Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura

Abstract

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% ؎ 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 com- IntroductionIdiopathic, or immune, thrombocytopenic purpura (ITP) is an autoimmune disease characterized by isolated thrombocytopenia in an otherwise healthy person. The thrombocytopenia in ITP is caused by accelerated platelet destruction due to the action of antiplatelet immunoglobulin G (IgG) autoantibodies that bind to antigens on the platelet cell membrane. The platelets are subsequently destroyed by tissue macrophages, predominantly in the spleen. 1 As a result of the accelerated destruction, platelet survival is usually greatly shortened and platelet production is thought to be compensatorily increased. 2,3 However, there is also evidence that platelet production can be impaired in ITP. This was demonstrated in platelet kinetic studies using radiolabeled platelets. [4][5][6] The reduced platelet production rate might be mediated by the action of antiplatelet antibodies, which can bind to megakaryocytes in the bone marrow. [7][8][9] Recent in vitro studies support this concept showing that human megakaryocyte colony formation and proplatelet formation is inhibited 10 and that a reduced expansion of megakaryocytic progenitors can be observed especially in the presence of certain antiplatelet glycoprotein antibodies. 11 However, despite the evidence of a reduced platelet production in several ITP patients, numbers of megakaryocytes in the bone marrow are usually normal or increased. 6 This is compatible with the finding that plasma thrombopoietin (TPO) levels in ITP patients are not significantly different from healthy controls, indicating that the total megakaryocytic mass has not been changed in ITP. Investigating the relationship between thrombokinetic parameters and the glycocalicin index (GCI), a parameter of platelet destruction, 12 we recently demonstrated that there is an inverse correlation between the platelet production rate and the GCI. 13 These results suggest that despite the normal number of megakaryocytes in the bone marrow an increased destruction of platelets and/or megakaryocytes might occur. These findings support the concept of ineffective thrombopoiesis in the bone marrow. To investigate whether apoptosis or other forms of programmed cell death are responsible for this ineffective thrombopoiesis, we examined the ultrastructure of bone marrow megakaryocytes from ITP patients with electron microscopy. The results demonstrate that, independent of the refractoriness of ITP to therapy, in all patients most bone ma...

Plant Sterols Cause Macrothrombocytopenia in a Mouse Model of Sitosterolemia

“…Electron Microscopy-For ultrastructural analyses of megakaryocytes, spleen was collected and fixed overnight at 4°C in 2% glutaraldehyde and processed for electron microscopy as described previously (26).…”

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

“…Ultrastructure Nuclear changes Nucleus in periphery of cell Chromatin condensation: Some MKs: crescent-shaped (apoptosis) Majority of MKs: without margination (compatible with para-apoptosis) 19 Nucleus centrally in the MK Non-condensed chromatin, partly lying in spherical speckles/small clumps; no nucleoli; smooth non-lobulated outlines 43 Cytoplasmic changes Vacuoles, swelling of endoplasmic reticulum, mitochondria with disrupted cristae and distended DMS; enlarged peripheral zone (para-apoptosis) 19 Cytoplasmic vacuoles; mitochondrial disruption reduced numbers of granules, disordered DMS 43 DNA fragmentation TUNEL/ISEL Normal in children with acute and chronic ITP (bone marrow aspirates) 48 Conflicting results (see text)…”

Platelet production rate predicts the response to prednisone therapy in patients with idiopathic thrombocytopenic purpura

“…In addition, a reduction of premature cell death of ITP megakaryocytes has been observed during in vivo prednisone therapy [3].…”

“…Additional support for a suppressed platelet production in ITP comes from in vitro studies showing that megakaryocyte production and maturation can be inhibited by antiplatelet antibodies [1,2,37]. Similar in vitro cultured megakaryocytes in the presence of ITP plasma show features compatible with premature cell death [3]. The variability in PPR among different individuals is therefore likely to be related to the degree of autoantibody binding to megakaryocyte antigens.…”

“…Premature cell death of megakaryocytes plays an important role in the reduced platelet production [3]. However, easily applicable tests that can make a distinction between a reduced platelet half life and a reduced PPR are lacking.…”