REVIEW Megakaryocytic Dysfunction in Myelodysplastic Syndromes And

Megakaryocytic dysfunction in myelodysplastic syndromes and idiopathic thrombocytopenic purpura is in part due to different forms of cell death

EJ Houwerzijl1, NR Blom1,2, JJL van der Want2, E Vellenga1 and JTM de Wolf1

1Department of Hematology, University Medical Center Groningen, Groningen, The Netherlands and 2Department of Cell Biology and Electron Microscopy, University Medical Center Groningen, Groningen, The Netherlands

Platelet production requires compartmentalized caspase acti- undergoes apoptosis, a common form of programmed cell death vation within megakaryocytes. This eventually results in (PCD).2,3 However, the apoptotic program is also required for platelet release in conjunction with apoptosis of the remaining megakaryocyte. Recent studies have indicated that in low-risk proplatelet formation and the release of mature platelets. myelodysplastic syndromes (MDS) and idiopathic thrombocy- Activation of caspase-3 and -9 has been demonstrated not only topenic purpura (ITP), premature cell death of megakaryocytes in senescent megakaryocytes but also in maturing and proplate- may contribute to thrombocytopenia. Different cell death let-bearing megakaryocytes.4 In contrast to senescent megakaryo- patterns have been identified in megakaryocytes in these cytes, no DNA fragmentation could be detected in maturing disorders. Growing evidence suggests that, besides apoptosis, megakaryocytes, suggesting that caspase activation is compart- necrosis and autophagic cell death, may also be programmed. Therefore, programmed cell death (PCD) can be classified in mentalized. Consistent with these findings, the cytoplasmic apoptosis, a caspase-dependent process, apoptosis-like, auto- distribution of activated caspase-3 showed a granular pattern in phagic and necrosis-like PCD, which are predominantly cas- maturing megakaryocytes, whereas in senescent megakaryo- pase-independent processes. In MDS, megakaryocytes show cytes a diffuse cytoplasmic staining pattern was observed. Thus, features of necrosis-like PCD, whereas ITP megakaryocytes after proplatelet formation caspase activation switches from a demonstrate predominantly characteristics of apoptosis-like circumscript to a diffuse form, thereby inducing apoptosis in PCD (para-apoptosis). Triggers for these death pathways are largely unknown. In MDS, the interaction of Fas/Fas-ligand senescent megakaryocytes. This notion is supported by observa- might be of importance, whereas in ITP antiplatelet autoanti- tions of diminished proplatelet formation by caspase inhibitors 4 bodies recognizing common antigens on megakaryocytes and and overexpression of antiapoptotic protein Bcl-2. platelets might be involved. These findings illustrate that Thus, in normal physiology platelet production and mega- cellular death pathways in megakaryocytes are recruited in karyocyte apoptosis are closely related events. In diseases, both physiological and pathological settings, and that different however, premature PCD might disrupt platelet formation. This forms of cell death can occur in the same cell depending on the stimulus and the cellular context. Elucidation of the underlying has been demonstrated in myelodysplastic syndromes (MDS) mechanisms might lead to novel therapeutic interventions. and idiopathic thrombocytopenic purpura (ITP). This review Leukemia (2006) 20, 1937–1942. doi:10.1038/sj.leu.2404385; discusses the role of PCD in these disorders and summarizes published online 7 September 2006 some recent developments concerning PCD. Keywords: apoptosis; programmed cell death; megakaryocytes; platelets; ITP; MDS Programmed cell death

