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

Nancy A Turner,Joel L Moake,Christopher B Doering

doi:10.1371/journal.pone.0140740

Nancy A Turner, Joel L Moake + Show 1 more

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https://doi.org/10.1371/journal.pone.0140740

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Journal: PloS one	Publication Date: Oct 16, 2015
Citations: 67	License type: CC BY 4.0

Affiliation: Rice University

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.

Highlights

Human factor VIII (FVIII) functions as a cofactor for activated factor IX, and mutations in the FVIII gene (F8) result in hemophilia A. [1] FVIII in plasma is proteolyzed and/or pinocytosed unless it is protected by non-covalent binding to soluble forms of von Willebrand factor (VWF) multimers. [2,3,4]VWF is synthesized in megakaryocytes and endothelial cells (ECs), and is stored in megakaryocyte/platelet alpha granules and EC Weibel-Palade bodies (WPBs). [5] VWF multimersPLOS ONE | DOI:10.1371/journal.pone.0140740 October 16, 2015Human Endothelial Cells Synthesize and Store FVIII are secreted from WPBs in ultra-long, hyper-adhesive strings anchored to EC surfaces
We show that FVIII is stored in WPBs along with VWF, in both EC types; and that stimulated glomerular microvascular endothelial cells (GMVECs) and Human umbilical vein endothelial cells (HUVECs) secrete cell-anchored ultra-large (UL) VWF strings with FVIII bound to them
We consider this likely because the findings by Dashty, et al, are not consistent with our findings that the F8 mRNA levels were expressed in the same order of magnitude in HUVECs, GMVECs and fibroblasts (S4 Table)

Summary

Introduction

Human Endothelial Cells Synthesize and Store FVIII are secreted from WPBs in ultra-long, hyper-adhesive strings anchored to EC surfaces. Platelets adhere to these secreted/anchored strings and initiate hemostasis. [15,16,17] Experimental proof of FVIII secretion with DDAVP stimulation has not been reported for unaltered (non-transfected) human ECs (or any other natural non-EC cell type). [19,20] These two articles are compatible with the previous human data reported by Shahani, et al, who used receptor-specific cell sorting to isolate LSECs from hepatocytes in liver tissues to show that LSECs are the primary source of FVIII synthesis within the liver. FVIII protein or activity levels, either released into culture medium or from cell lysates, have been measured in human liver sinusoidal (LS) ECs, cardiac microvascular (MV) ECs, intestinal MVECs, dermal MVECs, lung MVECs and pulmonary artery ECs. [11,12,13,14] Positive F8 mRNA expression has been reported for each of these EC types, except intestinal MVECs. [12,13,14] because of the variety of ECs, and even after extensive investigation, the precise nature of FVIII synthesis, storage and secretion in ECs remains unclear.

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