Asuman Mutlu

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.

Platelet activation and apoptosis are different phenomena: evidence from the sequential dynamics and the magnitude of responses during platelet storage

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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

Stored platelet concentrates produced by the platelet-rich plasma method are more resistant to apoptosis but more sensitive to activation than are platelets prepared by the buffy-coat and apheresis methods.

Recently, we studied the contributions of PLT activation and apoptosis to the PLT storage lesion during conventional (Days 2-5), extended (Days 6-8), and long-term (Days 11-16) storage of leukoreduced PCs produced by the PLT-rich plasma method (PRP-PCs). Four apoptotic parameters, including depolarization of mitochondrial inner membrane potential (DYm), caspase-3 activation, phosphatidylserine (PS) exposure, and release of PLT microparticles, were used for quantifying mitochondrial, cytoplasmic, plasma membrane, and cellular manifestations of PLT apoptosis, respectively, and PLT activation was determined by P-selectin (CD62) exposure. It was demonstrated that PLT activation and apoptosis responses are triggered sequentially, rather than in parallel, during storage of PRP-PCs. PLT activation was readily induced under the conventional and extended PLT storage, whereas triggering of PLT apoptosis required long-term storage. Furthermore, for all storage days, the level of PLT activation reached was significantly higher than the level of apoptosis. Different results have been reported in the subsequent publication of Albanyan and coworkers, in which PLT activation and apoptosis phenomena during storage for 1, 3, 5, and 7 days were studied in leukoreduced PCs produced by the buffy coat (BC-PCs) and apheresis (APPCs) methods. The aim of the current report is to compare PLT activation and apoptosis responses in PRP-PCs versus BC-PCs and AP-PCs by analyzing results of these closely related studies. Table 1 summarizes the reported data on the dynamics of DYm depolarization and caspase-3 activation in PRP-, BC-, and AP-PCs. In PRP-PCs, DYm depolarization and caspase-3 activation did not increase significantly even after long-term storage for up to 12 days; storage for 13-14 days was required for significant triggering of these apoptotic events. In contrast, much faster apoptotic changes were observed during storage of BC-PCs and AP-PCs: when DYm depolarization was measured by the red-to-green fluorescence ratio of the DYmdetecting dye JC-1, as was employed in our study, DYm depolarization was significantly increased by Days 5 and 7 (Table 1). However, when DYm depolarization was measured by an alternative JC-1 method, as the percentage of PLTs with depolarized DYm, which detects cells with full DYm depolarization, only BC-PCs, but not AP-PCs, showed this apoptotic alteration on Days 5 and 7 (Fig. 1B, right panel in Albanyan et al.). As evidenced from the data on the sequential dynamics (Table 1) and the magnitude of PLT apoptosis and activation responses, as measured by PS and CD62 exposure, respectively (Table 2), the ability of PLT apoptosis and activation to be induced by PLT storage differs significantly in PRP-PCs, in comparison to BC-PCs and AP-PCs. In PRP-PCs, PLT apoptosis is more resistant to PLT storage than PLT activation (Table 2, Column 2, p < 0.001 for Days 2, 5, and 7); in contrast, in BC-PCs and AP-PCs, PLT apoptosis and activation have approximately equal sensitivity to storage (Table 2, Columns 3 and 4, 18.6%-21.3% vs. 20%-25% for Day 7). Furthermore, PLT apoptosis in PRPPCs is more resistant to storage than is apoptosis in BC-PCs and AP-PCs (Tables 1 and 2A, Columns 2-4, p < 0.01 for Days 5 and 7); in contrast, activation in PRPPCs is more sensitive to storage than is activation in BC-PCs and AP-PCs (Table 2B, Columns 2-4, 65.8 9.7% vs. 20%-25% for Day 7). In conclusion, the analysis of these recently reported data indicates that PLT activation rather than apoptosis most contributes to the PLT storage lesion during storage of PRP-PCs for 7 days. Therefore, PLT activation, and not apoptosis, should be the main concern in maintaining quality of PRP-PCs during blood banking storage. In contrast, for BC-PCs and AP-PCs, both activation and

The level of matrix metalloproteinase-2 in donor plasma strongly correlates with post-transfusion recovery of stored platelet concentrates

Background and Objective  A simple test accurately predicting post‐transfusion recovery of stored platelet concentrates (PCs) is needed.