Armen V. Gyulkhandanyan

Markers of platelet apoptosis: methodology and applications

Apoptosis, or programmed cell death, is a physiological mechanism that serves for controlled deletion of damaged cells. While long attributed exclusively to nucleated cells, over recent years it has been recognized that apoptosis also occurs in anucleate platelets. We describe here experiences of determining markers of apoptosis in human platelets treated in vitro with pro-apoptotic chemical and physical stimuli. These include depolarization of mitochondrial inner membrane, cytochrome c release, expression of pro-apoptotic and anti-apoptotic proteins of Bcl-2 family, activation of apoptosis executioner caspase-3, exposure of phosphatidylserine, platelet shrinkage, fragmentation to microparticles, blebbing and filopod extrusion on the platelet surface. These assays serve to characterize platelet apoptosis in different cellular compartments (mitochondria, cytosol and plasma membrane) and at the whole-cell level. Methods commonly employed in studies of platelet apoptosis markers include flow cytometry, Western blot analysis and electron microscopy. An integrated methodological approach, based on determination of different platelet apoptosis markers, represents a useful tool for examining platelet apoptosis in various physiological and pathological settings.

Activation of caspases-9, -3 and -8 in human platelets triggered by BH3-only mimetic ABT-737 and calcium ionophore A23187: caspase-8 is activated via bypass of the death receptors

Platelet apoptosis and activation have been studied in human platelets treated with BH3‐only mimetic ABT‐737 and calcium ionophore A23187, agents triggering apoptosis through the intrinsic mitochondrial pathway. Platelet apoptosis was determined as activation of crucial apoptosis‐associated caspases, initiator caspase‐9 of intrinsic apoptosis pathway, executioner caspase‐3 and initiator caspase‐8 of extrinsic death receptor pathway, and platelet activation was detected by P‐selectin (CD62) exposure on the platelet surface. We found that ABT‐737 predominantly induced activation of caspases‐9, ‐3 and ‐8 rather than CD62 exposure, whereas A23187 induces both caspases activation and CD62 exposure. Caspase‐8 activation was stimulated independently of the extrinsic apoptosis pathway via mitochondrial membrane permeabilization and depolarization. These data suggest that (i) caspase‐8 activation is triggered in ABT‐737‐ and A23187‐treated anucleate platelets through the mitochondria‐initiated caspase activation cascade bypassing the death receptors, and (ii) ABT‐737‐treated platelets are a useful experimental tool for discerning the role of platelet apoptosis in platelet function and survival.

Selective triggering of platelet apoptosis, platelet activation or both

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.

Concurrent and separate inside-out transition of platelet apoptosis and activation markers to the platelet surface

The cell plasma membrane is tightly coupled with the vital processes of apoptosis and activation. In the current study, we investigated exposure of the apoptosis marker phosphatidylserine (PS) and activation marker P‐selectin (CD62) on the plasma membrane of anucleate platelets. We found that, depending on triggering stimuli, the plasma membrane of human platelets may exist in four states with predominant exposure of (i) PS but not CD62 (75·9 ± 2·8% of total cells), (ii) CD62 but not PS (86·2 ± 1·3%), (iii) both PS and CD62 (89·6 ± 1·0%) or (iv) neither PS nor CD62 (87·9–97·5%), when platelets were treated at optimal conditions with pro‐apoptotic BH3 mimetic ABT‐737, thrombin, calcium ionophore A23187 or control diluents, respectively. The dynamics of PS exposure induced by ABT‐737 is a slow temperature‐dependent process requiring 90 min treatment at 37°C rather than at room temperature for obtaining high levels of PS exposure. In contrast, thrombin‐induced CD62 exposure and A23187‐induced PS and CD62 exposure showed fast temperature‐independent dynamics. This model of selective and concurrent stimulation of PS and/or CD62 transition to the platelet surface provides an experimental horizon for elucidating the roles of plasma membrane markers of platelet apoptosis and activation in platelet clearance.

Mitochondrial permeability transition pore (MPTP)-dependent and -independent pathways of mitochondrial membrane depolarization, cell shrinkage and microparticle formation during platelet apoptosis.

