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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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Intraflagellar transport proteins are involved in Thrombocyte filopodia formation and secretion
2017Co-Authors: Uvaraj Radhakrishnan, Abdullah Alsrhani, Hemalatha Sundaramoorthi, Jannon L Fuchs, Gauri Khandekar, Brian D. Perkins, Yoshihiro Omori, Meghana Kashyap, Pudur JagadeeswaranAbstract:Intraflagellar 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 agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by Thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant Thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type Thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in Thrombocyte Function.
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Zebrafish Thrombocyte aggregation by whole blood aggregometry and flow cytometry
Platelets, 2015Co-Authors: Hemalatha Sundaramoorthi, Rekha Panapakam, Pudur JagadeeswaranAbstract:Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established Thrombocyte qualitative and quantitative Functional assays, quantifying Thrombocyte Function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate Thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of Thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for Thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with Thrombocyte Functional defects or to identify genes involved in Thrombocyte Function by large scale knockdowns.
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Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish Thrombocytes
Blood cells molecules & diseases, 2014Co-Authors: Hemalatha Sundaramoorthi, Gauri Khandekar, Seongcheol Kim, Pudur JagadeeswaranAbstract:Morpholino and vivo-morpholino gene knockdown methods have been used to study Thrombocyte Function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study Thrombocyte Function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish Thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the Thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to Thrombocyte Functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study Thrombocyte Function in zebrafish.
Gauri Khandekar - 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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Intraflagellar transport proteins are involved in Thrombocyte filopodia formation and secretion
2017Co-Authors: Uvaraj Radhakrishnan, Abdullah Alsrhani, Hemalatha Sundaramoorthi, Jannon L Fuchs, Gauri Khandekar, Brian D. Perkins, Yoshihiro Omori, Meghana Kashyap, Pudur JagadeeswaranAbstract:Intraflagellar 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 agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by Thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant Thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type Thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in Thrombocyte Function.
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Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish Thrombocytes
Blood cells molecules & diseases, 2014Co-Authors: Hemalatha Sundaramoorthi, Gauri Khandekar, Seongcheol Kim, Pudur JagadeeswaranAbstract:Morpholino and vivo-morpholino gene knockdown methods have been used to study Thrombocyte Function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study Thrombocyte Function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish Thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the Thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to Thrombocyte Functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study Thrombocyte Function in zebrafish.
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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.
Hemalatha Sundaramoorthi - One of the best experts on this subject based on the ideXlab platform.
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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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Intraflagellar transport proteins are involved in Thrombocyte filopodia formation and secretion
2017Co-Authors: Uvaraj Radhakrishnan, Abdullah Alsrhani, Hemalatha Sundaramoorthi, Jannon L Fuchs, Gauri Khandekar, Brian D. Perkins, Yoshihiro Omori, Meghana Kashyap, Pudur JagadeeswaranAbstract:Intraflagellar 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 agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by Thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant Thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type Thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in Thrombocyte Function.
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Zebrafish Thrombocyte aggregation by whole blood aggregometry and flow cytometry
Platelets, 2015Co-Authors: Hemalatha Sundaramoorthi, Rekha Panapakam, Pudur JagadeeswaranAbstract:Zebrafish has become an excellent model system to study mammalian hemostasis. Despite our extensive efforts to develop technologies to measure zebrafish hemostasis and even with previously established Thrombocyte qualitative and quantitative Functional assays, quantifying Thrombocyte Function for high throughput applications has been a challenge. In this paper, we have developed two quantitative methods to estimate Thrombocyte aggregation: one by whole blood aggregometry and the other by flow cytometry. We found that it is possible to conduct whole blood aggregometry using only 2 µl of blood and the currently available aggregometer. Each of three agonists, arachidonic acid, ADP, and collagen yielded impedance curves similar to those obtained with human blood. We were also able to use flow cytometry to indirectly quantify the extent of Thrombocyte aggregation by labeling whole blood with mepacrine, aggregating in the presence of each of the above agonists, separating the aggregates from the white blood cells by centrifugation, and then sorting the resulting white cell fraction for Thrombocyte numbers. These methods have high throughput capabilities and have the potential to be used in large scale screens to detect and characterize mutants with Thrombocyte Functional defects or to identify genes involved in Thrombocyte Function by large scale knockdowns.
