Platelet Apoptosis Can Be Triggered Bypassing the Death Receptors.

In nucleated cells, the extrinsic pathway of the programmed cell death (apoptosis) is triggered by interaction of death ligands of the tumor necrosis factor superfamily with the death receptors on external cell surface membrane. In this review, we present evidence that, in contrast to nucleated cells, apoptosis in anucleate platelets can be induced through bypassing the death receptors, using instead specific receptors on the platelet surface mediating platelet activation, aggregation, and blood coagulation. These platelet surface receptors include the protease-activated receptor 1 of thrombin and glycoproteins IIbIIIa and Ibα, receptors of fibrinogen, and von Willebrand factor. The pro-apoptotic BH3 mimetic ABT-737 and calcium ionophore A23187 also trigger platelet apoptosis without using death receptors. These agents induce the intrinsic pathway of platelet apoptosis by direct targeting mitochondrial and extra-mitochondrial apoptotic responses.

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.

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.

Desialylation is a mechanism of Fc-independent platelet clearance and a therapeutic target in immune thrombocytopenia.

Immune thrombocytopenia (ITP) is a common bleeding disorder caused primarily by autoantibodies against platelet GPIIbIIIa and/or the GPIb complex. Current theory suggests that antibody-mediated platelet destruction occurs in the spleen, via macrophages through Fc-FcγR interactions. However, we and others have demonstrated that anti-GPIbα (but not GPIIbIIIa)-mediated ITP is often refractory to therapies targeting FcγR pathways. Here, we generate mouse anti-mouse monoclonal antibodies (mAbs) that recognize GPIbα and GPIIbIIIa of different species. Utilizing these unique mAbs and human ITP plasma, we find that anti-GPIbα, but not anti-GPIIbIIIa antibodies, induces Fc-independent platelet activation, sialidase neuraminidase-1 translocation and desialylation. This leads to platelet clearance in the liver via hepatocyte Ashwell-Morell receptors, which is fundamentally different from the classical Fc-FcγR-dependent macrophage phagocytosis. Importantly, sialidase inhibitors ameliorate anti-GPIbα-mediated thrombocytopenia in mice. These findings shed light on Fc-independent cytopenias, designating desialylation as a potential diagnostic biomarker and therapeutic target in the treatment of refractory ITP.

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.

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.

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.

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.

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.

Apoptosis in the anucleate platelet.

For many years, programmed cell death, known as apoptosis, was attributed exclusively to nucleated cells. Currently, however, apoptosis is also well-documented in anucleate platelets. This review describes extrinsic and intrinsic pathways of apoptosis in nucleated cells and in platelets, platelet apoptosis induced by multiple chemical stimuli and shear stresses, markers of platelet apoptosis, mitochodrial control of platelet apoptosis, and apoptosis mediated by platelet surface receptors PAR-1, GPIIbIIIa and GPIbα. In addition, this review presents data on platelet apoptosis provoked by aging of platelets in vitro during platelet storage, platelet apoptosis in pathological settings in humans and animal models, and inhibition of platelet apoptosis by cyclosporin A, intravenous immunoglobulin and GPIIbIIIa antagonist drugs.

Mitochondrial control of platelet apoptosis: effect of cyclosporin A, an inhibitor of the mitochondrial permeability transition pore.

The role of the mitochondrial permeability transition pore (MPTP) in apoptosis of nucleated cells is well documented. In contrast, the role of MPTP in apoptosis of anucleated platelets is largely unknown. The aim of this study was to elucidate the contribution of MPTP in the control of different manifestations of platelet apoptosis by analyzing the effect of cyclosporin A (CsA), a potent inhibitor of MPTP formation. Using flow cytometry, we studied the effect of pretreatment of platelets with CsA on apoptotic responses in human platelets stimulated with calcium ionophore A23187. We found that CsA inhibited A23187-stimulated platelet apoptosis, completely preventing (i) depolarization of mitochondrial inner membrane potential (DeltaPsim), (ii) activation of cytosolic apoptosis executioner caspase-3, (iii) platelet shrinkage, and (iv) fragmentation of platelets to microparticles, but (v) only partially (approximately 25%), inhibiting phosphatidylserine (PS) exposure on the platelet surface. This study shows that MPTP formation is upstream of DeltaPsim depolarization, caspase-3 activation, platelet shrinkage and microparticle formation, and stringently controls these apoptotic events in A23187-stimulated platelets but is less involved in PS externalization. These data also indicate that CsA may rescue platelets from apoptosis, preventing caspase-3 activation and inhibiting the terminal cellular manifestations of platelet apoptosis, such as platelet shrinkage and degradation to microparticles. Furthermore, the results suggest a novel potentially useful application of CsA as an inhibitor of platelet demise through apoptosis in thrombocytopenias associated with enhanced platelet apoptosis.

