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Abstract
BackgroundADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13+/+) and deficient (Adamts13−/−) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells.Methodology/Principal FindingsImmunohistochemistry was performed on kidney sections from ADAMTS13 wild-type and ADAMTS13-deficient mice. Phenotypic differences were examined by ultramorphology. ADAMTS13 expression in human glomerular endothelial cells and dermal microvascular endothelial cells was investigated by real-time PCR, flow cytometry, immunofluorescence and immunoblotting. VWF cleavage was demonstrated by multimer structure analysis and immunoblotting. ADAMTS13 was demonstrated in glomerular endothelial cells in Adamts13+/+ mice but no staining was visible in tissue from Adamts13−/− mice. Thickening of glomerular capillaries with platelet deposition on the vessel wall was detected in Adamts13−/− mice. ADAMTS13 mRNA and protein were detected in both human endothelial cells and the protease was secreted. ADAMTS13 activity was demonstrated in glomerular endothelial cells as cleavage of VWF.Conclusions/SignificanceGlomerular endothelial cells express and secrete ADAMTS13. The proteolytic activity could have a protective effect preventing deposition of platelets along capillary lumina under the conditions of high shear stress present in glomerular capillaries.
Highlights
The known substrate for ADAMTS13 is von Willebrand Factor (VWF) [1], a glycoprotein, that induces platelet adhesion and aggregation at sites of vascular injury and high-shear stress [2]
Media taken from these stimulated cells exhibited weak bands at 140 kDa and 176 kDa (Figure 8D, lane 4 shows media from cells stimulated with estradiol), albeit not more than media from unstimulated cells. These experiments were not carried out using media from HMVEC. These results indicate that active ADAMTS13 is present inside glomerular endothelial cells and a small amount is secreted into the cell media but the activity detected in the medium was very low
In this study we show that glomerular endothelial cells are capable of producing biologically active ADAMTS13, and that when ADAMTS13 is lacking, as in the ADAMTS13-deficient mouse, the glomerular capillary vessel wall is thickened and irregular
Summary
The known substrate for ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motifs) is von Willebrand Factor (VWF) [1], a glycoprotein, that induces platelet adhesion and aggregation at sites of vascular injury and high-shear stress [2]. ULVWF multimers are cleaved on the surface of endothelial cells into smaller multimers by ADAMTS13 [7]. Apart from ADAMTS13, four other proteases, elastase, proteinase 3, cathepsin G and matrix metalloprotease 9 (MMP9), have been shown to cleave VWF at sites identical with, or near, the ADAMTS13 cleavage site [9]. ADAMTS13 is, considered most important for cleavage of VWF under physiological conditions and conditions of increased shear stress [7]. ADAMTS13 is the physiological von Willebrand factor (VWF)-cleaving protease. The aim of this study was to examine ADAMTS13 expression in kidneys from ADAMTS13 wild-type (Adamts13+/+) and deficient (Adamts132/2) mice and to investigate the expression pattern and bioactivity in human glomerular endothelial cells
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