The Experts below are selected from a list of 243 Experts worldwide ranked by ideXlab platform
Pudur Jagadeeswaran - One of the best experts on this subject based on the ideXlab platform.
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g protein coupled receptor gpr34l mutation affects Thrombocyte function in zebrafish
British Journal of Haematology, 2018Co-Authors: Abdullah Alsrhani, Gauri Khandekar, Lala Zafreen, Florence L Marlow, Elliott W Abrams, Mary C Mullins, Pudur JagadeeswaranAbstract:: Haemostasis is a defence mechanism that has evolved to protect organisms from losing their circulating fluid. We have previously introduced zebrafish as a model to study the genetics of haemostasis to identify novel genes that play a role in haemostasis. Here, we identify a zebrafish mutant that showed prolonged time to occlusion (TTO) in the laser injury venous thrombosis assay. By linkage analysis and fine mapping, we found a mutation in the orphan G protein-coupled receptor 34 like gene (gpr34l) causing a change of Val to Glu in the third external loop of Gpr34l. We have shown that injection of zebrafish gpr34l RNA rescues the prolonged TTO defect. The Thrombocytes from the mutant showed elevated levels of cAMP that supports the defective Thrombocyte function. We also have demonstrated that knockdown of this gene by intravenous Vivo-Morpholino injections yielded a phenotype similar to the gpr34l mutation. These results suggest that the lack of functional Gpr34l leads to increased cAMP levels that result in defective Thrombocyte aggregation.
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Intraflagellar transport proteins are involved in Thrombocyte filopodia formation and secretion
Platelets, 2017Co-Authors: Uvaraj P. Radhakrishnan, Abdullah Alsrhani, Hemalatha Sundaramoorthi, Meghana V. Kashyap, Jannon L Fuchs, Gauri Khandekar, Brian D. Perkins, Yoshihiro Omori, Pudur JagadeeswaranAbstract:AbstractIntraflagellar transport (IFT) proteins are vital for the genesis and maintenance of cilia. Our identification of ift122 transcripts in zebrafish Thrombocytes that lack primary cilia was unexpected. IFT proteins serve transport in cilia, whose narrow dimensions may have necessitated the evolution of IFT from vesicular transport in ancestral eukaryotes. We hypothesized that IFTs might also facilitate transport within the filopodia that form when Thrombocytes are activated. To test this possibility, we knocked down ift122 expression by injecting antisense Morpholino oligonucleotides (MOs) into zebrafish embryos. Laser-induced arterial thrombosis showed prolonged time to occlusion (TTO) of the vessel, as would be expected with defective Thrombocyte function. Acute effects in adult zebrafish were evaluated by Vivo-Morpholino (Vivo-MO) knockdown of ift122. Vivo-MO morphants showed a prolonged time to Thrombocyte aggregation (TTA) in the plate tilt assay after Thrombocyte activation by the following ago...
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dioxin induced Thrombocyte aggregation in zebrafish
Blood Cells Molecules and Diseases, 2015Co-Authors: Hemalatha Sundaramoorthi, Pudur JagadeeswaranAbstract:Abstract 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a canonical member of a group of dioxins which are byproducts of industrial combustion and are dangerous environmental pollutants. TCDD has been shown to cause several abnormalities in humans and wildlife, and recently, some dioxins have been found to activate platelets. However, TCDD-mediated platelet activation pathways are elusive and virtually nothing is known about TCDD activation of fish Thrombocytes. To investigate TCDD effect on Thrombocyte function, we tested zebrafish blood in presence of TCDD using a Thrombocyte functional assay. We found that TCDD activated Thrombocytes. Further experiments showed that Thrombocytes of fish treated with TCDD formed both aggregates and filopodia. To investigate the mechanism of TCDD-mediated activation of Thrombocytes we used inhibitors for Gq, cyclooxygenase-1, aryl hydrocarbon receptor (AHR), c-src, Akt, and ERK1/2. We found that TCDD induces AHR which activates c-src and signals the activation of Akt and ERK1/2 which are ultimately involved in generation of thromboxane A2. Furthermore, we found that ADP potentiates TCDD action, which led to the discovery that ADP itself activates AHR in the absence of TCDD. Taken together, these results resolved the pathway of TCDD activation of Thrombocytes and led to the finding that ADP is an activator of AHR.
