Apoptotic Processes in Megakaryocytes and Platelets

doi:10.1053/j.seminhematol.2010.03.006

Seminars in Hematology

Volume 47, Issue 3, July 2010, Pages 227-234

https://doi.org/10.1053/j.seminhematol.2010.03.006 Get rights and content

It is becoming increasingly clear that most mammalian cells are capable of undergoing apoptosis and that, within particular lineages, specific apoptotic pathways have evolved to regulate survival and turnover. The role of apoptosis in the megakaryocyte lineage is an intriguing one. Various insults, such as chemotherapeutics, autoantibodies, and human immunodeficiency virus (HIV), have been suggested to induce the apoptotic death of megakaryocytes and/or their progenitors. Conversely, apoptotic processes have been implicated in megakaryocyte development and platelet production. Platelets also contain functional apoptotic pathways, which circumscribe their survival. It has even been suggested that platelet activation responses involve components of the apoptotic machinery, highlighting a potential role for apoptotic processes in hemostasis and thrombosis. This review discusses the current state of knowledge about how apoptosis and apoptotic proteins contribute to the generation and function of megakaryocytes and platelets.

Section snippets

Apoptosis: Two Biochemical Pathways to Cell Death

Metazoan cells possess two highly conserved pathways that regulate their apoptotic death (Figure 1).⁶ Both pathways share a common endpoint: the activation of proteolytic enzymes called caspases, which mediate the rapid dismantling of cells. In viable cells, caspases normally reside in the cytosol as inactive precursors. The initiator/apical caspases, such as caspase-9 and caspase-8, are autocatalytically activated and proteolytically activate the effector/executioner caspases-3 and -7, which

Apoptotic Death of Megakaryocytes

Megakaryocytes presumably need to prevent the inadvertent activation of the apoptotic machinery in order to survive and produce platelets. This may be relatively straightforward at steady state, but in a range of pathophysiological settings, megakaryocytes and their progenitors are targets of signals that appear to induce apoptotic death. A prime example is cancer chemotherapy. Since the 1960s, when the link was first established, a wide range of chemotherapeutic agents have been demonstrated

Apoptotic Processes in Megakaryocyte Development and Platelet Production

Inextricably linked to the notion of apoptotic death induced by a pathogenic or pharmacological insult, yet conceptually distinct, is the idea that megakaryocytes might deliberately employ the apoptotic machinery to facilitate platelet production (Figure 2). Since the initial observation that platelet release from mature megakaryocytes in culture corresponded to the onset of apoptotic morphology,39, 40 an increasing body of evidence has suggested that platelet shedding is an apoptotic process.

Apoptosis in the Regulation of Platelet Life and Death

Once released into the circulation, platelets encounter one of two fates: consumption in a hemostatic process, or removal by the reticuloendothelial system in the liver and spleen. Since only a fraction of the circulating platelet population is required to maintain hemostasis at steady state, the vast majority of platelets die via the second route, being cleared after approximately 10 days in humans⁵⁰ and 5 days in mice.⁵¹ The mechanism underpinning this finite existence is apoptosis. First

Conclusions

There is still much to be learned about the role of apoptotic pathways and processes in megakaryocytes and platelet biology. In particular, it remains to be established how megakaryocyte survival is regulated at steady state and in response to insult, what contribution the apoptotic machinery makes to platelet production, and whether apoptotic pathways play a role in hemostasis and thrombosis. In addition, there is preliminary evidence that alternative cell death pathways, such as autophagy⁷⁴