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BH3-mimetic ABT-737 induces strong mitochondrial membrane depolarization in platelets but only weakly stimulates apoptotic morphological changes, platelet shrinkage and microparticle formation

BACKGROUND Depolarization of mitochondrial inner transmembrane potential (ΔΨm) is a key biochemical manifestation of the intrinsic apoptosis pathway in anucleate platelets. Little is known, however, about the relationship between ΔΨm depolarization and downstream morphological manifestations of platelet apoptosis, cell shrinkage and microparticle (MP) formation. OBJECTIVES To elucidate this relationship in human platelets. MATERIALS AND METHODS Using flow cytometry, we analyzed ΔΨm depolarization, platelet shrinkage and MP formation in platelets treated with BH3-mimetic ABT-737 and calcium ionophore A23187, well-known inducers of intrinsic platelet apoptosis. RESULTS We found that at optimal treatment conditions (90min, 37°C) both ABT-737 and A23187 induce ΔΨm depolarization in the majority (88-94%) of platelets and strongly increase intracellular free calcium. In contrast, effects of A23187 and ABT-737 on platelet shrinkage and MP formation are quite different. A23187 strongly stimulates cell shrinkage and MP formation, whereas ABT-737 only weakly induces these events (10-20% of the effect seen with A23187, P<0.0001). CONCLUSIONS These data indicate that a high level of ΔΨm depolarization and intracellular free calcium does not obligatorily ensure strong platelet shrinkage and MP formation. Since ABT-737 efficiently induces clearance of platelets from the circulation, our results suggest that platelet clearance may occur in the absence of the morphological manifestations of apoptosis.

The GPIIbIIIa antagonist drugs eptifibatide and tirofiban do not induce activation of apoptosis executioner caspase-3 in resting platelets but inhibit caspase-3 activation in platelets stimulated with thrombin or calcium ionophore A23187

Platelet surface receptor, glycoprotein (GP) IIbIIIa (integrin αIIbβ3), mediates platelet aggregation and plays a key role in hemostasis and thrombosis.[1][1],[2][2] Numerous GPIIbIIIa antagonists have been designed and tested as inhibitors of platelet aggregation.[3][3] Two of these antagonists

Mitochondrial Inner Membrane Depolarization as a Marker of Platelet Apoptosis : Disclosure of Nonapoptotic Membrane Depolarization

Availability of universal marker for the diagnosis of platelet apoptosis is an important but currently unresolved goal of platelet physiology investigations. Mitochondrial inner transmembrane potential (▵Ψm) depolarization is frequently used as a marker of apoptosis in nucleated cells and anucleate platelets. Since ▵Ψm depolarization in platelets is also frequently associated with concurrent induction of other apoptotic responses, it may appear that ▵Ψm depolarization is a good universal marker of platelet apoptosis. However, data presented in the current study indicate that this is incorrect. We report here fundamental differences in the effects of potassium ionophore valinomycin and calcium ionophore A23187 on human platelet apoptosis. Although both A23187-triggered and valinomycin-triggered ▵Ψm depolarization are strongly induced, the former is dependent on the opening of mitochondrial permeability transition pore (MPTP) and the latter is MPTP-independent. Furthermore, effects of calcium and potassium ionophores on other apoptotic events are also basically different. A23187 induces caspase-3 activation, proapoptotic Bax and Bak protein expression, phosphatidylserine exposure, and microparticle formation, whereas valinomycin does not induce these apoptotic manifestations. Discovery of targeted ▵Ψm depolarization not associated with apoptosis in valinomycin-treated platelets indicates that this marker should not be used as a single universal marker of platelet apoptosis in unknown experimental and clinical settings as it may lead to a false-positive apoptosis diagnosis.

Role of mitochondrial membrane permeabilization and depolarization in platelet apoptosis

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How to Avoid False-Negative and False-Positive Diagnoses of Platelet Apoptosis: Illustrative Experimental and Clinically Relevant Cases:

Platelets may selectively execute apoptosis (PL-Apo), activation (PL-Act), and both or no responses when exposed to different chemical agents, shear stresses, and stored under blood banking conditions. Appropriate diagnosis of PL-Apo is an important issue of platelet physiology investigations. However, in diagnosing PL-Apo, there is a risk of a false-negative or false-positive diagnosis. The goal of the current review is to present recommendations that may help to avoid incorrect PL-Apo diagnosis. Analyzing reported studies, we recommend (1) using platelet-rich plasma rather than isolated platelets to minimize artificial stimulation of PL-Apo during platelet isolation, (2) using established optimal conditions for stimulation of PL-Apo and/or PL-Act, (3) using a panel of PL-Apo and PL-Act markers, and (4) appropriate positive and negative controls for quantification of PL-Apo and PL-Act responses.