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Patrick J Skelly - One of the best experts on this subject based on the ideXlab platform.

  • schistosoma mansoni glyceraldehyde 3 phosphate dehydrogenase enhances formation of the Blood Clot Lysis protein plasmin
    Biology Open, 2020
    Co-Authors: David Pirovich, Akram A Dadara, Patrick J Skelly
    Abstract:

    Schistosomes are intravascular Blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic anaLysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNAi significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to about 8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in E. coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite9s hosts, and has a Km for GAP of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thromboLysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade Blood Clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.

  • schistosoma mansoni glyceraldehyde 3 phosphate dehydrogenase enhances formation of the Blood Clot Lysis protein plasmin
    Biology Open, 2020
    Co-Authors: David Pirovich, Akram A Dadara, Patrick J Skelly
    Abstract:

    Schistosomes are intravascular Blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic anaLysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNAi significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to about 8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in E. coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite9s hosts, and has a Km for GAP of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thromboLysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade Blood Clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.

  • Schistosoma mansoni glyceraldehyde-3-phosphate dehydrogenase enhances formation of the Blood-Clot Lysis protein plasmin
    'The Company of Biologists', 2020
    Co-Authors: David Pirovich, Akram A. Da'dara, Patrick J Skelly
    Abstract:

    Schistosomes are intravascular Blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic anaLysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichia coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis–Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thromboLysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade Blood Clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo. This article has an associated First Person interview with the first author of the paper

David Pirovich - One of the best experts on this subject based on the ideXlab platform.

  • schistosoma mansoni glyceraldehyde 3 phosphate dehydrogenase enhances formation of the Blood Clot Lysis protein plasmin
    Biology Open, 2020
    Co-Authors: David Pirovich, Akram A Dadara, Patrick J Skelly
    Abstract:

    Schistosomes are intravascular Blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic anaLysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNAi significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to about 8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in E. coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite9s hosts, and has a Km for GAP of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thromboLysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade Blood Clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.

  • schistosoma mansoni glyceraldehyde 3 phosphate dehydrogenase enhances formation of the Blood Clot Lysis protein plasmin
    Biology Open, 2020
    Co-Authors: David Pirovich, Akram A Dadara, Patrick J Skelly
    Abstract:

    Schistosomes are intravascular Blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic anaLysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNAi significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to about 8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in E. coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite9s hosts, and has a Km for GAP of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thromboLysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade Blood Clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo.

  • Schistosoma mansoni glyceraldehyde-3-phosphate dehydrogenase enhances formation of the Blood-Clot Lysis protein plasmin
    'The Company of Biologists', 2020
    Co-Authors: David Pirovich, Akram A. Da'dara, Patrick J Skelly
    Abstract:

    Schistosomes are intravascular Blood flukes that cause the parasitic disease schistosomiasis. In agreement with Schistosoma mansoni (Sm) proteomic anaLysis, we show here that the normally intracellular glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is also found at the parasite surface; live worms from all intravascular life stages display GAPDH activity. Suppressing GAPDH gene expression using RNA interference significantly lowers this live worm surface activity. Medium in which the worms are cultured overnight displays essentially no activity, showing that the enzyme is not shed or excreted but remains associated with the worm surface. Immunolocalization experiments confirm that the enzyme is highly expressed in the parasite tegument (skin). Surface activity in schistosomula amounts to ∼8% of that displayed by equivalent parasite lysates. To address the functional role of SmGAPDH, we purified the protein following its expression in Escherichia coli strain DS113. The recombinant protein displays optimal enzymatic activity at pH 9.2, shows robust activity at the temperature of the parasite's hosts, and has a Michaelis–Menten constant for glyceraldehyde-3-phosphate (GAP) of 1.4 mM±0.24. We show that recombinant SmGAPDH binds plasminogen (PLMG) and promotes PLMG conversion to its active form (plasmin) in a dose response in the presence of tissue plasminogen activator. Since plasmin is a key mediator of thromboLysis, our results support the hypothesis that SmGAPDH, a host-interactive tegumental protein that can enhance PLMG activation, could help degrade Blood Clots around the worms in the vascular microenvironment and thus promote parasite survival in vivo. This article has an associated First Person interview with the first author of the paper

Xingwei Ding - One of the best experts on this subject based on the ideXlab platform.

  • localized light au hyperthermia treatment for precise rapid and drug free Blood Clot Lysis
    ACS Applied Materials & Interfaces, 2019
    Co-Authors: Lina Dong, Bixing Fang, Jinghuang Chen, Xinxin Miao, Xingwei Ding, Fen Yu, Tian Wang, Chen Li, Kui Hong, Xiaolei Wang
    Abstract:

    Thrombus diseases, induced by Blood stasis or vascular embolization normally, frequently occur with high disability and mortalities worldwide. At present, drug thromboLysis, a primary clinical ther...

  • localized light au hyperthermia treatment for precise rapid and drug free Blood Clot Lysis
    ACS Applied Materials & Interfaces, 2019
    Co-Authors: Lina Dong, Bixing Fang, Jinghuang Chen, Xinxin Miao, Tian Wang, Kui Hong, Xiao Liu, Chaochao Wei, Hongbo Xin, Xingwei Ding
    Abstract:

    Thrombus diseases, induced by Blood stasis or vascular embolization normally, frequently occur with high disability and mortalities worldwide. At present, drug thromboLysis, a primary clinical therapy for Blood Clot Lysis, could increase the lethal risk for hemorrhage when thromboLysis agents are overused in the whole body. Therefore, a novel and advanced therapy for Blood Clot Lysis, based on remote physical signals, is helpful for assisting clinical therapy. Here, we used the localized light-Au-hyperthermia (LAH) treatment, induced by gold nanorods (Au NRs) irradiated with near-infrared light (808 nm), for precise, rapid, and drug-free Blood Clot Lysis. The LAH technology was first introduced in the murine hematoma model and the murine myocardial infarction model for Blood Clot Lysis. Compared with traditional therapy, LAH was assured to shorten the time of detumescence in the murine hematoma model owing to their precise and localized hyperthermia. Meanwhile, we also discovered that LAH was a benefit to vascular recanalization in the murine myocardial infarction model. In addition, the Au NRs used in LAH present ideal biocompatibility in the murine model, which endows it to be suitable for Blood Clot Lysis in vivo.

