Bone Marrow Megakaryocytes and Platelet Kinetics in Systemic Lupus Erythematosus

ABSTRACT. The megakaryocyte number and mean megakaryocyte area were determined in histological sections of sternal bone marrow from 26 patients with systemic lupus erythematosus (SLE). Also 20 platelet survival studies were carried out in these patients. The results were analyzed with respect to corticosteroid (CS) and CS+azathioprine (AT) therapy. The mean bone marrow megakaryocyte number was highest in untreated SLE patients, slightly lower in patients receiving CSs and lowest in those receiving CSs + AT. The difference was, however, not significant. The mean megakaryocyte areas were smallest in untreated SLE patients, slightly larger in those treated with CSs and significantly (p<0.05) larger in patients who received CSs + AT than in untreated patients. Platelet production rate was normal in all 3 groups of SLE patients. The results suggest that CS and AT therapy in SLE intervenes with the bone marrow megakaryopoiesis without affecting the production rate of platelets.     

Circulating megakaryocytes

Cells resembling the smaller variants of the Warthin-Finkeldey giant cells of measles were observed in the pulmonary alveolar capillaries in a high percentage of primates and humans apparently free from pathological changes associated with measles. Cells resembling megakaryocytes were found also in the liver and spleen in both groups but only in a small minority of cases. The relative appearances and distribution of these two types of cells pointed to their both being circulating megakaryocytes, the majority being filtered off in the pulmonary alveolar capillaries, where they become compressed. The paucity of these cells in other tissues is a reflection of the small number of megakaryocytes capable of passing along the alveolar capillaries. The presence of these cells in the lungs and to a lesser extent elsewhere appears to be a frequent occurrence in both diseased and healthy subjects and the factors involved in their appearance are discussed.     

Intact Megakaryocytes in the Venous Blood as a Marker for Thrombopoiesis

A total of 110 children, aged 0–15 years, were investigated for circulating megakaryocytes in cubital venous blood using the saponin-haemolysis leucoconcentration technique. The average number of megakaryocytes decreased from 17.8 per ml blood in the first year of life to 5.5 after the 6th year, which is the same value as in adult humans. The intact thrombo-cytogenic megakaryocyte value decreases from about 40 % in the first year of life to only a few percent (< 5 %) after the 10th year, the same value as in adult humans. There was a significantly higher number of megakaryocytes in children aged 0–6 years than in those aged 7–15 years. We concluded that occurrence of intact megakaryocytes ≥ 25 % in the venous blood is a sign of a normal thrombopoietic activity in the bone marrow, and the percentage of intact megakaryocytes in cubital venous blood reflects the decrease in thrombopoiesis in the bone marrow of fingers, hands and forearms during childhood. The decline in thrombopoietic activity is concentrated in three periods: 1–3, 6–7 and 10–11 years. An occurrence of intact megakaryocytes > 5 % in venous blood draining organs or bone marrow is a sign of some thrombopoietic activity.     

Peromyscus leucopus mice: a potential animal model for haematological studies

Peromyscus leucopus mice share physical similarities with laboratory mice Mus musculus (MM) but have higher agility and longer lifespan. We compared domesticated P. leucopus linville (PLL) and M. musculus C57BL/6 (MMB6) mice for cellular composition of peripheral blood (PB), bone marrow (BM) and spleen. PLL mice had significantly fewer platelets and significantly more monocytes in the blood, and notably fewer megakaryocytes in the BM. Spleens of PLL mice were significantly smaller, with 50% fewer cells and reduced ‘red pulp’. There was no obvious haematological change in PLL mice between 2–8 and 16–26 months of age, except for a significant increase in blood monocytes. Cellular reactive oxygen species (ROS) content showed no change with age but differed significantly between different cell types. Treating two to eight month‐old PLL mice with antioxidant N‐acetylcysteine in drinking water for three  months did not affect cellular ROS content, but increased blood leucocytes especially the concentration of monocytes. The low platelets, low megakaryocytes, high monocytes and low splenic erythropoiesis in PLL mice resemble human measurements better than the values seen in MMB6.     

How many Ca2+ATPase isoforms are expressed in a cell type? A growing family of membrane proteins illustrated by studies in platelets

Ca²⁺ signaling plays a key role in normal and abnormal platelet functions. Understanding platelet Ca²⁺ signaling requires the knowledge of proteins involved in this process. Among these proteins are Ca²⁺ATPases or Ca²⁺ pumps that deplete the cytosol of Ca²⁺ ions. Here, we will particularly focus on two Ca²⁺ pump families: the plasma membrane Ca²⁺ATPases (PMCAs) that extrude cytosolic Ca²⁺ towards the extracellular medium and the sarco/endoplasmic reticulum Ca²⁺ATPases (SERCAs) that pump Ca²⁺ into the endoplasmic reticulum (ER). In the present review, we will summarize data on platelet Ca²⁺ATPases including their identification and biogenesis. First of all, we will present the Ca²⁺ATPase genes and their isoforms expressed in platelets. We will especially focus on a member of the SERCA family, SERCA3, recently found to give rise to a number of species-specific isoforms. Next, we will describe the differences in Ca²⁺ATPase patterns observed in human and rat platelets. Last, we will analyze how the expression of Ca²⁺ATPase isoforms changes during megakaryocytic maturation and show that megakaryocytopoiesis is associated with a profound reorganization of the expression and/or activity of Ca²⁺ATPases. Taken together, these data provide new aspects of investigations to better understand normal and abnormal platelet Ca²⁺ signaling.     