PCD is defined as an active, controlled, process of sequential, in Introduction principle reversible, events leading to cell death. PCD requires the activity of specific genes, is not associated with an Megakaryocytes arise from pluripotent hematopoietic stem cells inflammatory response and plays a major role in both normal that undergo lineage commitment, proliferation and differentia- development and disease. These characteristics distinguish PCD tion under the influence of cytokines, in particular thrombo- from accidental necrosis. Apoptosis is often used as a synonym of poietin (TPO). As a result of endomitosis, megakaryocytes PCD. However, certain forms of necrosis and autophagic cell become polyploid cells with a large cytoplasmic mass. This death are also programmed events.5–8 Therefore, PCD can be enables each megakaryocyte to produce 1000–3000 platelets. subdivided into apoptosis (type I), autophagic cell death (type II) The exact mechanism of platelet production remains unclear. and necrosis-like PCD (type III).6,8 This classification is primarily Current evidence supports the mechanism initially suggested by based on morphology. However, many other techniques to assess Wright in 1906, that platelets bud off from cytoplasmic cell death exist. Most of these methods are not sensitive and pseudopodial extensions (proplatelets). These proplatelets are specific enough to diagnose cell death independently. Therefore, created from an abundant cytoplasmic reservoir of membranes it is advised to use multiple techniques simultaneously.9,10 A 1 (demarcation membrane system). After the release of platelets, summary of frequently used techniques is given in Table 1.9,10 the remaining senescent megakaryocyte, consisting of a nucleus and a thin margin of cytoplasm (denuded megakaryocyte), Apoptosis (type I PCD) Correspondence: Dr EJ Houwerzijl, Department of Hematology, Apoptosis is morphologically characterized by chromatin University Medical Center Groningen, Hanzeplein 1, PO Box condensation, nuclear fragmentation, cell surface blebbing, cell 30001, 9700 RB Groningen, The Netherlands. E-mail: [email protected] shrinkage and the formation of apoptotic bodies. The induction Received 17 February 2006; revised 24 July 2006; accepted 4 August of classic apoptosis involves the sequential activation of initiator 2006; published online 7 September 2006 and effector caspases, which are proteolytic enzymes that Megakaryocytic cell death in MDS and ITP EJ Houwerzijl et al 1938 Table 1 A selection of methods of cell death detection applicable to tissue sections9,10

Methods Type of cell death Stage of cell death Comments

Morphology Electron microscopy Apoptosis Late Gold standard, but morphology alone is insensitive Autophagic cell death Autophagosome Gold standard (two-membraned vacuoles) Necrosis Late Gold standard, not distinguishable from necrosis-like PCD

Histochemistry TUNEL/ISEL Apoptosis Intermediate and late Pretreatment that deteriorates DNA can reduce sensitivity False-positivity may occur in: necrosis, cells in process of DNA repair, pretreatment that induces DNA breaks Helpful in diagnosing apoptosis when used in combination with morphology Applicable to archival material Caspases Apoptosis Intermediate and late Helpful in diagnosing apoptosis when used in combination with morphology Correlates well with morphology and TUNEL Applicable to archival material LC-3 Autophagic cell death Autophagosome Correlates well with morphology of autophagosomes Abbreviations: ISEL, in situ end labeling; LC-3, light chain-3; PCD, programmed cell death; TUNEL, TdT-mediated dUTP-biotin nick end labeling.

Figure 1 Schematic model of pathways leading to cell death. Predominantly mitochondrial pathways are depicted. Classical apoptosis can be triggered by the extrinsic (A) and intrinsic (mitochondrial) pathway (B). Both pathways can be triggered by death receptor-mediated caspase-8 activity. Numerous other stress responses converge on the mitochondria and can promote the onset of MPT. MPT can subsequently lead to MOMP. Depending on multiple factors, such as the speed of MPT, the number of mitochondria involved and the availability of adenosine triphosphate (ATP), either apoptosis, necrosis or autophagy occurs. The green ovals represent a reﬁnement of the traditional classiﬁcation of cell death.7 Abbreviations: AIF, apoptosis inducing factor; CD, cell death; EndoG, endonuclease G; MPT, mitochondrial permeability transition; MOMP, mitochondrial outer membrane permeabilization; ROS, reactive oxygen species.

degrade essential cellular targets. Caspase-3, the main effector frequently autophagy, at least initially, functions as a protective caspase, can be activated by an extrinsic pathway involving mechanism for cell destruction. In situations of starvation, the cellular death receptors, and an intrinsic (mitochondrial) path- cells maintains ATP production by reducing the amount of way involving cytochrome c, Apaf-1 and caspase 9. The cytoplasm and organelles into autophagosomes.11 Autophagic mitochondrial pathway can also induce caspase-independent cell death has predominantly been observed when the apoptotic apoptosis (also called apoptosis-like PCD) (Figure 1).7 pathway is blocked, suggesting that cells die preferentially by apoptosis and that apoptosis is a faster process than autophagic cell death.12 Autophagic cell death (type II PCD) Type II PCD can develop from autophagy (self eating), Necrosis-like PCD (type III cell death) characterized by sequestration of cellular organelles in dou- Necrosis is an unregulated process, characterized by rapid cell ble-membraned vacuoles (autophagosomes), which fuse with and organelle swelling, loss of plasma membrane integrity, ATP lysosomes leading to degradation of their content. However, depletion, ion deregulation and activation of degradative

