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Loss of ATP diphosphohydrolase activity with endothelial cell activation.

Robson SC, Kaczmarek E, Siegel JB, Candinas D, Koziak K, Millan M, Hancock WW, Bach FH - J. Exp. Med. (1997)

Bottom Line: This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines.Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature.We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sandoz Center for Immunobiology, Boston, Massachusetts, USA.

ABSTRACT
Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

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Effect of EC activation upon ATPDase  antigen expression. (a) Western blotting. The polyclonal antibody recognized ATPDase in human EC  preparations purified from quiescent- and TNFαstimulated cells by Western blotting. We did not observe significant diminution of ATPDase antigen expression, nor evidence for proteolytic degradation,  despite the documented reduction in enzyme activity  at this time point. (b) Immunocytofluorometric analysis. These plots demonstrate high levels of surface  expression of CD39 on quiescent HUVEC (bold line,  anti-CD39; faint line, isotype control mAb) (A). The  expression of CD39 epitopes on the EC surface was  largely unaltered after TNFα stimulation (10 ng/ml;  B) or direct oxidative stress with H2O2 (100 μM; C).  Cells were analyzed by flow cytometry as described  in Materials and Methods.
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Figure 5: Effect of EC activation upon ATPDase antigen expression. (a) Western blotting. The polyclonal antibody recognized ATPDase in human EC preparations purified from quiescent- and TNFαstimulated cells by Western blotting. We did not observe significant diminution of ATPDase antigen expression, nor evidence for proteolytic degradation, despite the documented reduction in enzyme activity at this time point. (b) Immunocytofluorometric analysis. These plots demonstrate high levels of surface expression of CD39 on quiescent HUVEC (bold line, anti-CD39; faint line, isotype control mAb) (A). The expression of CD39 epitopes on the EC surface was largely unaltered after TNFα stimulation (10 ng/ml; B) or direct oxidative stress with H2O2 (100 μM; C). Cells were analyzed by flow cytometry as described in Materials and Methods.

Mentions: The polyclonal antibody directed to peptide sequences from porcine aortic ATPDase did not react with ATPDase antigen as expressed by intact EC by immunocytochemistry (data not shown). However, this antibody did recognize denatured ATPDase from human EC lysates from quiescent and TNFα stimulated cells analyzed by Western blotting. Using this technique, we did not observe either diminution of ATPDase antigen expression, or evidence for protein degradation, despite the reduction in enzyme activity in EC stimulated with TNFα at 2 h. (Fig. 5 a).


Loss of ATP diphosphohydrolase activity with endothelial cell activation.

Robson SC, Kaczmarek E, Siegel JB, Candinas D, Koziak K, Millan M, Hancock WW, Bach FH - J. Exp. Med. (1997)

Effect of EC activation upon ATPDase  antigen expression. (a) Western blotting. The polyclonal antibody recognized ATPDase in human EC  preparations purified from quiescent- and TNFαstimulated cells by Western blotting. We did not observe significant diminution of ATPDase antigen expression, nor evidence for proteolytic degradation,  despite the documented reduction in enzyme activity  at this time point. (b) Immunocytofluorometric analysis. These plots demonstrate high levels of surface  expression of CD39 on quiescent HUVEC (bold line,  anti-CD39; faint line, isotype control mAb) (A). The  expression of CD39 epitopes on the EC surface was  largely unaltered after TNFα stimulation (10 ng/ml;  B) or direct oxidative stress with H2O2 (100 μM; C).  Cells were analyzed by flow cytometry as described  in Materials and Methods.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196106&req=5

Figure 5: Effect of EC activation upon ATPDase antigen expression. (a) Western blotting. The polyclonal antibody recognized ATPDase in human EC preparations purified from quiescent- and TNFαstimulated cells by Western blotting. We did not observe significant diminution of ATPDase antigen expression, nor evidence for proteolytic degradation, despite the documented reduction in enzyme activity at this time point. (b) Immunocytofluorometric analysis. These plots demonstrate high levels of surface expression of CD39 on quiescent HUVEC (bold line, anti-CD39; faint line, isotype control mAb) (A). The expression of CD39 epitopes on the EC surface was largely unaltered after TNFα stimulation (10 ng/ml; B) or direct oxidative stress with H2O2 (100 μM; C). Cells were analyzed by flow cytometry as described in Materials and Methods.
Mentions: The polyclonal antibody directed to peptide sequences from porcine aortic ATPDase did not react with ATPDase antigen as expressed by intact EC by immunocytochemistry (data not shown). However, this antibody did recognize denatured ATPDase from human EC lysates from quiescent and TNFα stimulated cells analyzed by Western blotting. Using this technique, we did not observe either diminution of ATPDase antigen expression, or evidence for protein degradation, despite the reduction in enzyme activity in EC stimulated with TNFα at 2 h. (Fig. 5 a).

Bottom Line: This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines.Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature.We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo.

View Article: PubMed Central - PubMed

Affiliation: Sandoz Center for Immunobiology, Boston, Massachusetts, USA.

ABSTRACT
Quiescent endothelial cells (EC) regulate blood flow and prevent intravascular thrombosis. This latter effect is mediated in a number of ways, including expression by EC of thrombomodulin and heparan sulfate, both of which are lost from the EC surface as part of the activation response to proinflammatory cytokines. Loss of these anticoagulant molecules potentiates the procoagulant properties of the injured vasculature. An additional thromboregulatory factor, ATP diphosphohydrolase (ATPDase; designated as EC 3.6.1.5) is also expressed by quiescent EC, and has the capacity to degrade the extracellular inflammatory mediators ATP and ADP to AMP, thereby inhibiting platelet activation and modulating vascular thrombosis. We describe here that the antithrombotic effects of the ATPDase, like heparan sulfate and thrombomodulin, are lost after EC activation, both in vitro and in vivo. Because platelet activation and aggregation are important components of the hemostatic changes that accompany inflammatory diseases, we suggest that the loss of vascular ATPDase may be crucial for the progression of vascular injury.

Show MeSH
Related in: MedlinePlus