- Top of page
- Materials and methods
- Author contributions
- Conflicts of interest
- Supporting Information
Anucleate platelets perform two fundamental processes, activation and apoptosis. We elaborated an approach for selective and concurrent stimulation of platelet apoptosis and/or activation, processes important in haemostasis and platelet clearance. Human platelets were treated with BH3 mimetic ABT-737, thrombin, calcium ionophore A23187 and matched diluents. Apoptosis was determined as mitochondrial inner membrane potential (ΔΨm) depolarization and activation as P-selectin exposure. At optimal treatment conditions (90–180 min, 37°C), ABT-737 predominantly induced apoptosis, when 77–81% platelets undergo only ΔΨm depolarization. The ABT-737 impact on ΔΨm depolarization is strongly time- and temperature-dependent, and much higher at 37°C than at room temperature. In contrast, when platelets were treated with thrombin for 15–90 min at either temperature, activation-only was predominantly (79–85%) induced, whereas A23187 triggers both apoptosis and activation (73–81%) when platelets were treated for 15–60 min at 37°C or 15–90 min at room temperature. These data demonstrate that, depending on the triggering stimulus, platelets predominantly undergo ΔΨm depolarization-only, P-selectin exposure-only, or both responses, indicating that platelet apoptosis and activation are different phenomena driven by different mechanisms. The described model provides a basis for studying differential pharmacological manipulation of platelet apoptosis and activation and their role in haemostasis, thrombosis and platelet clearance.
Platelet activation is an essential reaction contributing to multiple platelet functions (Smyth et al, 2009; Semple et al, 2011). Over the past fifteen years, it has been also recognized that, as with nucleated cells (Kroemer & Reed, 2000; Ashkenazi, 2002; Danial & Korsmeyer, 2004; Kroemer et al, 2007; Hotchkiss et al, 2009), anucleate platelets are able to undergo programmed cell death, apoptosis, in response to diverse stimuli (Vanags et al, 1997; Brown et al, 2000; Li et al, 2000; Leytin & Freedman, 2003; Gyulkhandanyan et al, 2012; Leytin, 2012). These fundamental platelet responses, activation and apoptosis, may play a key role in the major platelet-dependent processes, haemostasis and thrombosis, and in regulation of the platelet lifespan. However, the absence of a valid model for selective stimulation of platelet apoptosis and activation, or both responses, is a serious limitation for studying the role of apoptosis and activation in platelet clearance and haemostatic function.
To develop an approach to examining platelet activation versus apoptosis, we treated human platelets with three agents, a pro-apoptotic BH3-only mimetic, the anti-cancer drug ABT-737 (Oltersdorf et al, 2005; Mason et al, 2007; Zhang et al, 2007; Dasgupta et al, 2010; Mutlu et al, 2012), the potent platelet agonist thrombin (Coughlin, 2005; Lundblad & White, 2005; Leytin et al, 2006a, 2007; Bahou, 2007; Lopez et al, 2008) and calcium ionophore A23187 (Leytin et al, 2004, 2006a, 2009; Rand et al, 2004; Mutlu et al, 2012), known inducers of apoptosis and/or activation in nucleated cells and platelets. We concurrently determined an apoptosis marker, depolarization of mitochondrial inner transmembrane potential (ΔΨm) (Kroemer & Reed, 2000; Leytin et al, 2004, 2006a, 2007, 2009; Rand et al, 2004; Gyulkhandanyan et al, 2012), and a marker of platelet activation, exposure of P-selectin (CD62) on the platelet surface (Stenberg et al, 1985; Michelson et al, 1991; Leytin et al, 2000a, 2007). As the result of this study, we elaborated a model for selective and simultaneous triggering of platelet apoptotic and/or activation responses. This model can serve as an important experimental tool for elucidating the role of platelet apoptosis and activation in platelet clearance and haemostatic function.
- Top of page
- Materials and methods
- Author contributions
- Conflicts of interest
- Supporting Information
Despite the lack of a nucleus, platelets are capable of performing multiple vital functions of nucleated cells and are involved in diverse physiological and pathological processes beyond haemostasis and thrombosis (Smyth et al, 2009; Semple et al, 2011). Furthermore, platelets have the essential machinery for executing programmed cell death, i.e., apoptosis (Vanags et al, 1997; Li et al, 2000; Leytin & Freedman, 2003; Mason et al, 2007; Kile, 2009; Leytin, 2012).