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Zebrafish Thrombocytes: Functions and Origins
Advances in Hematology, 2012Co-Authors: Gauri Khandekar, Pudur JagadeeswaranAbstract:Platelets play an important role in mammalian hemostasis. Thrombocytes of early vertebrates are functionally equivalent to mammalian platelets. A substantial amount of research has been done to study platelet function in humans as well as in animal models. However, to date only limited functional genomic studies of platelets have been performed but are low throughput and are not cost-effective. Keeping this in mind we introduced zebrafish, a vertebrate genetic model to study platelet function. We characterized zebrafish Thrombocytes and established functional assays study not only their hemostatic function but to also their production. We identified a few genes which play a role in their function and production. Since we introduced the zebrafish model for the study of hemostasis and thrombosis, other groups have adapted this model to study genes that are associated with Thrombocyte function and a few novel genes have also been identified. Furthermore, transgenic zebrafish with GFP-tagged Thrombocytes have been developed which helped to study the production of Thrombocytes and their precursors as well as their functional roles not only in hemostasis but also hematopoiesis. This paper integrates the information available on zebrafish Thrombocyte function and its formation.
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role of zebrafish Thrombocyte and non Thrombocyte microparticles in hemostasis
Blood Cells Molecules and Diseases, 2012Co-Authors: Maira Carrillo, Uvaraj P. Radhakrishnan, Pudur JagadeeswaranAbstract:Hemostasis is a defense mechanism that protects an organism from bleeding in the event of injury. We have previously demonstrated the utility of the zebrafish as a model to study human hemostasis. However, there are no studies on the role of microparticles in hemostasis in early vertebrates. Studying microparticles in zebrafish may provide insight into the evolution of microparticle function in hemostasis and may lead to direct observation of these microparticles in zebrafish larvae due to transparency of the vessels. In this investigation we demonstrate the presence of cellular microparticles in fish blood by both immunostaining as well as by using zebrafish whose Thrombocytes are labeled with green fluorescent protein. Further investigation showed that microparticles were also labeled by fluorescein isothiocyanate annexin V, suggesting that these particles are derived via apoptosis. A portion of the fluorescein isothiocyanate annexin V labeled microparticles was also labeled by DiI-C18. Labeling by DiI-C18 suggests that some microparticles are derived from young Thrombocytes. Additionally, GpIIb antibody labels almost all Thrombocyte-derived microparticles and a greater percentage of microparticles are labeled by GpIIb antibody than by DiI-C18. This suggests that Thrombocyte microparticles are derived from both young and mature Thrombocytes. Furthermore, the increase of microparticles by adding excessive microparticles into blood in vitro and through intravenous injections led to an increased hemostatic response. In addition, treatment with tumor necrosis factor alpha resulted in an increased number of Thrombocyte microparticles and enhanced hemostasis; in contrast, treatment with zVAD-FMK, a caspase inhibitor, resulted in a decrease in Thrombocyte microparticles and decreased hemostasis. We also found that Thrombocyte microparticles agglutinate, along with other cells and cellular microparticles, in the presence of an excess of either ristocetin or ultra-large von Willebrand factor. Also, stimulation of von Willebrand factor release in vivo resulted in clusters of Thrombocyte microparticles in the veins. Moreover, Thrombocyte microparticles were the first to appear at the site of arterial injury. We found that Thrombocyte microparticles are functionally equivalent to platelet microparticles. The microparticles initiate arterial thrombus formation in a von Willebrand factor-dependent manner and further enhance thrombus formation by forming clusters of microparticles in venous thrombosis. This finding may have applications for understanding the role of platelet microparticles in humans and may have diagnostic applications.
Robert I Handin - One of the best experts on this subject based on the ideXlab platform.