Leukemia Megakaryocytic cell death in MDS and ITP EJ Houwerzijl et al 1939 enzymes. However, regulated forms of necrotic cell death tion of megakaryocytes and their precursors.15 Morphological (called necrosis-like PCD or programmed necrosis) have also studies of MDS bone marrow have shown increased numbers of been observed, especially under conditions (i.e. viral infections) abnormal megakaryocytes, in particular micromegakaryocytes in which apoptosis is inhibited.5–8 with a low peak ploidy number (4–8N), suggesting an expansion of megakaryocytic precursors, an arrest in terminal megakaryocyte differentiation and impaired nuclear development. Other Cross-talk between types of cell death and mitochondria dysplastic features include large mononuclear forms, multiple Between the different types of cell death, flexibility exists separate nuclei, dissociation between cytoplasmic and nuclear making it possible that apoptosis can switch to necrosis and maturation and megakaryocytic hypogranulation.22 Although autophagy depending on the cellular context. Several studies not all megakaryocytes in MDS appear abnormal on light have shown that the inhibition of the apoptotic machinery can microscopy, cytogenetic studies show that the majority of 6,8 11 trigger a switch from apoptosis to necrosis or autophagy. In megakaryocytes are involved in the MDS clone, even when these processes, mitochondrial dysfunction plays an important micromegakaryocytes are not analyzed.23 7 role. Mitochondria can integrate cell death signals and Several studies have shown a defective in vitro megakaryo- 12 represent a nexus at which different pathways interact. Crucial cytopoiesis in MDS. In many MDS cases, megakaryocyte events at the mitochondrial level are: opening of mitochondrial progenitor growth was unresponsive to recombinant TPO.22,24 permeability transition (MPT) pores in the inner mitochondrial This defective response is probably due to deregulated TPO- membrane, loss of the mitochondrial transmembrane potential receptor-mediated signaling pathways,25 as a lack of serum and mitochondrial outer membrane permeabilization TPO,26 a decreased expression of c-mpl (TPO-receptor)22 or 7,8,13 (MOMP) (Figure 1). The intensity of the stimulus leading mutations in c-mpl25 have been excluded. to MPT and the cellular context determines which type of cell death develops.7,8 PCD in MDS megakaryocytes. Studies on apoptosis in MDS megakaryocytes are scarce compared to erythroid and PCD in MDS myeloid precursors. This is in part due to the low number of megakaryocytes and their vulnerability during bone marrow Apoptosis in MDS: evidence and controversies sample processing. Elevated numbers of denuded megakaryo- MDS are a heterogeneous group of clonal hematopoietic stem cytes in bone marrow biopsies from MDS patients have been cell disorders, characterized by a dysplastic and ineffective reported27 and have been ascribed to apoptosis on the basis of hematopoiesis. The alterations in the hematopoietic cells are earlier reports concerning the ultrastructure of senescent murine thought to result from irreversible DNA damage within a megakaryocytes.2 The observations in MDS, however, were hematopoietic stem cell and an accumulation of multiple solely based on light microscopy.27 Several studies using genetic lesions during hematopoiesis. The precise pathogenesis electron microscopy,28–30 TdT-mediated dUTP-biotin nick end of MDS is not elucidated, but numerous studies (reviewed by 29,31 14 15 16 labeling (TUNEL) or in situ end labeling (ISEL) have reported Parker and Mufti; Liesveld et al.; Yoshida and Mufti ) apoptosis in megakaryocytes. Other authors,32,33 however, indicate that enhanced intramedullary apoptosis may be an reported that they could not identify apoptosis in megakaryo- important disease mechanism, especially in explaining the cytes, perhaps as a result of bone marrow sample preparation paradox between bone marrow hypercellularity and peripheral techniques or owing to a lineage-restricted propensity for cytopenias, in particular in low-risk MDS. However, studies on apoptosis. Similarly, a recent study34 using ISEL demonstrated apoptosis have also been conflicting and disagreement regard- apoptosis in only 4% of megakaryocytes, which were pre- ing the degree and extent of apoptosis, the involvement dominantly micromegakaryocytes. These findings are largely of stromal cells and the clinical implications of apoptosis in consistent with results from our own study,19 in which MDS MDS bone marrow, remain. The evidence and controversies megakaryocytes were negative for activated caspase-3 and regarding apoptosis in MDS have been reviewed extensively 15–17 showed no ultrastructural features of apoptosis. The megakaryo- elsewhere. cytes demonstrated ultrastructural changes resembling necrosis- like cell death, including clumping and random degradation of Etiology of thrombocytopenia in low-risk MDS and PCD nuclear chromatin and cytoplasmic vacuoles (Figure 2c and of MDS megakaryocytes Table 2). How these megakaryocytic changes arise, is not clear. Preliminary results suggest a role for the Fas/Fas-ligand Thrombocytopenia occurs in 30–50% of patients with MDS and (FasL) system. Immunohistochemical staining of MDS may result in serious bleeding complications. Isolated thrombo- megakaryocytes showing necrosis-like morphology was cytopenia is the presenting manifestation in 5–10% of MDS positive for Fas and FasL (Blom et al., unpublished observations, patients, and may then be mistaken for ITP.18 2005). Increased death signals, in particular the Fas/FasL system might play an important role in inducing intramedullary Increased platelet destruction? Up to 50% of patients have apoptosis in MDS.35 Apart from inducing apoptosis, stimulation a decreased platelet lifespan, suggesting that increased periph- of Fas and other death receptors (i.e. tumor necrosis eral platelet destruction mediated by (non-)immune mechan- factor-receptor 1) can also trigger caspase-independent 19 isms might contribute to thrombocytopenia in MDS. Findings pathways leading to necrosis-like PCD.36 Crucial in that several immunomodulatory agents and splenectomy lead to Fas-mediated signaling to either apoptotic or necrosis-like an increase in platelet counts in some MDS patients may support PCD is the Fas-associated death domain (FADD). When 20,21 this notion. caspases are inhibited, signaling via FADD leads to necrosis- like PCD. Alternatively, FADD-induced necrotic PCD can be Decreased platelet production. However, thrombocyto- reverted to apoptosis by degradation of receptor-interacting penia in MDS is mainly caused by ineffective platelet protein 1.36 Thus, as it is the cellular context that determines production resulting from impaired proliferation and differentia- whether stimulation of Fas triggers apoptotic or necrosis-like