The role of platelet apoptosis, based on the determination of different apoptosis markers, has been studied in human diseases and animal models of diseases (reviewed by Gyulkhandanyan et al, 2012) including immune thrombocytopenia (Piguet & Vesin, 2002; Catani et al, 2006; Leytin et al, 2006b; Winkler et al, 2012), Bernard–Soulier syndrome (Rand et al, 2010), chronic uraemia (Bonomini et al, 2004), bacterial infection (Yeh et al, 2010), malaria (Piguet et al., 2002) and type 2 diabetes (Cohen et al, 2002). Interestingly, murine (Piguet & Vesin, 2002; Leytin et al, 2006b) and human immune thrombocytopenia (Winkler et al, 2012) and thrombocytopenia in a murine model of malaria (Piguet et al., 2002) were ameliorated by treatment with anti-apoptotic agents concurrent with inhibition of apoptosis in platelets. Platelet apoptosis was also studied in stored platelet concentrates (Li et al, 2000; Bertino et al, 2003; Leytin & Freedman, 2003; Perrotta et al, 2003; Leytin et al, 2008).
Animal models have been further used for tracking apoptotic platelets during ageing of platelets in the normal rabbit circulation (Rand et al, 2004) and in a canine model of suppressed thrombopoiesis (Pereira et al, 2002). Apoptotic status of circulating platelets in these studies was detected by ΔΨm depolarization and phosphatidylserine exposure on the cell surface (Pereira et al, 2002; Rand et al, 2004). Another approach is genetic mutations of Bcl-2 family proteins in mice: deletion of the anti-apoptotic Bcl-XL protein reduces the half-life of murine platelets and causes thrombocytopenia, whereas in pro-apoptotic Bak deficient mice platelet life-span is longer than normal (Mason et al, 2007). Furthermore, injection of the pro-apoptotic BH3 mimetic ABT-737 induces platelet clearance from the circulation in mice (Mason et al, 2007) and dogs (Zhang et al, 2007). Infusion of rabbit platelets treated in vitro with calcium ionophore A23187 into recipient rabbits resulted in platelet clearance (Rand et al, 2004); in contrast, in vitro treatment with thrombin did not induce clearance of rabbit platelets (Rand et al, 2004).
Although the role of platelet activation in haemostasis and thrombosis is well-documented, the role of platelet apoptosis in these vital processes and of platelet apoptosis versus activation in platelet clearance are still to be elucidated. To provide a methodological basis for investigation of these unresolved questions, the current study established a model for selective targeted stimulation in vitro of platelet apoptosis without affecting activation, platelet activation without affecting apoptosis, as well as both responses. Furthermore, we determined the conditions when neither apoptosis nor activation was affected.
In contrast to previous reports (Leytin et al, 2004, 2006a, 2007, 2009; Mason et al, 2007; Zhang et al, 2007; Dasgupta et al, 2010; Vogler et al, 2011; Mutlu et al, 2012), this study establishes a model that permits dissociation of platelet apoptosis and activation. For this, we elaborated a simple and effective method of preparation of three platelet populations that are highly enriched for apoptotic-only, activated-only, or both apoptotic and activated platelets. This novel methodology involves: (i) concurrent use of three platelet agonists in one study: BH3 mimetic ABT-737 for selective induction of platelet apoptosis, thrombin for selective induction of platelet activation and calcium ionophore A23187 for selective induction of apoptosis plus activation, as well as matched control diluent buffers for determining conditions when neither apoptosis nor activation are induced, (ii) concurrent flow cytometric analysis of the crucial manifestation of intrinsic pathway of platelet apoptosis, ΔΨm depolarization, together with the platelet activation event, P-selectin (CD62) exposure on the cell surface, (iii) detailed statistical analysis of platelet distribution between four platelet subpopulations (Apo+Act−, Apo−Act+, Apo+Act+ and Apo−Act−), and (iv) direct comparison of the sensitivity of platelet apoptotic and activation responses to time- and temperature-titration of platelets with ABT-737, thrombin and calcium ionophore.
This study design permitted us to define the optimal conditions for selective stimulation of the fundamental platelet processes of apoptosis and activation, separately or together. Maximal stimulation of apoptosis-only platelet response at the level of approximately 80% was achieved by ABT-737 treatment for 90 min at 37°C (Fig 2A, Table 2); shorter treatment at 37°C or treatment at RT for 15–180 min did not cause maximal stimulation (Table 1). However, if required by a particular study design, longer treatment with ABT-737 for 180 min at 37°C could be used, also yielding an Apo+Act− subpopulation at the level of about 80% (Fig 2A, Table 1). In contrast, predominant stimulation of an activation-only platelet response (by thrombin) and stimulation of both apoptosis and activation in the same cells (by A23187) was achieved by a shorter treatment for 15–30 min at 37°C or RT (Fig 2B, C); if required, a prolonged treatment up to 90 min at 37°C or RT could be used for thrombin, or up to 60 min at 37°C and up to 90 min at RT for A23187 (Tables 1 and 2). For obtaining the maximal level (88–97%) of non-apoptotic non-activated cells, platelets can be treated for 15–90 min at 37°C or RT with DMSO-free diluent buffer A or diluent buffer B containing DMSO (Table 2).