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Knockdown of αIIb by RNA degradation by delivering deoxyoligonucleotides piggybacked with control vivo-morpholinos into zebrafish Thrombocytes
Blood cells molecules & diseases, 2014Co-Authors: Hemalatha Sundaramoorthi, Gauri Khandekar, Seongcheol Kim, Pudur JagadeeswaranAbstract:Morpholino and vivo-morpholino gene knockdown methods have been used to study Thrombocyte Function in zebrafish. However, a large-scale knockdown of the entire zebrafish genome using these technologies to study Thrombocyte Function is prohibitively expensive. We have developed an inexpensive gene knockdown method, which uses a hybrid of a control vivo-morpholino and a standard antisense oligonucleotide specific for a gene. This hybrid molecule is able to deliver antisense deoxyoligonucleotides into zebrafish Thrombocytes because it piggybacks on a control vivo-morpholino. To validate use of this hybrid molecule in gene knockdowns, we targeted the Thrombocyte specific αIIb gene with a hybrid of a control vivo-morpholino and an oligonucleotide antisense to αIIb mRNA. The use of this piggyback technology resulted in degradation of αIIb mRNA and led to Thrombocyte Functional defect. This piggyback method to knockdown genes is inexpensive since one control vivo-morpholino can be used to target many different genes by making many independent gene-specific oligonucleotide hybrids. Thus, this novel piggyback technology can be utilized for cost-effective large-scale knockdowns of genes to study Thrombocyte Function in zebrafish.
Uvaraj P. Radhakrishnan - One of the best experts on this subject based on the ideXlab platform.
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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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vivo morpholino knockdown of αiib a novel approach to inhibit Thrombocyte Function in adult zebrafish
Blood Cells Molecules and Diseases, 2010Co-Authors: Uvaraj P. Radhakrishnan, Surendra Kumar Rajpurohit, Vrinda Kulkarni, Pudur JagadeeswaranAbstract:Abstract Knockdown of protein Function by antisense oligonucleotides has been used to understand the protein Function not only in development but also in human diseases. Recently, Vivo-Morpholinos, chemically modified morpholinos which penetrate the cells, have been used in adult experimental animal models to alter the splicing and thereby change the protein expression. Until now, there have been no such studies using Vivo-Morpholinos to evaluate hemostatic Function in adult animals. We injected αIIb Vivo-Morpholinos intravenously into adult zebrafish. Thrombocyte Function was assayed by time to aggregation assay of the citrated blood, annexin V binding to Thrombocytes, and gill bleeding. The Thrombocyte Functional inhibition occurred in 24 h after αIIb Vivo-Morpholinos injection and reached a maximum in 48 h. However, in 72 h, the inhibition was no longer observed. Reduction of annexin V binding to Thrombocytes and increased gill bleeding were observed 48 h after αIIb Vivo-Morpholino injections. The action of the αIIb Vivo-Morpholino was demonstrated by the presence of an alternatively spliced αIIb mRNA and the reduction of αIIb in Thrombocytes of fish treated with αIIb Vivo-Morpholino. These results provide the first proof of principle that Thrombocyte Function can be inhibited by Thrombocyte-specific Vivo-Morpholinos in adult zebrafish and presents an approach to knockdown Thrombocyte-specific genes to conduct biochemical studies in Thrombocytes. This study also provides the first antisense antithrombotic approach to inhibit Thrombocyte Function in adult zebrafish.
Abdullah Alsrhani - 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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Intraflagellar transport proteins are involved in Thrombocyte filopodia formation and secretion
2017Co-Authors: Uvaraj Radhakrishnan, Abdullah Alsrhani, Hemalatha Sundaramoorthi, Jannon L Fuchs, Gauri Khandekar, Brian D. Perkins, Yoshihiro Omori, Meghana Kashyap, Pudur JagadeeswaranAbstract:Intraflagellar 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 agonists: ADP, collagen, PAR1 peptide, and epinephrine. A luminescence assay for ATP revealed that ATP secretion by Thrombocytes was reduced in collagen-activated blood of Vivo-MO ift122 morphants. Moreover, DiI-C18 labeled morphant Thrombocytes exposed to collagen showed reductions in filopodia number and length. Analysis of ift mutants, in which cilia defects have been noted, also showed prolongation of TTO in our arterial laser thrombosis assay. Additionally, collagen activation of wild-type Thrombocytes led to a concentration of IFT122 both within and at the base of filopodia. Taken together these results, suggest that IFT proteins are involved in both the extension of filopodia and secretion of ATP, which are critical in Thrombocyte Function.