Generation of platelet progeny

Platelets are derived from the cytoplasm of bone marrow megakaryocytes through a process referred to as proplatelet formation. Recent studies demonstrate that circulating anucleate mouse and human platelets retain the capacity to form bulbar extensions, which resemble megakaryocyte-derived proplatelets. Newly-formed platelets display typical morphology, possess classic platelet constituents, and functional normally. Here we review what is currently known about the formation of platelet progeny.     

CD34+ megakaryocytes (≥30%) are associated with megaloblastic anaemia and non‐acute myeloid neoplasia

Insuasti‐Beltran G, Steidler N L, Kang H & Reichard K K
(2012) Histopathology  61, 694–701 CD34+ megakaryocytes (≥30%) are associated with megaloblastic anaemia and non‐acute myeloid neoplasia Aims: To evaluate the sensitivity and specificity of CD34 staining of megakaryocytes (MKs), in order to distinguish non‐neoplastic and neoplastic bone marrows (BMs). Methods and results: Three hundred BMs (120 non‐neoplastic and 180 neoplastic) were evaluated for percentage and intensity of CD34 staining of MKs. The selected non‐neoplastic cases included anaemia, autoimmune conditions, immune thrombocytopenia (ITP), and staging BMs. The neoplastic cases included myelodysplastic syndromes and/or myeloproliferative neoplasms (MDS, MPN, MDS/MPN). Eight per cent of non‐neoplastic (9/120) cases and 13% of neoplastic (24/180) cases showed ≥30% CD34+ MKs, and these were essentially restricted to cases of megaloblastic anaemia (MBA) and non‐acute myeloid neoplasms. The finding of ≥30% CD34+ MKs did not distinguish between categories of non‐acute myeloid neoplasms. MDS cases with ≥30% CD34+ MKs had lower platelet counts than cases with <30% (P = 0.03). Conclusions: In complex BM cases, the presence of ≥30% CD34+ MKs constitutes a potentially useful diagnostic tool with which to distinguish non‐acute myeloid neoplasms and MBA from non‐MBA reactive conditions, for minimal additional cost.     

Effects of thrombopoietin on the proliferation and differentiation of primitive and mature haemopoietic progenitor cells in cord blood

Thrombopoietin (TPO) is considered to be the primary growth factor for regulating megakaryopoiesis and thrombopoiesis. In this study we investigated the in vitro effect of TPO on relatively immature and mature CD34⁺ progenitor cells in cord blood. Cells were cultured in both liquid and semi-solid cultures containing 50 ng/ml TPO. The CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions in cord blood were both enriched for megakaryocyte progenitors as determined in a semisolid CFU-meg assay. Progenitor cells derived from the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions showed high proliferative capacity in liquid cultures. We observed a mean 19-fold expansion of the total CD34⁺ cell fraction, whereas in the CD34⁺/CD45RA⁻ and CD34⁺/CD38⁻ subfractions the mean expansion was 23- and 50-fold respectively. The expansion of the immature progenitor cell subfractions resulted in a highly purified megakaryocyte suspension containing > 80% megakaryocytes after 14 d in culture. However, these expanded megakaryocytes remained in a diploid (2N) and tetraploid (4N) state. Maturation could not be further induced by low concentration of TPO (0.1 ng/ml). The majority of the cells were 2N (80%) and 4N (15%) and only 5% of the cells had a ploidy of more than 4N. These results indicate that megakaryocyte progenitor cells in cord blood residing in the immature stem cell fraction exhibit a high proliferative capacity when cultured in the presence of TPO as the single growth factor, without maturation to hyperploid megakaryocytes.     

Ultrastructural localization of the small GTP-binding protein Rap 1 in human platelets and megakaryocytes

Summary. Several functions have been proposed for Rap 1B in human platelets, including the regulation of phospholipase (PL) Cγ and Ca²⁺ ATPase. However, its localization is largely unknown. In the present study we have investigated the subcellular distribution of Rap1 by immunocytochemical techniques using affinity purified polyclonal antibodies raised against residues 121–137 common to the 95% homologous Rap1A and Rap1B proteins. By immuno-fluorescence, a positive labelling was obtained on intact resting platelets and was abolished after adsorption of the antibodies with the control peptide. Immunoelectron microscopy was then used to further define the subcellular localization of Rap1B in platelets and megakaryocytes (MK). In resting cells, immunolabelling for Rap1B was associated with the plasma membrane, mostly at its inner face, and lined the membrane of the open canalicular system (OCS). Some labelling was also found outlining the α-granules, identified as such by a double labelling with an anti-GPIIb-IIIa. On thrombasthenic platelets the same localization was observed. When platelets were stimulated by thrombin, immunolabelling for Rap1B was redistributed to the zones of fusion of the granules with the OCS, and to the plasma membrane with a higher concentration on pseudopods. Human MK expressed Rap1 and the staining revealed the association of the protein with the demarcation membranes and α-granules.
This study presents a first approach to the localization of a small GTP binding-protein Rap1B in whole platelets and MK, and shows its association with both the plasma and OCS membranes, as well as with the α-granule membranes.