Leukemia Megakaryocytic cell death in MDS and ITP EJ Houwerzijl et al 1940

Figure 2 The ultrastructure of megakaryocytes. ITP megakaryocyte showing features of apoptosis (a; Â 5000) and apoptosis-like cell death (para- apoptosis) (b; Â 7000). (c) A megakaryocyte from a patient with MDS (refractory anemia), demonstrating characteristics of necrosis-like cell death. An apoptotic neutrophilic granulocyte is visible in the upper part of the image ( Â 5000).

Table 2 Characteristics of megakaryocytic alterations in ITP and MDS

ITP MDS

Ultrastructure Nuclear changes Nucleus in periphery of cell Nucleus centrally in the MK Chromatin condensation: Non-condensed chromatin, partly lying in spherical Some MKs: crescent-shaped (apoptosis) speckles/small clumps; Majority of MKs: without margination no nucleoli; smooth non-lobulated outlines43 (compatible with para-apoptosis)19 Cytoplasmic changes Vacuoles, swelling of endoplasmic Cytoplasmic vacuoles; mitochondrial disruption reticulum, mitochondria with disrupted cristae reduced numbers of granules, disordered DMS43 and distended DMS; enlarged peripheral zone (para-apoptosis)19

DNA fragmentation TUNEL/ISEL Normal in children with acute and chronic ITP Conﬂicting results (see text) (bone marrow aspirates)48

Caspase activity Immunohistochemistry Megakaryocytes with apoptotic morphology Caspase-3: conﬂicting results: negative43/positive50 were caspase-3 positive19 Caspase-8: minority (o10%) positive19 Possible stimuli Factors in ITP plasma induce abnormalities Fas/Fas-L?a compatible with PCD19 Possible inhibitors Prednisolon, IVIG19 Unknown Abbreviations: DMS, demarcation membrane system; Fas-L, Fas-ligand; ISEL, in situ endlabeling; ITP, idiopathic thrombocytopenic purpura; IVIG, intravenous immunoglobulins; MDS, myelodysplastic syndromes; MK, megakaryocyte; PCD, programmed cell death; TUNEL, TdT-mediated dUTP-biotin nick end labeling. aBlom et al., unpublished results.