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Megakaryocytopoiesis involves the process of the development of hematopoietic stem cells into megakaryocytes (MKs), which are the specialized cells responsible for the production of blood platelets. Platelets are one of the crucial factors for hemostasis and thrombosis. In terminally differentiated MKs, many molecular process such as caspase activation and a massive cytoskeletal rearrangement drive the formation of cytoplasmic extensions called proplatelets. These cytoplasmic extensions packed with granules and organelles are then released from the bone marrow into the blood circulation as platelets. Classically, caspase activation is associated with apoptosis and recent reports suggest their involvement in cell differentiation and maturation. There is no clear evidence about the stimulus for caspase activation during megakaryocyte development. In the current study, we attempted to understand the importance of endoplasmic reticulum stress in the caspase activation during megakaryocyte maturation. We used human megakaryoblstic cell line (Dami cells) as an experimental model. We used PMA (Phorbol 12-myristate 13 acetate) to induce megakaryocytic differentiation to understand the involvement of ER stress and caspase activation during MK maturation. Further, we used Thapsigargin, a non-competitive inhibitor of the sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) as a positive control to induce ER stress. We observed larger and adherent cells with the increased expression of megakaryocytic markers (CD41 and CD61) and UPR markers in PMA or Thapsigargin treated cells as compared to control. Also, Thapsigargin treatment induced increased caspase activity and PARP cleavage. The increased expression of megakaryocyte maturation markers alongside with ER stress and caspase activation suggests the importance of ER stress in caspase activation during MK maturation.
Busulfan Triggers Intrinsic Mitochondrial-Dependent Platelet Apoptosis Independent of Platelet Activation
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Citation Excerpt :
Apoptosis, a process of programmed cell death, is considered to be a main mechanism responsible for the regulation of life span and elimination of infected or damaged nucleated cells. As enucleated cells, platelets have also been observed to undergo apoptotic-like events as demonstrated by activation and translocation of Bcl-2 family members, release of cytochrome c, and activation of caspase-3 [17,18]. Consistent with this, degradation of antiapoptotic Bcl-xL represents a molecular clock to regulate platelet life span through controlling the prodeath activity of proapoptotic Bak, leading to platelet apoptosis [19].
As a nonspecific alkylating antineoplastic agent, busulfan has been widely used in the treatment of patients with chronic myeloid leukemia. In vitro and in vivo studies demonstrated busulfan-induced cell apoptosis. Whether busulfan triggers platelet apoptosis remains unclear. This study aimed to evaluate the role of busulfan in platelet apoptosis. Isolated human platelets were incubated with busulfan followed by analysis of platelet apoptosis by flow cytometry or western blot, including mitochondrial depolarization, expression of Bcl-2, and Bax and caspase 3 activation. Meanwhile, platelet activation, expression of glycoprotein Ibα (GPIbα), glycoprotein VI (GPVI), and <alpha>IIb<beta>3 and platelet aggregation in response to collagen and adenosine diphosphate (ADP) were measured. Additionally, busulfan was injected into mice with or without administration of caspase inhibitor QVD-Oph to investigate its effect on platelet lifespan. Our results showed that busulfan-treated platelets displayed increased mitochondrial membrane depolarization, decreased expression of Bcl-2, increased expression of Bax and caspase 3 activation in dose-dependent manner, which were inhibited by QVD-Oph. Platelet activation was not observed in busulfan-treated platelets as showed by no increased P-selectin expression and PAC-1 binding. However, busulfan reduced collagen- or ADP-induced platelet aggregation without affecting expression of GPIbα, GPVI, and <alpha>IIb<beta>3. Furthermore, busulfan reduced circulating platelet lifespan which was ameliorated by QVD-Oph in mice. In conclusion, busulfan triggers mitochondrial-dependent platelet apoptosis and reduces platelet lifespan in mice. These data suggest targeting caspase activation might be beneficial in the prophylaxis of platelet apoptosis-associated thrombocytopenia after administration of busulfan.
Linkage between the mechanisms of thrombocytopenia and thrombopoiesis
2016, Blood
Citation Excerpt :
Indeed, in a reconstitution study, IL-1α administration rapidly induced MK rupture-dependent thrombopoiesis and increased platelet counts in mice.49 Although there has long been controversy regarding caspase activation or caspase-mediated apoptosis during platelet biogenesis,50 reports finally showed that proplatelet-based platelet biogenesis is independent of the caspase-induced apoptosis pathway in MKs.51,52 However, Nishimura et al revealed that IL-1α/IL-1 type 1 receptor signaling leads to caspase-3 activation, which reduces plasma membrane stability and ultimately leads to MK rupture in a process distinct from typical Fas ligand–induced apoptosis.49
Thrombocytopenia is defined as a status in which platelet numbers are reduced. Imbalance between the homeostatic regulation of platelet generation and destruction is 1 potential cause of thrombocytopenia. In adults, platelet generation is a 2-stage process entailing the differentiation of hematopoietic stem cells into mature megakaryocytes (MKs; known as megakaryopoiesis) and release of platelets from MKs (known as thrombopoiesis or platelet biogenesis). Until recently, information about the genetic defects responsible for congenital thrombocytopenia was only available for a few forms of the disease. However, investigations over the past 15 years have identified mutations in genes encoding >20 different proteins that are responsible for these disorders, which has advanced our understanding of megakaryopoiesis and thrombopoiesis. The underlying pathogenic mechanisms can be categorized as (1) defects in MK lineage commitment and differentiation, (2) defects in MK maturation, and (3) defect in platelet release. Using these developmental stage categories, we here update recently described mechanisms underlying megakaryopoiesis and thrombopoiesis and discuss the association between platelet generation systems and thrombocytopenia.
Platelet Storage
2014, Pathobiology of Human Disease: A Dynamic Encyclopedia of Disease Mechanisms
Severely thrombocytopenic patients are at risk for catastrophic hemorrhage and cannot safely undergo complex lifesaving therapies without access to transfusion. Stepwise improvements in platelet collection techniques and storage conditions have increased both the availability and use of platelet concentrates over the last 60 years. A brief history of platelet transfusion is followed by current utilization statistics. In the context of outlining collection methods by apheresis or whole blood separation, relevant product standards are presented. Platelet metabolic function constrains container composition, nutritive media, and the overall storage environment. Accordingly, an overview of metabolism provides the necessary background to understand progress to date and improvements still to be made in platelet storage. A review of quality assessment techniques provides additional insight into how the safety and efficacy of platelet products continue to evolve. Causes for the slow deterioration of quality on blood bank shelves are discussed as a gateway to research into storage lesion mitigation. This article concludes with mention of unique alternatives promising to further advance the science of platelet transfusion.
JAK2V617F leads to intrinsic changes in platelet formation and reactivity in a knock-in mouse model of essential thrombocythemia
2013, Blood
Citation Excerpt :
Pecam1 was overexpressed in JAK2V617F MKs, and there is ample evidence that Pecam1 plays a role in fine-tuning megakaryopoiesis, in particular regulating MK migration.32,53 Programmed cell death, or apoptosis, is a regulator of proplatelet formation (reviewed by Geddis54) as well as platelet count by influencing platelet survival in circulation.55 Several apoptosis-related genes were found to be less expressed in JAK2V617F MKs, among them Phlda3, a negative regulator of AKT signaling, a pathway now recognized as a therapeutic target in myeloproliferative disorders.56
The principal morbidity and mortality in patients with essential thrombocythemia (ET) and polycythemia rubra vera (PV) stems from thrombotic events. Most patients with ET/PV harbor a JAK2V617F mutation, but its role in the thrombotic diathesis remains obscure. Platelet function studies in patients are difficult to interpret because of interindividual heterogeneity, reflecting variations in the proportion of platelets derived from the malignant clone, differences in the presence of additional mutations, and the effects of medical treatments. To circumvent these issues, we have studied a JAK2V617F knock-in mouse model of ET in which all megakaryocytes and platelets express JAK2V617F at a physiological level, equivalent to that present in human ET patients. We show that, in addition to increased differentiation, JAK2V617F-positive megakaryocytes display greater migratory ability and proplatelet formation. We demonstrate in a range of assays that platelet reactivity to agonists is enhanced, with a concomitant increase in platelet aggregation in vitro and a reduced duration of bleeding in vivo. These data suggest that JAK2V617F leads to intrinsic changes in both megakaryocyte and platelet biology beyond an increase in cell number. In support of this hypothesis, we identify multiple differentially expressed genes in JAK2V617F megakaryocytes that may underlie the observed biological differences.
Stimulation of platelet apoptosis by balhimycin
2013, Biochemical and Biophysical Research Communications
Glycopeptides, such as vancomycin, are powerful antibiotics against methicillin-resistant Staphylococcus aureus. Balhimycin, a glycopeptide antibiotic isolated from Amycolatopsis balhimycina, is similarly effective as vancomycin. Side effects of vancomycin include triggering of platelet apoptosis, which is characterized by cell shrinkage and by cell membrane scrambling with phosphatidylserine exposure at the cell surface. Stimulation of apoptosis may involve increase of cytosolic Ca²⁺ activity, ceramide formation, mitochondrial depolarization and/or caspase activation. An effect of balhimycin on apoptosis has, however, never been reported. The present study thus tested whether balhimycin triggers platelet apoptosis. Human blood platelets were treated with balhimycin and cell volume was estimated from forward scatter, phosphatidylserine exposure from annexin V-binding, cytosolic Ca²⁺ activity from fluo-3AM fluorescence, ceramide formation utilizing antibodies, mitochondrial potential from DiOC₆ fluorescence, and caspase-3 activity utilizing antibodies. As a result, a 30 min exposure to balhimycin significantly decreased cell volume (⩾1 μg/ml), triggered annexin V binding (⩾1 μg/ml), increased cytosolic Ca²⁺ activity (⩾1 μg/ml), stimulated ceramide formation (⩾10 μg/ml), depolarized mitochondria (⩾1 μg/ml) and activated caspase-3 (⩾1 μg/ml). Cell membrane scrambling and caspase-3 activation were virtually abrogated by removal of extracellular Ca²⁺. Cell membrane scrambling was not significantly blunted by pancaspase inhibition with zVAD-FMK (1 μM). In conclusion, balhimycin triggers cell membrane scrambling of platelets, an effect dependent on Ca²⁺, but not on activation of caspases.