Table 2. Optimal conditions for the maximal stimulation of predominant apoptotic and/or activation platelet responses triggered by treatment of platelets with BH3 mimetic ABT-737, thrombin and calcium ionophore A23187 or the absence of apoptotic and activation responses in platelets treated with diluents A and B
|Predominant platelet subpopulation||Trigger||Optimal conditions||Cells in subpopulation (% of total)a|
| ||ABT-737||90 min, 37°C||76·7 ± 2·9|
| ||Thrombin|| || |
| ||A23187|| || |
| ||Diluent A/B||15–90 min, 37°C/RT||88·2–97·0|
Time- and temperature-dependency experiments indicate that the platelet activation response to thrombin is strong (84–85% CD62-positive cells) and fast (15 min at 37°C or RT) (Table 1). In contrast, the apoptotic response to thrombin required a much longer time (180 min at 37°C or RT), when not more than 20% cells undergo ΔΨm depolarization (Table 1). A dose-titration study of CD62- and ΔΨm-responses also demonstrated much higher sensitivity of platelet activation to thrombin compared to platelet apoptosis (Leytin et al, 2007).
Characterizing effects of an RT environment on platelet apoptosis and activation is important for the clinically relevant setting of platelet transfusion therapy (Leytin et al, 2008), because conventional blood banking storage of platelets for transfusion is at 22°C (Perrotta & Snyder, 2007). Furthermore, platelets traverse central and peripheral circulations, undergoing repeated exposure to lower temperatures at the body surface (Hoffmeister et al, 2003) and this periodic exposure of platelets to environmental temperatures may impact platelet apoptosis, activation and clearance.
Presented data demonstrate that BH3 mimetic ABT-737 much more efficiently triggers the mitochondrial apoptotic event ΔΨm depolarization in platelets at 37°C than at RT. In contrast, the platelet agonist thrombin almost equally induces CD62 exposure from α-granules to the platelet surface at both temperatures, indicating different temperature dependencies of ΔΨm and CD62 markers of platelet apoptosis and activation respectively. The stimulatory effects of elevated (37°C and 43°C) temperatures on intrinsic apoptosis pathway have been also shown in mitochondria isolated from nucleated cells (Pagliari et al, 2005; Lazarou et al, 2010). It was reported that prolonged (30–60 min) but not early (5–15 min) incubation of Bak with isolated mitochondria of HeLa cells at 37°C results in Bak activation, mitochondrial outer membrane permeabilization (MOMP) and cytochrome c release (Lazarou et al, 2010). Activation of Bak and Bax by elevated temperatures is downregulated with anti-apoptotic Bcl-2 and Bcl-XL proteins and upregulated with pro-apoptotic BH3-only proteins (Pagliari et al, 2005; Lazarou et al, 2010).
Our study also shows that, depending on the triggering stimulus, a high level of platelet apoptosis is not necessarily associated with platelet activation, a high level of platelet activation is not necessarily associated with platelet apoptosis, and that the apoptotic and activation responses may be concomitant. The different effects of ABT-737, A23187 and thrombin as inducers of apoptosis and/or activation may reflect the different actions of these agonists on mitochondrial membrane permeabilization and ΔΨm depolarization.
The biomedical significance of the current study includes several aspects. Firstly, the seminal methodological aspect consists of elaboration of a simple and effective method of preparation of three platelet populations which are highly enriched for apoptotic-only, activated-only, or both apoptotic and activated platelets. These specific populations, which otherwise are not easily isolated by flow cytometric sorting, can be readily obtained by treatment of platelets with appropriate inducers (ABT-737, thrombin and A23187). Secondly, this study, taken together with previously reported data (Leytin et al, 2007, 2008; Schoenwaelder et al, 2009), demonstrates that platelet apoptosis and activation are different phenomena, and opens possibilities for targeted differential pharmacological manipulation of platelet apoptosis and activation. Third, selective stimulation of apoptosis or activation enables the study of the mechanisms of these fundamental cellular responses in anucleate platelets. Fourth, the current study provides a basis for future investigations on the roles of platelet apoptosis and activation in platelet clearance, haemostasis, thrombosis and other platelet-associated diseases.