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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

Specificity of antibodies to human FVIII and VWF.Denatured, non-reduced samples of recombinant (r) FVIII [Helixate FS, 130 ng per lane (1.30 IU)] and denatured, reduced samples of plasma purified VWF (90–130 ng per lane) were separated by 4–15% sodium dodecyl sulfate (SDS)-PAGE. Lanes containing rFVIII are marked as F8 and MW indicates molecular weight markers in kDa. (A) Arrows on the Coomassie stained gels show the ~230 and ~80 kDa bands for rFVIII and the monomer subunit of reduced VWF at ~250 kDa. (B, C and D) Western blots of gels shown in (A) were detected in (B) with goat anti-human VWF plus donkey anti-goat-HRP, in (C) with rabbit anti-human VWF plus donkey anti-rabbit-HRP, and in (D) with mouse monoclonal anti-human FVIII plus goat anti-mouse-HRP. Panel (E) is a schematic drawing illustrating the interpretation of the Coomassie stained bands and anti-FVIII detected bands generated from denatured rFVIII. The addition of SDS to rFVIII results in the dissociation of copper (Cu) ions [30,31] (or other ions that may be involved, such as the calcium and manganese ions required for FVIII activation) that bridge the heavy chain (~150 kDa) and light chain (~80 kDa) of the rFVIII protein (~230 kDa). [1] The rFVIII was produced using a ~90 kDa B domain. (The B domain was cleaved within the Golgi of the producing BHK cells prior to processing and metal coordination, resulting in a heavy chain of ~150 kDa.) [29].
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pone.0140740.g001: Specificity of antibodies to human FVIII and VWF.Denatured, non-reduced samples of recombinant (r) FVIII [Helixate FS, 130 ng per lane (1.30 IU)] and denatured, reduced samples of plasma purified VWF (90–130 ng per lane) were separated by 4–15% sodium dodecyl sulfate (SDS)-PAGE. Lanes containing rFVIII are marked as F8 and MW indicates molecular weight markers in kDa. (A) Arrows on the Coomassie stained gels show the ~230 and ~80 kDa bands for rFVIII and the monomer subunit of reduced VWF at ~250 kDa. (B, C and D) Western blots of gels shown in (A) were detected in (B) with goat anti-human VWF plus donkey anti-goat-HRP, in (C) with rabbit anti-human VWF plus donkey anti-rabbit-HRP, and in (D) with mouse monoclonal anti-human FVIII plus goat anti-mouse-HRP. Panel (E) is a schematic drawing illustrating the interpretation of the Coomassie stained bands and anti-FVIII detected bands generated from denatured rFVIII. The addition of SDS to rFVIII results in the dissociation of copper (Cu) ions [30,31] (or other ions that may be involved, such as the calcium and manganese ions required for FVIII activation) that bridge the heavy chain (~150 kDa) and light chain (~80 kDa) of the rFVIII protein (~230 kDa). [1] The rFVIII was produced using a ~90 kDa B domain. (The B domain was cleaved within the Golgi of the producing BHK cells prior to processing and metal coordination, resulting in a heavy chain of ~150 kDa.) [29].

Mentions: Similar amounts of non-reduced recombinant (r) FVIII protein and reduced purified plasma VWF were analyzed by Western blotting using mouse monoclonal anti-human FVIII and polyclonal rabbit and goat anti-human VWF antibodies. Bands for both proteins were visible on Coomassie stained gels (Fig 1A). The mouse monoclonal antibody, produced using purified human FVIII as the immunogen, did not detect plasma-purified VWF (Fig 1D). The polyclonal anti-VWF antibodies did not detect the rFVIII protein (Fig 1B and 1C). The schematic drawing in panel (E) shows the generation of the rFVIII bands detected on the Coomassie stained gel and Western blot (Fig 1D). [29]


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)

Specificity of antibodies to human FVIII and VWF.Denatured, non-reduced samples of recombinant (r) FVIII [Helixate FS, 130 ng per lane (1.30 IU)] and denatured, reduced samples of plasma purified VWF (90–130 ng per lane) were separated by 4–15% sodium dodecyl sulfate (SDS)-PAGE. Lanes containing rFVIII are marked as F8 and MW indicates molecular weight markers in kDa. (A) Arrows on the Coomassie stained gels show the ~230 and ~80 kDa bands for rFVIII and the monomer subunit of reduced VWF at ~250 kDa. (B, C and D) Western blots of gels shown in (A) were detected in (B) with goat anti-human VWF plus donkey anti-goat-HRP, in (C) with rabbit anti-human VWF plus donkey anti-rabbit-HRP, and in (D) with mouse monoclonal anti-human FVIII plus goat anti-mouse-HRP. Panel (E) is a schematic drawing illustrating the interpretation of the Coomassie stained bands and anti-FVIII detected bands generated from denatured rFVIII. The addition of SDS to rFVIII results in the dissociation of copper (Cu) ions [30,31] (or other ions that may be involved, such as the calcium and manganese ions required for FVIII activation) that bridge the heavy chain (~150 kDa) and light chain (~80 kDa) of the rFVIII protein (~230 kDa). [1] The rFVIII was produced using a ~90 kDa B domain. (The B domain was cleaved within the Golgi of the producing BHK cells prior to processing and metal coordination, resulting in a heavy chain of ~150 kDa.) [29].
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4608722&req=5

pone.0140740.g001: Specificity of antibodies to human FVIII and VWF.Denatured, non-reduced samples of recombinant (r) FVIII [Helixate FS, 130 ng per lane (1.30 IU)] and denatured, reduced samples of plasma purified VWF (90–130 ng per lane) were separated by 4–15% sodium dodecyl sulfate (SDS)-PAGE. Lanes containing rFVIII are marked as F8 and MW indicates molecular weight markers in kDa. (A) Arrows on the Coomassie stained gels show the ~230 and ~80 kDa bands for rFVIII and the monomer subunit of reduced VWF at ~250 kDa. (B, C and D) Western blots of gels shown in (A) were detected in (B) with goat anti-human VWF plus donkey anti-goat-HRP, in (C) with rabbit anti-human VWF plus donkey anti-rabbit-HRP, and in (D) with mouse monoclonal anti-human FVIII plus goat anti-mouse-HRP. Panel (E) is a schematic drawing illustrating the interpretation of the Coomassie stained bands and anti-FVIII detected bands generated from denatured rFVIII. The addition of SDS to rFVIII results in the dissociation of copper (Cu) ions [30,31] (or other ions that may be involved, such as the calcium and manganese ions required for FVIII activation) that bridge the heavy chain (~150 kDa) and light chain (~80 kDa) of the rFVIII protein (~230 kDa). [1] The rFVIII was produced using a ~90 kDa B domain. (The B domain was cleaved within the Golgi of the producing BHK cells prior to processing and metal coordination, resulting in a heavy chain of ~150 kDa.) [29].
Mentions: Similar amounts of non-reduced recombinant (r) FVIII protein and reduced purified plasma VWF were analyzed by Western blotting using mouse monoclonal anti-human FVIII and polyclonal rabbit and goat anti-human VWF antibodies. Bands for both proteins were visible on Coomassie stained gels (Fig 1A). The mouse monoclonal antibody, produced using purified human FVIII as the immunogen, did not detect plasma-purified VWF (Fig 1D). The polyclonal anti-VWF antibodies did not detect the rFVIII protein (Fig 1B and 1C). The schematic drawing in panel (E) shows the generation of the rFVIII bands detected on the Coomassie stained gel and Western blot (Fig 1D). [29]

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