In vitro large scale production of megakaryocytes to functional platelets from human hematopoietic stem cells.

Megakaryocytopoiesis results in the formation of platelets, which are essential for hemostasis. Decreased production or increased destruction of platelets can cause thrombocytopenia, in which platelet transfusion is the mode of treatment. The present study is aimed in generation of megakaryocytes (MKs) and platelet from human hematopoietic stem cells (HSCs). The purity of HSCs was assessed through Flow cytometry and immunocytochemistry (ICC) studies. These pure HSCs were induced with thrombopoietin (TPO), similarly with Andrographis paniculata extract (APE) for 21 days to generate MKs. The APE is mainly composed of andrographolide which stimulates TPO from the liver, and this binds to CD110 present on the surface of HSCs and triggers the proliferation of HSCs and initiate higher MKs population subsequently, a large number of platelets. The results of the present study showed increased proliferation of HSCs grown in the presence of APE and revealed a high population of CD41a and CD42b positive MKs as enumerated by Flow cytometry compared with TPO induced MKs. These results also concurred with qRT-PCR and western blot analysis. The scanning electron microscopy (SEM) revealed the morphology of differentiated MKs and platelets were similar to human blood platelets. The differentiated MKs in APE exhibited polyploidy up to 32 N while TPO induced MKs showed polyploidy of 8 N, these results corroborated with colony forming unit assay. On thrombin stimulation, high expression of P-selectin (CD62p) and fibrinogen binding were detected in APE induced platelets. Autologous transplantation of platelets generated from APE may be a useful option in thrombocytopenia condition.

Detachment of surface membrane invagination systems by cationic amphiphilic drugs.

Several cell types develop extensive plasma membrane invaginations to serve a specific physiological function. For example, the megakaryocyte demarcation membrane system (DMS) provides a membrane reserve for platelet production and muscle transverse (T) tubules facilitate excitation:contraction coupling. Using impermeant fluorescent indicators, capacitance measurements and electron microscopy, we show that multiple cationic amphiphilic drugs (CADs) cause complete separation of the DMS from the surface membrane in rat megakaryocytes. This includes the calmodulin inhibitor W-7, the phospholipase-C inhibitor U73122, and anti-psychotic phenothiazines. CADs also caused loss of T tubules in rat cardiac ventricular myocytes and the open canalicular system of human platelets. Anionic amphiphiles, U73343 (a less electrophilic U73122 analogue) and a range of kinase inhibitors were without effect on the DMS. CADs are known to accumulate in the inner leaflet of the cell membrane where they bind to anionic lipids, especially PI(4,5)P2. We therefore propose that surface detachment of membrane invaginations results from an ability of CADs to interfere with PI(4,5)P2 interactions with cytoskeletal or BAR domain proteins. This establishes a detubulating action of a large class of pharmaceutical compounds.

Kv1.3 is the exclusive voltage-gated K+ channel of platelets and megakaryocytes: roles in membrane potential, Ca2+ signalling and platelet count.

A delayed rectifier voltage-gated K(+) channel (Kv) represents the largest ionic conductance of platelets and megakaryocytes, but is undefined at the molecular level. Quantitative RT-PCR of all known Kv alpha and ancillary subunits showed that only Kv1.3 (KCNA3) is substantially expressed in human platelets. Furthermore, megakaryocytes from Kv1.3(/) mice or from wild-type mice exposed to the Kv1.3 blocker margatoxin completely lacked Kv currents and displayed substantially depolarised resting membrane potentials. In human platelets, margatoxin reduced the P2X(1)- and thromboxaneA(2) receptor-evoked [Ca(2+)](i) increases and delayed the onset of store-operated Ca(2+) influx. Megakaryocyte development was normal in Kv1.3(/) mice, but the platelet count was increased, consistent with a role of Kv1.3 in apoptosis or decreased platelet activation. We conclude that Kv1.3 forms the Kv channel of the platelet and megakaryocyte, which sets the resting membrane potential, regulates agonist-evoked Ca(2+) increases and influences circulating platelet numbers.

Assessment of the toxicity of Artemisia inculta extract on the bone marrow of mice infected with schistosomiasis.

UNLABELLED: The discovery of the crude extract of Artemisia inculta (an Egyptian species of the Artemisia plant) for the therapy of schistosomiasis has evoked the study of its long-term toxicity produced by antischistosomal doses. Hence, an assessment of the noxious effects on bone marrow was attempted in this study. Transmission electron microscopy of the bone marrow was conducted on infected mice receiving a crude ethanolic extract from Artemisia inculta by newly designed dose regimens (500 or 800 mg/kg body weight in the 7th, 14th and 21st weeks post infection). This regimen was found to produce an efficient therapeutic effect in decreasing worm load and causing tegumental damage of juvenile and adult worms. The ultrastructural features of all cell lines in the bone marrow were comparable in both treated (500 and 800 mg/kg body weight dose levels) and untreated groups showing normal cellularity, maturation and morphology. This denotes that the drug is non-toxic to the hemopoietic tissue. IN CONCLUSION: Electron microscopy has provided a direct and accurate evidence of the nontoxic property of the plant extract on bone marrow using therapeutic doses. However, this study should be extended to other vital organs such as the liver or brain to establish the safety of this drug.