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analysis of Thrombocyte development in cd41 gfp transgenic zebrafish
Blood, 2005Co-Authors: David Traver, Kimberly Dooley, Robert I HandinAbstract:Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (αIIb, glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)–tagged Thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase–polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of Thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFPhigh subset, which had the morphologic appearance of mature Thrombocytes, and a GFPlow subset with an immature appearance, suggesting that they may be Thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating Thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify Thrombocytes, Thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.
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analysis of Thrombocyte development in cd41 gfp transgenic zebrafish commentary
Blood, 2005Co-Authors: Pudur Jagadeeswaran, David Traver, Kimberly Dooley, Robert I HandinAbstract:Thrombocytes are the nucleated equivalent of platelets In nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (α IIb , glycoprotein IIb [GPllb]) and its promoterandhavegeneratedtransgeniczebrafish lines with green fluorescent protein (GFP)-tagged Thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of Thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP high subset, which had the morphologic appearance of mature Thrombocytes, and a GFP low subset with an immature appearance, suggesting that they may be Thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP + cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating Thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP + cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify Thrombocytes, Thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.
Yuta Tanizaki - One of the best experts on this subject based on the ideXlab platform.
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Thrombopoietin induces production of nucleated Thrombocytes from liver cells in Xenopus laevis
Scientific Reports, 2015Co-Authors: Yuta Tanizaki, Takako Ishida-iwata, Miyako Obuchi-shimoji, Megumi Ichisugi, Ayaka Tahara-mogi, Mizue Meguro-ishikawa, Takashi KatoAbstract:The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated Thrombocytes instead of platelets, the features of nucleated Thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO ( xl TPO). Hepatic and splenic cells were cultured in liquid suspension with recombinant xl TPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X . laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xl TPO produced mature, spindle-shaped Thrombocytes that were activated by thrombin, thereby altering their morphology. Xl TPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xl TPO enhanced viability of peripheral Thrombocytes, indicating the xl TPO-Mpl pathway stimulates anti-apoptotic in peripheral Thrombocytes. The development of Thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200).
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Cellular characterization of Thrombocytes in Xenopus laevis with specific monoclonal antibodies.
Experimental Hematology, 2014Co-Authors: Yuta Tanizaki, Takako Ishida-iwata, Miyako Obuchi-shimoji, Takashi KatoAbstract:Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped Thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as Thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog ( Xenopus laevis ), we needed to distinguish nucleated Thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated Thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis Thrombocytes. Whereas T5 recognized both Thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped Thrombocytes. The T12 + Thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl 2 , adenosine diphosphate, thrombin, or various collagens, T12 + Thrombocytes exhibited aggregation. These Thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating Thrombocytes exhibited lower DNA ploidy than hepatic Thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating Thrombocytes. Although differences between MKs and Thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.
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proliferation and differentiation of Thrombocyte progenitors in the liver and the spleen in xenopus laevis under the stimulation of thrombopoietin
