platelets are not yet fully delineated.4 However, it is clear that production of coated-platelets requires a number of distinct activation events to produce the proposed end product with its strongly bound -granule proteins and exposed PS.4 The data presented here indicate that MPTP formation is a critical event in synthesis of these unusual cells, and this conclusion leads to an interesting observation concerning coated-platelet formation and the loss of m catalyzed by MPTP. As noted by Helestrap et al,17 there is an "all or nothing" aspect to mitochondrial swelling and MPTP formation; there are no intermediate steps or partially depolarized cells observed. A similar situation exists with coated-platelets. Coated platelets have high levels of bound adhesive proteins,4 whereas the noncoated-platelet population has very low levels of bound proteins; there are no intermediate species. Perhaps MPTP formation during platelet activation is the critical step that commits a given cell to becoming a coated-platelet. Regardless of whether MPTP formation is the determining step in coated-platelet formation, these findings indicate the essential role that MPTP can play in platelet activation, and future work will focus on the exact consequences of this pore opening.
Acknowledgments
This work was supported by the National Institutes of Health (HL68129) and the Warren Medical Research Institute.
Received September 27, 2004; accepted November 24, 2004.
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