Barbara M Alving - One of the best experts on this subject based on the ideXlab platform.

  • inhibitory effects of lysine analogues on t pa induced whole Blood Clot Lysis
    Thrombosis Research, 1994
    Co-Authors: Chitra Krishnamurti, Svetislava J Vukelja, Barbara M Alving
    Abstract:

    Abstract The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting Lysis of Clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized Blood from normal volunteers and 125I-fibrinogen in a dilute whole Blood Clot assay to determine the relative concentrations of lysine analogues required for inhibition of Clot Lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging Clot Lysis if (1) whole Blood Clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole Blood Clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if Clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole Blood Clot Lysis by several mechanisms.

  • inhibitory effects of lysine analogues on t pa induced whole Blood Clot Lysis
    Thrombosis Research, 1994
    Co-Authors: Chitra Krishnamurti, Svetislava J Vukelja, Barbara M Alving
    Abstract:

    Abstract The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting Lysis of Clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized Blood from normal volunteers and 125I-fibrinogen in a dilute whole Blood Clot assay to determine the relative concentrations of lysine analogues required for inhibition of Clot Lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging Clot Lysis if (1) whole Blood Clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole Blood Clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if Clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole Blood Clot Lysis by several mechanisms.

David R E Macallan - One of the best experts on this subject based on the ideXlab platform.

  • a reconstituted dilute Blood Clot Lysis assay for the medium throughput screening of thrombolytic compounds
    Analytical Biochemistry, 1999
    Co-Authors: Albert P Gadbut, John R Schullek, Arthur M Hanel, David R E Macallan
    Abstract:

    Abstract Antithrombotic and Clotting factors have long been targets for drug discovery, necessitating the development of Blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted Blood Clot Lysis assay which eliminates the need for on-site donors. The assay uses whole Blood stored at 4°C obtained from a local Blood bank, diluted 1:10 in phosphate buffer. This Blood was supplemented with 125 I-labeled fibrinogen and the release of radioactive fibrinopeptides from formed Clots was measured. The whole Blood used in this assay, which had been stored at 4°C for several days, no longer formed solid or retracting Clots. Thus, platelets 5–7 days ex vivo (165 × 10 6 platelets) were added to the whole Blood in the presence of thrombin (0.80 IU/ml) to form Clots. Solid Clots formed within 2 min of thrombin addition and began retracting shortly thereafter. In the absence of any thrombolytic agent, Clots fully retracted within 2.5 h and remained stable. Thrombin-stimulated Clot formation was completely inhibited by heparin. Clots could be lysed in a dose-dependent fashion in the presence of tissue-type plasminogen activator. Clot Lysis could be completely inhibited in a dose-dependent fashion with plasminogen activator inhibitor type 1. To demonstrate the utility of this assay as a screen for thrombolytic agents, a 14-amino-acid PAI-1-inhibitory peptide relieved the PAI-1 effect on tPA in a dose-dependent fashion. These data describe an assay for the screening of potential pro-fibrinolytic agents that target PAI-1 inhibition in a human plasma-based system that is versatile, cost-effective, and physiologically relevant and does not rely on the availability of on-site Blood donors.

  • a reconstituted dilute Blood Clot Lysis assay for the medium throughput screening of thrombolytic compounds
    Analytical Biochemistry, 1999
    Co-Authors: Albert P Gadbut, John R Schullek, Arthur M Hanel, David R E Macallan
    Abstract:

    Antithrombotic and Clotting factors have long been targets for drug discovery, necessitating the development of Blood assays to determine the efficacy of lead compounds prior to animal testing. We have developed a reconstituted Blood Clot Lysis assay which eliminates the need for on-site donors. The assay uses whole Blood stored at 4 degrees C obtained from a local Blood bank, diluted 1:10 in phosphate buffer. This Blood was supplemented with 125I-labeled fibrinogen and the release of radioactive fibrinopeptides from formed Clots was measured. The whole Blood used in this assay, which had been stored at 4 degrees C for several days, no longer formed solid or retracting Clots. Thus, platelets 5-7 days ex vivo (165 x 10(6) platelets) were added to the whole Blood in the presence of thrombin (0.80 IU/ml) to form Clots. Solid Clots formed within 2 min of thrombin addition and began retracting shortly thereafter. In the absence of any thrombolytic agent, Clots fully retracted within 2.5 h and remained stable. Thrombin-stimulated Clot formation was completely inhibited by heparin. Clots could be lysed in a dose-dependent fashion in the presence of tissue-type plasminogen activator. Clot Lysis could be completely inhibited in a dose-dependent fashion with plasminogen activator inhibitor type 1. To demonstrate the utility of this assay as a screen for thrombolytic agents, a 14-amino-acid PAI-1-inhibitory peptide relieved the PAI-1 effect on tPA in a dose-dependent fashion. These data describe an assay for the screening of potential pro-fibrinolytic agents that target PAI-1 inhibition in a human plasma-based system that is versatile, cost-effective, and physiologically relevant and does not rely on the availability of on-site Blood donors.