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

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 clearanc...

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Autores principales: Turner, Nancy A., Moake, Joel L.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Public Library of Science 2015
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608722/
https://www.ncbi.nlm.nih.gov/pubmed/26473492
http://dx.doi.org/10.1371/journal.pone.0140740
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author Turner, Nancy A.
Moake, Joel L.
author_facet Turner, Nancy A.
Moake, Joel L.
author_sort Turner, Nancy A.
collection PubMed
description 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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spelling pubmed-46087222015-10-29 Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings Turner, Nancy A. Moake, Joel L. PLoS One Research Article 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. Public Library of Science 2015-10-16 /pmc/articles/PMC4608722/ /pubmed/26473492 http://dx.doi.org/10.1371/journal.pone.0140740 Text en © 2015 Turner, Moake http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.
spellingShingle Research Article
Turner, Nancy A.
Moake, Joel L.
Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings
title Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings
title_full Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings
title_fullStr Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings
title_full_unstemmed Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings
title_short Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings
title_sort factor viii is synthesized in human endothelial cells, packaged in weibel-palade bodies and secreted bound to ulvwf strings
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4608722/
https://www.ncbi.nlm.nih.gov/pubmed/26473492
http://dx.doi.org/10.1371/journal.pone.0140740
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