Blood, 2010Co-Authors: Yuta Tanizaki, Ayaka Tahara, Sayaka Kinoshita, Motoki Yamauchi, Mizue Meguro, Shun Maekawa, Kazumichi Nagasawa, Takako Ishidaiwata, Miyako Obuchishimoji, Nami NogawakosakaAbstract:Abstract 2012 In the biology of thrombopoiesis, several challenging issues such as polyploidy induction, proplatelet formation with endomitotic maturation and tubular cytoplasmic projections, and ability of cell division as reported in human platelets, have not been elucidated sufficiently. Comparative characterization of Thrombocyte developments in animals may bring about a new perspective. Characteristics of Thrombocyte precursors as megakaryocytes (MKs) and mature Thrombocytes in most vertebrates, however, remain poorly defined. Most non-mammalian vertebrates have nucleated and spindle Thrombocytes instead of platelets. Since african clawed frog, Xenopus laevis, is one of the most popular species providing various animal models in embryology and physiology, we attempt to establish an adult Xenopus model for analyses of hematopoiesis. We clarified peripheral Thrombocytes by various staining methods, and searched immature thrombocytic cells in Xenopus organs. When peripheral blood cells were subjected to acetylcholinesterase staining, Thrombocytes in the circulation, i.e. mature Thrombocytes were positively identified. The size of elliptical mature Thrombocytes was approx. 20.5±0.6 μm by 7.6±1.1 μm in diameters on cytocentrifuge preparations. We produced monoclonal antibody to Xenopus mature Thrombocytes (T12) previously. The subsequent flow cytometry with a FACSAria II cell sorter revealed that the proportion of the peripheral T12-positive Thrombocytes in lower FSC and SSC ranges were 1.5±0.3% of whole peripheral blood cells, and the expression of Xenopus c-Mpl (xlMpl) mRNA in the sorted cells was detected by RT-PCR. The mRNA expressions of Xenopus TPO (xlTPO) and xlMpl were also detected predominantly in the spleen and the liver, indicating that the sites of Thrombocyte progenitor-residing organ and thrombopoietic activity-releasing organ were coincident in adult Xenopus. This resembled the relationship between Xenopus erythropoietin (EPO) and EPO receptor-expressing erythrocytic progenitors, as we have reported (Nogawa-Kosaka et al, 2010, Exp Hematol). Next, immunohistochemical analysis with T12 antibody revealed that thrombocytic cells were localized in sinusoid of the liver and the spleen. We then performed a thrombocytic colony assay in the presence of recombinant xlTPO expressed in E. coli. Hepatic and splenic cells composed of respective 80,000 cells in 1mL were incubated in 35mm dishes at 23°C under 5% CO2 with 0.87% methylcellulose-based semi-solid medium containing 20% FCS and xlTPO (5 ng/ml). The xlTPO-induced colonies derived from the spleen, including T12 positive thrombocytic colonies, emerged after 2 days, and the number reached to 65±2 in the culture (1 mL). The number of liver-derived colonies was smaller than that of spleen-derived ones, indicating that the density of Thrombocyte progenitors in Xenopus was higher in the spleen, but the total mass of Thrombocyte progenitors in the body is mostly distributed in the liver based on ratio by organ weights. In Xenopus, moderate thrombocytopenia, as well as anemia, was induced by phenylhydrazine (PHZ). The nadir of circulating Thrombocyte counts was observed 4 days after PHZ-administration. When we culture cells of the liver or the spleen in the presence of the PHZ-induced thrombocytopenic serum, colonies composed of white cells and red cells were developed, suggesting that multiple or bipotent hematopoietic progenitors existed. When the hepatic cells were stimulated by xlTPO (5 ng/ml) for 2 days in the liquid culture, T12-positive megakaryocytic larger cells with multinucleated spherical shapes (approx. 30 ±3 μm in diameter) appeared, and such cells did not appear under EPO stimulation. On the other hand, the size of megakaryocytic cells derived from the spleen was smaller. Regardless of the origin of the Thrombocyte progenitors, the cells stimulated by xlTPO in the liquid cultures expressed mRNAs of c-Mpl, CD41 and Fli-1, demonstrating that Thrombocyte progenitors at different development stages resided in the liver and the spleen. It is still a missing piece of the puzzle whether Xenopus Thrombocyte progenitors or mature Thrombocytes undergo endomitosis to generate higher polyploid cells under the stimulation by TPO; however the unique megakaryocytic cells observed in this study have a clue to reveal the cellular evolution of platelets/MKs. Disclosures: No relevant conflicts of interest to declare.
David Traver - One of the best experts on this subject based on the ideXlab platform.
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analysis of Thrombocyte development in cd41 gfp transgenic zebrafish
Blood, 2005Co-Authors: David Traver, Kimberly Dooley, Robert I HandinAbstract:Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (αIIb, glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)–tagged Thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase–polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of Thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFPhigh subset, which had the morphologic appearance of mature Thrombocytes, and a GFPlow subset with an immature appearance, suggesting that they may be Thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating Thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify Thrombocytes, Thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.