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: This work was supported by the Australian National Health and Medical Research Council (Project Grants No. 516725 and 575535), the Sylvia and Charles Viertel Charitable Foundation (Fellowship to B.T.K.), and the Leukaemia Foundation of Australia (Scholarship to M.J.W.).

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Apoptotic Processes in Megakaryocytes and Platelets

Section snippets

Apoptosis: Two Biochemical Pathways to Cell Death

Apoptotic Death of Megakaryocytes

Apoptotic Processes in Megakaryocyte Development and Platelet Production

Apoptosis in the Regulation of Platelet Life and Death

Conclusions

Cell

Mol Cell

Molecular cell

Cell

Cell

Cell

Cell

Cell

Cell

J Biol Chem

Cell

Cell

Semin Hematol

Blood

Blood

Blood

Exp Hematol

J Thromb Haemost

Blood

Blood

Blood

FEBS Lett

Cell

J Biol Chem

Transfus Apher Sci

Blood

Blood

Blood

Blood

Blood

Biochim Biophys Acta

J Thromb Haemost

Blood

Blood

Deranged removal of apoptotic cells: its role in the genesis of lupus

Nephrol Dial Transplant

Apoptotic mechanisms after cerebral ischemia

Stroke

Role of apoptosis in cardiovascular disease

Apoptosis