PCD, increased Fas/FasL expression in bone marrow cells of PCD of megakaryocytes in ITP MDS patients might also be related to the presence of necrosis- like PCD. A similar switch in PCD might occur dependent on in Is platelet production impaired in ITP? vivo or in vitro conditions. Caspase-independent cell death In classical ITP, platelet’s lifespan is greatly reduced owing to might occur primarily in bone marrow megakaryocytes, but may accelerated immune-mediated peripheral platelet destruction, switch to apoptosis when the cells are taken from their predominantly in the spleen. As a result, platelet production is microenvironment. Although some in vitro studies suggest that considered to be compensatory increased, reﬂected by an MDS stromal cells induce apoptosis in hematopoietic cells,14 in elevated number of megakaryocytes in the bone marrow vivo it may be possible that prosurvival signals provided by the and by an increased platelet turnover determined with microenvironment inhibit the apoptotic pathway. These signals radiolabeled platelet studies.38 However, many observations might, in concert with the intrinsic cellular defects in signaling have questioned this traditional view on ITP. Early morpho- pathways,37 make the MDS megakaryocytes vulnerable for logical studies revealed that the number of megakaryocytes necrosis-like PCD. When the remaining prosurvival signals in ITP patients is often normal instead of increased, and that an disappear owing to the detachment of megakaryocytes from the increased number of megakaryocytes does not always microenvironment, the cells might become programmed to the imply increased platelet production.39 Most megakaryocytes apoptotic route. in these observations were morphologically altered and In summary, the mechanisms underlying defective megakar- surrounded by a greatly diminished number of platelets. In yocytopoiesis in MDS are not completely elucidated. PCD of addition, platelet kinetic studies have identiﬁed large sub- mature megakaryocytes probably contributes to thrombocyto- groups of ITP patients with a decreased or normal platelet penia; however, apoptosis may play a secondary role. turnover.40,41

Leukemia Megakaryocytic cell death in MDS and ITP EJ Houwerzijl et al 1941 Morphological alterations of megakaryocytes Concluding remarks Morphological changes in ITP megakaryocytes, such as extensive cytoplasmic vacuolization, hypogranularity and smoothing In the last decade, the knowledge on PCD has expanded of the cell membrane, were already described by Frank substantially. The classical division of cell death in apoptosis, in 1915, and later confirmed by others.39 It was argued that which is often used as a synonym of PCD, and necrosis has these alterations were artifacts induced by fixation and/or evolved to a broad spectrum of PCD in which both apoptosis, staining methods. Similar abnormalities, however, were found necrosis-like PCD, autophagic cell death and mixed forms have using phase-contrast microscopy, by which cells can be their place. PCD is an essential element of normal and examined in the living and unstained state.42 Ultrastructurally, pathological cell physiology. This also accounts for the a majority of ITP megakaryocytes show alterations,43 including megakaryocytopoiesis. Like many cells, megakaryocytes possess cytoplasmic vacuoles owing to swelling of mitochondria and several pathways that lead to cell death. In normal physiological endoplasmic reticulum and chromatin condensation. This conditions, elements of the apoptotic system are required for the morphology resembles features of PCD, including apoptosis formation of platelets; in diseases, such as MDS and ITP, (confirmed by detection of caspase-3 activity) and para- inappropriate, apoptotic and non-apoptotic, PCD of megakaryo- apoptosis, a form of active caspase-3-negative,43 TUNEL-44 cytes occurs. In ITP, megakaryocytes are intrinsically normal and ISEL-45 negative apoptosis-like PCD (Figure 2a–b and cells and PCD is induced by external factors. MDS megakar- Table 2). Para-apoptotic megakaryocytes have also been yocytes undergo increased PCD probably as a consequence of described in idiopathic myelofibrosis45 and GATA-1low mice.44 extensive intrinsic defects and a perturbed response to (possible In these mice, blocked megakaryocyte maturation results in an aberrant) extrinsic factors from the bone marrow microenviron- accumulation of defective megakaryocytes showing increased ment. Future research has to focus on the exact triggers, neutrophil emperipolesis. Subsequent release of neutrophilic mechanisms and clinical implications of PCD in these disorders, proteases in the megakaryocyte cytoplasm might induce para- in order to generate more insight into the pathophysiology of apoptosis.44 thrombocytopenia in ITP and MDS and to develop new An alternative explanation for the extensive cytoplasmic treatment strategies, especially for patients with refractory vacuoles in ITP megakaryocytes might be autophagy. Consider- disease and symptomatic thrombocytopenia. Although blocking ing a state of compensatory increased megakaryocytopoiesis caspases often stops the apoptotic process, PCD is frequently not and therefore a state of increased metabolic demand and prevented and other, caspase-independent, forms of cell death relative nutrient deficiency, autophagy might be a mechanism develop instead. As mitochondrial dysfunction (especially for generating enough energy to maintain cell metabolism. MOMP) represents the ‘point of no return’ of cell death, Alternatively, autophagy might be a way of sequestering and preventing mitochondrial dysfunction or common regulatory degrading specific pathogens, such as immunoglobulins in the mechanisms upstream of mitochondrial dysfunction might be case of ITP. In this situation, autophagy may end in type II PCD promising for novel therapeutic interventions. Although a major or perhaps apoptosis, when autophagy has reached its limits. problem in developing these strategies is drug specificity, so that The observed cytoplasmic vacuoles in megakaryocytes of ITP interventions do not influence normal cell physiology, numer- patients, however, appear of non-lysosomal origin. They are ous strategies of inhibition of cell death that target death mainly not double membraned, mostly empty and appear to receptors and their ligands, p53, stress kinases and proteases, originate predominantly from dilated organelles. and MOMP, are currently investigated and in some instances already available for clinical use.13

