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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.

No MeSH data available.


Related in: MedlinePlus

FVIII in HUVEC WPBs does not overlap with β-actin or Factor H.HUVECs were fixed and treated with Triton-X prior to staining with mouse monoclonal anti-FVIII plus donkey anti-mouse IgG AF-488 (green). This was followed by staining either with goat anti-β-actin plus chicken anti-goat IgG AF-647 (red) or with goat anti-Factor H plus chicken anti-goat IgG AF-647 (red). Merged images and the corresponding intensity scatter plots are: (A and C) FVIII and β-actin at 60×, N = 4; (B and D) FVIII and β-actin at 100×, N = 4; (E and G) FVIII and Factor H at 60×, N = 3; and (F and H) FVIII and Factor H at 100×, N = 3. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.
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pone.0140740.g006: FVIII in HUVEC WPBs does not overlap with β-actin or Factor H.HUVECs were fixed and treated with Triton-X prior to staining with mouse monoclonal anti-FVIII plus donkey anti-mouse IgG AF-488 (green). This was followed by staining either with goat anti-β-actin plus chicken anti-goat IgG AF-647 (red) or with goat anti-Factor H plus chicken anti-goat IgG AF-647 (red). Merged images and the corresponding intensity scatter plots are: (A and C) FVIII and β-actin at 60×, N = 4; (B and D) FVIII and β-actin at 100×, N = 4; (E and G) FVIII and Factor H at 60×, N = 3; and (F and H) FVIII and Factor H at 100×, N = 3. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.

Mentions: Fluorescent images of HUVECs with simultaneous detection of FVIII and either β-actin or Factor H, produced PCC values that were below the values for correlation (Table 3, Fig 6 and S2 Dataset). Mean PCC values for β-actin (0.35 and 0.38) and Factor H (0.42 and 0.44) indicate that detection signals for these two proteins are not spatially correlated with FVIII detection signals in HUVECs (Table 3). Intensity scatter plots display separate populations and the merged fluorescent images do not show FVIII in overlapping locations with β-actin or Factor H (Fig 6).


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 in HUVEC WPBs does not overlap with β-actin or Factor H.HUVECs were fixed and treated with Triton-X prior to staining with mouse monoclonal anti-FVIII plus donkey anti-mouse IgG AF-488 (green). This was followed by staining either with goat anti-β-actin plus chicken anti-goat IgG AF-647 (red) or with goat anti-Factor H plus chicken anti-goat IgG AF-647 (red). Merged images and the corresponding intensity scatter plots are: (A and C) FVIII and β-actin at 60×, N = 4; (B and D) FVIII and β-actin at 100×, N = 4; (E and G) FVIII and Factor H at 60×, N = 3; and (F and H) FVIII and Factor H at 100×, N = 3. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0140740.g006: FVIII in HUVEC WPBs does not overlap with β-actin or Factor H.HUVECs were fixed and treated with Triton-X prior to staining with mouse monoclonal anti-FVIII plus donkey anti-mouse IgG AF-488 (green). This was followed by staining either with goat anti-β-actin plus chicken anti-goat IgG AF-647 (red) or with goat anti-Factor H plus chicken anti-goat IgG AF-647 (red). Merged images and the corresponding intensity scatter plots are: (A and C) FVIII and β-actin at 60×, N = 4; (B and D) FVIII and β-actin at 100×, N = 4; (E and G) FVIII and Factor H at 60×, N = 3; and (F and H) FVIII and Factor H at 100×, N = 3. Values for Pearson’s correlation coefficient (PCC) are on each scatter plot.
Mentions: Fluorescent images of HUVECs with simultaneous detection of FVIII and either β-actin or Factor H, produced PCC values that were below the values for correlation (Table 3, Fig 6 and S2 Dataset). Mean PCC values for β-actin (0.35 and 0.38) and Factor H (0.42 and 0.44) indicate that detection signals for these two proteins are not spatially correlated with FVIII detection signals in HUVECs (Table 3). Intensity scatter plots display separate populations and the merged fluorescent images do not show FVIII in overlapping locations with β-actin or Factor H (Fig 6).

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