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Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

Turner NA, Moake JL - PLoS ONE (2015)

Bottom Line: In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs.The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H.Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Rice University, Houston, Texas, United States of America.

ABSTRACT
The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

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Related in: MedlinePlus

FVIII is secreted from stimulated GMVECs bound to ULVWF strings.GMVECs were stimulated with 100 μM histamine for 2 min. Cells were then stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green), washed and fixed. Following fixation, the cells were stained with mouse monoclonal anti-FVIII plus goat anti-mouse IgG AF-647 (red). Panels (A, C and E) show representative ULVWF strings with bound FVIII from merged images. Dashed lines (that were moved away to not obscure the string image) indicate the measured portions of the string. Corresponding graphs (B from image A, D from image C, and F from image E) show the intensities from the 488-nm (VWF, green) and 647-nm (FVIII, red) channels measured along the ULVWF string (in pixels) in the merged image. In the 60× images (A and E) 100 pixels = 11.4 μm and in the 100× image (C) 200 pixels = 11.8 μm. The ratio of FVIII intensity/VWF intensity is shown for each ULVWF string. Images are representative of 9–11 experiments.
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pone.0140740.g009: FVIII is secreted from stimulated GMVECs bound to ULVWF strings.GMVECs were stimulated with 100 μM histamine for 2 min. Cells were then stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green), washed and fixed. Following fixation, the cells were stained with mouse monoclonal anti-FVIII plus goat anti-mouse IgG AF-647 (red). Panels (A, C and E) show representative ULVWF strings with bound FVIII from merged images. Dashed lines (that were moved away to not obscure the string image) indicate the measured portions of the string. Corresponding graphs (B from image A, D from image C, and F from image E) show the intensities from the 488-nm (VWF, green) and 647-nm (FVIII, red) channels measured along the ULVWF string (in pixels) in the merged image. In the 60× images (A and E) 100 pixels = 11.4 μm and in the 100× image (C) 200 pixels = 11.8 μm. The ratio of FVIII intensity/VWF intensity is shown for each ULVWF string. Images are representative of 9–11 experiments.

Mentions: GMVECs and HUVECs stimulated with histamine resulted in WPB secretion from the two types of ECs. The EC-secreted and anchored ULVWF strings had bound FVIII (GMVECs Fig 9 and HUVECs Fig 10). The only possible source of this bound FVIII in these experiments was the EC WPB. Intensity measurements made along the secreted/anchored ULVWF strings from merged images demonstrate both FVIII and VWF. Intensity ratios of FVIII to VWF determined that ULVWF strings secreted by GMVECs have ~2-fold more FVIII attached than the strings secreted from HUVECs (Table 4, S5 and S6 Datasets).


Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

Turner NA, Moake JL - PLoS ONE (2015)

FVIII is secreted from stimulated GMVECs bound to ULVWF strings.GMVECs were stimulated with 100 μM histamine for 2 min. Cells were then stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green), washed and fixed. Following fixation, the cells were stained with mouse monoclonal anti-FVIII plus goat anti-mouse IgG AF-647 (red). Panels (A, C and E) show representative ULVWF strings with bound FVIII from merged images. Dashed lines (that were moved away to not obscure the string image) indicate the measured portions of the string. Corresponding graphs (B from image A, D from image C, and F from image E) show the intensities from the 488-nm (VWF, green) and 647-nm (FVIII, red) channels measured along the ULVWF string (in pixels) in the merged image. In the 60× images (A and E) 100 pixels = 11.4 μm and in the 100× image (C) 200 pixels = 11.8 μm. The ratio of FVIII intensity/VWF intensity is shown for each ULVWF string. Images are representative of 9–11 experiments.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4608722&req=5

pone.0140740.g009: FVIII is secreted from stimulated GMVECs bound to ULVWF strings.GMVECs were stimulated with 100 μM histamine for 2 min. Cells were then stained with rabbit anti-VWF plus chicken anti-rabbit IgG AF-488 (green), washed and fixed. Following fixation, the cells were stained with mouse monoclonal anti-FVIII plus goat anti-mouse IgG AF-647 (red). Panels (A, C and E) show representative ULVWF strings with bound FVIII from merged images. Dashed lines (that were moved away to not obscure the string image) indicate the measured portions of the string. Corresponding graphs (B from image A, D from image C, and F from image E) show the intensities from the 488-nm (VWF, green) and 647-nm (FVIII, red) channels measured along the ULVWF string (in pixels) in the merged image. In the 60× images (A and E) 100 pixels = 11.4 μm and in the 100× image (C) 200 pixels = 11.8 μm. The ratio of FVIII intensity/VWF intensity is shown for each ULVWF string. Images are representative of 9–11 experiments.
Mentions: GMVECs and HUVECs stimulated with histamine resulted in WPB secretion from the two types of ECs. The EC-secreted and anchored ULVWF strings had bound FVIII (GMVECs Fig 9 and HUVECs Fig 10). The only possible source of this bound FVIII in these experiments was the EC WPB. Intensity measurements made along the secreted/anchored ULVWF strings from merged images demonstrate both FVIII and VWF. Intensity ratios of FVIII to VWF determined that ULVWF strings secreted by GMVECs have ~2-fold more FVIII attached than the strings secreted from HUVECs (Table 4, S5 and S6 Datasets).

Bottom Line: In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs.The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H.Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioengineering, Rice University, Houston, Texas, United States of America.

ABSTRACT
The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

No MeSH data available.


Related in: MedlinePlus