Intravenous immunoglobulin inhibits anti-glycoprotein IIb-induced platelet apoptosis in a murine model of immune thrombocytopenia.

We have previously shown that injection of anti-glycoprotein (GP) IIb induces murine immune thrombocytopenia (ITP) and that intravenous immunoglobulin (IVIg) ameliorates ITP. We hypothesise that murine ITP may be associated with platelet apoptosis, which is upregulated by anti-GPIIb and downregulated by IVIg. The current study demonstrated that anti-GPIIb injection induced three critical apoptosis manifestations in platelets: (i) mitochondrial inner transmembrane potential (delta psi m) depolarisation; (ii) caspase-3 activation; and (iii) phosphatidylserine (PS) exposure. IVIg administration inhibited caspase-3 activation and PS exposure, but not delta psi m-depolarisation, in anti-GPIIb-treated platelets, demonstrating that IVIg ameliorates thrombocytopenia concomitantly with inhibiting late, but not early mechanisms of platelet apoptosis.

Role of platelet surface glycoprotein Ibalpha and P-selectin in the clearance of transfused platelet concentrates.

BACKGROUND: Role of P-selectin (CD62) and glycoprotein (GP) Ibalpha in posttransfusion clearance of platelet concentrates (PCs) is unclear. STUDY DESIGN AND METHODS: Platelet (PLT) activation in vitro was determined by flow cytometry using anti-CD62 and anti-GPIbalpha. PC clearance in vivo was evaluated in an animal model using rabbits with an inhibited reticuloendothelial system, as measured by 0.5-hour (R(0.5)), 24-hour (R(24)), and total (R( summation operator )) PLT recoveries, and survival time (ST). Correlations were analyzed between in vitro assays of PLT activation and in vivo clearance of conventional (Days 2-5), outdated (Days 7-8), and refrigerated PCs. RESULTS: Binding of anti-CD62 to the PLT surface was significantly increased and of anti-GPIbalpha decreased in outdated and refrigerated PCs compared to conventional PCs. Negative correlation was observed between in vitro anti-CD62 binding and the fast (R(0.5)) PLT clearance, but not with delayed (R(24) and ST) clearance. In contrast, anti-GPIbalpha binding showed positive correlations with delayed but not with fast PLT clearance. Overall (R( summation operator )) clearance correlated better with anti-GPIbalpha than with anti-CD62 binding. CD62 density on the PLT surface was decreased after PC transfusion, whereas GPIbalpha density remained unchanged. CONCLUSION: These data suggest that CD62 exposure on the PLT surface during PC storage triggers fast CD62-mediated PC clearance, whereas in vitro GPIbalpha changes are involved in delayed GPIbalpha-mediated PC clearance.

Pathologic high shear stress induces apoptosis events in human platelets.

Recently, it has been discovered that apoptosis of anucleate platelets can be induced by chemical agonists. Other studies demonstrated that mechanical forces (shear stresses) stimulate platelet activation and signaling in the absence of exogenous chemical stimuli. We analyzed whether shear stresses can trigger platelet apoptosis, a question that has not yet been studied. Using a cone-and-plate viscometer, we exposed human platelet-rich plasma to different shear stresses, ranging from physiologic arterial and arteriole levels (10-44 dyn/cm2) to pathologic high levels (117-388 dyn/cm2) occurring in stenotic vessels. We found that pathologic shear stresses induce not only platelet activation (P-selectin upregulation and GPIbalpha downregulation) but also trigger apoptosis events, including mitochondrial transmembrane potential depolarization, caspase 3 activation, phosphatidylserine exposure, and platelet shrinkage and fragmentation, whereas physiological shear stresses are not effective. This novel finding suggests that shear-induced platelet apoptosis can be mediated by mechanoreceptors, does not require nuclear participation, and may affect platelet clearance.