Acknowledgements

The etiology of the megakaryocyte alterations in ITP This work was supported by a grant from the JK de Cock Some have ascribed these megakaryocytic changes to increased Foundation and the Dutch Cancer Society (2003–2920). compensatory megakaryocytopoiesis, as similar abnormalities have been described in megakaryocytes from animals made thrombocytopenic by thrombocytopheresis.46 Increased thrombo- References cytopenia-induced thrombopoiesis should theoretically lead to elevated numbers of denuded megakaryocytes.3 To our knowl- 1 Patel SR, Hartwig JH, Italiano Jr JE. The biogenesis of platelets from edge, this has not been reported in ITP bone marrow.47,48 megakaryocyte proplatelets. J Clin Invest 2005; 115: 3348–3354. 43 2 Radley JM, Haller CJ. Fate of senescent megakaryocytes in the Furthermore, the damaged megakaryocytes found in ITP bone marrow. Br J Haematol 1983; 53: 277–287. show no ultrastructural resemblance to these denuded mega- 3 Zauli G, Vitale M, Falcieri E, Gibellini D, Bassini A, Celeghini C karyocytes. et al. In vitro senescence and apoptotic cell death of human There is evidence that factors in ITP plasma, possibly megakaryocytes. Blood 1997; 90: 2234–2243. antiplatelet autoantibodies, are responsible for the megakaryo- 4 De Botton S, Sabri S, Daugas E, Zermati Y, Guidotti JE, Hermine O cyte alterations in ITP. Morphological alterations resembling et al. Platelet formation is the consequence of caspase activation within megakaryocytes. Blood 2002; 100: 1310–1317. those found in ITP megakaryocytes could be induced in 5 Edinger AL, Thompson CB. Death by design: apoptosis, necrosis megakaryocytes from healthy persons within 2 h after intra- and autophagy. Curr Opin Cell Biol 2004; 16: 663–669. venous injections of plasma from ITP patients.42 We found 6 Kitanaka C, Kuchino Y. Caspase-independent programmed corresponding results with electron microscopy in megakaryo- cell death with necrotic morphology. Cell Death Differ 1999; 6: cytes cultivated in the presence of ITP plasma.43 It has been 508–515. shown that antiplatelet autoantibodies can recognize antigens 7 Leist M, Ja¨a¨ttela¨ M. Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2001; 2: 589–598. on megakaryocytes and can suppress the in vitro production 8 Assunc¸ao Guimaraes C, Linden R. Programmed cell deaths. 49 ˜ ˜ and maturation of megakaryocytes, suggesting that platelet Apoptosis and alternative deathstyles. Eur J Biochem 2004; 271: production in many ITP patient is suppressed. 1638–1650.

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