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analysis of Thrombocyte development in cd41 gfp transgenic zebrafish commentary
Blood, 2005Co-Authors: Pudur Jagadeeswaran, David Traver, Kimberly Dooley, Robert I HandinAbstract:Thrombocytes are the nucleated equivalent of platelets In nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (α IIb , glycoprotein IIb [GPllb]) and its promoterandhavegeneratedtransgeniczebrafish lines with green fluorescent protein (GFP)-tagged Thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of Thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP high subset, which had the morphologic appearance of mature Thrombocytes, and a GFP low subset with an immature appearance, suggesting that they may be Thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP + cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating Thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP + cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify Thrombocytes, Thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.
Takashi Kato - One of the best experts on this subject based on the ideXlab platform.
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Thrombopoietin induces production of nucleated Thrombocytes from liver cells in Xenopus laevis
Scientific Reports, 2015Co-Authors: Yuta Tanizaki, Takako Ishida-iwata, Miyako Obuchi-shimoji, Megumi Ichisugi, Ayaka Tahara-mogi, Mizue Meguro-ishikawa, Takashi KatoAbstract:The development of mammalian megakaryocytes (MKs) and platelets, which are thought to be absent in non-mammals, is primarily regulated by the thrombopoietin (TPO)/Mpl system. Although non-mammals possess nucleated Thrombocytes instead of platelets, the features of nucleated Thrombocyte progenitors remain to be clarified. Here, we provide the general features of TPO using Xenopus laevis TPO ( xl TPO). Hepatic and splenic cells were cultured in liquid suspension with recombinant xl TPO. These cells differentiated into large, round, polyploid CD41-expressing cells and were classified as X . laevis MKs, comparable to mammalian MKs. The subsequent culture of MKs after removal of xl TPO produced mature, spindle-shaped Thrombocytes that were activated by thrombin, thereby altering their morphology. Xl TPO induced MKs in cultured hepatic cells for at least three weeks; however, this was not observed in splenic cells; this result demonstrates the origin of early haematopoietic progenitors in the liver rather than the spleen. Additionally, xl TPO enhanced viability of peripheral Thrombocytes, indicating the xl TPO-Mpl pathway stimulates anti-apoptotic in peripheral Thrombocytes. The development of Thrombocytes from MKs via the TPO-Mpl system in X. laevis plays a crucial role in their development from MKs, comparable to mammalian thrombopoiesis. Thus, our results offer insight into the cellular evolution of platelets/MKs in vertebrates. (200/200).
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Cellular characterization of Thrombocytes in Xenopus laevis with specific monoclonal antibodies.
Experimental Hematology, 2014Co-Authors: Yuta Tanizaki, Takako Ishida-iwata, Miyako Obuchi-shimoji, Takashi KatoAbstract:Platelets are produced from megakaryocytes (MKs) in the bone marrow. In contrast, most nonmammalian vertebrates have nucleated and spindle-shaped Thrombocytes instead of platelets in their circulatory systems, and the presence of MKs as Thrombocyte progenitors has not been verified. In developing a new animal model in adult African clawed frog ( Xenopus laevis ), we needed to distinguish nucleated Thrombocytes and their progenitors from other blood cells, because the cellular morphology of activated Thrombocytes resembles lymphocytes and other cells. We initially generated two monoclonal antibodies, T5 and T12, to X. laevis Thrombocytes. Whereas T5 recognized both Thrombocytes and leukocytes, T12 specifically reacted to spindle-shaped Thrombocytes. The T12 + Thrombocytes displayed much higher DNA ploidy than nucleated erythrocytes, and they expressed CD41 and Fli-1. In the presence of CaCl 2 , adenosine diphosphate, thrombin, or various collagens, T12 + Thrombocytes exhibited aggregation. These Thrombocytes were located predominantly in the hepatic sinusoids and the splenic red pulp, suggesting that both organs are the sites of thrombopoiesis. Notably, circulating Thrombocytes exhibited lower DNA ploidy than hepatic Thrombocytes. Intraperitoneal administration of T12 produced immune thrombocytopenia in frogs, which reached a nadir 4 days postinjection, followed by recovery, suggesting that humoral regulation maintained the number of circulating Thrombocytes. Although differences between MKs and Thrombocytes in X. laevis remain to be defined, our results provide further insight into MK development and thrombopoiesis in vertebrates.
