FV is synthesized in hepatocytes as well as megakaryocytes in the mouse. However, the primary cellular source of FVIII biosynthesis is controversial, with contradictory evidence supporting an endothelial or hepatocyte origin. In order to generate mice with Lman1 expression specifically deleted in the endothelium or the hepatocytes, either a Tie2- Cre or Albumin- Cre transgene was crossed into Lman1 conditional mice Lman1 fl.
RiboTag mice carry a hemaglutinin-tagged ribosomal protein that can be used for cell-type specific immunoprecipitation of polyribosomes and subsequent RNA analysis when crossed with a Cre -recombinase expressing animal.
In contrast, this analysis demonstrated a statistically significant depletion fold of transcripts from many known hepatocyte-specific genes, including multiple coagulation factor genes. Similar examination of kidney endothelial cell RNA also demonstrated enrichment for FVIII transcripts, thereby demonstrating that endothelial cells from multiple tissues and vascular beds contribute to the plasma FVIII pool in the mouse.
These results explain the successful reversal of hemophilia A by both liver and kidney transplants. Taken together, these results definitively demonstrate that endothelial cells are the primary source of FVIII biosynthesis in the mouse, and that hepatocytes make no significant contribution to the plasma FVIII pool.
Sign In or Create an Account. From Genetics Home Reference. More About This Health Condition. Analysis of 18 novel mutations in the factor VIII gene. Br J Haematol. Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A. Hum Mutat. Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A. Haemophilias A and B. Haemophilia A and haemophilia B: molecular insights. Mol Pathol.
VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. Editor: Christopher B.
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 credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Mary R. Competing interests: The authors have declared that no competing interests exist. Although the synthesis of FVIII has been demonstrated in many human tissue types, including liver, spleen, kidney and lymphatic tissue, [ 8 — 10 ] ECs are the only specific human cell type where direct evidence of FVIII production has been obtained.
The Rice Institutional Review Board IRB approved of the human tissue collection and the experiments on human endothelial cells in this study. The unidentified tissue samples designated for disposal were collected under a protocol approved by the Rice IRB. The Rice IRB waived the need for consent. GMVECs were used in experiments at passages 3—6 and not stained earlier than 11 days post seeding. Umbilical cords were washed with phosphate buffer mM NaCl, 0. The cords were rinsed with ml of phosphate buffer, and solutions containing endothelial cells were centrifuged at g for 10 min.
HUVECs were used at passages 2—7 in these studies, and not stained earlier than 6 days post-seeding. The fibroblasts were studied in parallel with the endothelial cells as negative controls. Cell type was confirmed by the presence of Fibroblast Surface Protein by fluorescent microscopy.
Fibroblasts in these experiments were used at passages 4—6, and cells were not stained earlier than 4 days post-seeding. The relative quantification of gene expression levels were calculated as described in the Applied Biosystems User Bulletin No. The quantification of each gene in each cell type relative to the expression in GMVECs was calculated using the following expressions:. Standard Deviation SD Calculations. Fluorescent images were acquired using IP Lab software version 3. The cells were stained for 15 min with the relevant primary and fluorescent antibody pairs.
The cell nuclei were detected with 1. In experiments where only cell surfaces were detected with antibodies, the treatment with Triton-X was omitted. Both coverslips were imaged at nm and at nm and merged with DAPI images.
Fibroblast cell surfaces were stained with Fibroblast Surface Protein FSP separately or in conjunction with internal cell staining to confirm cell type. In surface staining followed by internal staining, the cells were fixed again to retain antibodies to surface proteins before treatment with Triton-X and then stained with detection antibody pairs.
A plus secondary antibody chicken anti-goat IgG AF cat. The polyclonal antibody made against human Factor H has previously been shown to be monospecific in detecting Factor H protein in human serum and in Factor H-depleted serum by Western blot techniques. The PCC provides shape correlation by comparing the intensity distribution of the two channels. PCC values are independent of high background levels.
In most cases, scatter plots of green channel intensity y-axis verses red channel intensity x-axis from a merged image of 2 spatially correlated proteins should show a single linear relationship between the two channels. Values for PCC between 0.
Plots of the green intensity values and the red intensity values at the same locations allowed a visual demonstration of signal colocalization.
The graphs are intended to be visual aids showing for FVIII and VWF that the red and green intensities change in value in a synchronized manner at each image location.
In these experiments, GMVECs at passage 4 and 6 were stimulated and stained from 8 to 10 days after seeding. HUVECs at passage 2 and 4 were stimulated and stained from 6 to 7 days after seeding.
The intensity graphs are the intensity values from each channel measured at locations traced along the VWF strings in the green-channel fluorescent image. Graphs were generated of the red and green intensities versus the distance along ULVWF strings in pixels to demonstrate the process used to obtain the intensity data. The intensity graphs of measurements made along VWF strings with shorter lengths appear to be lower in resolution. Absorbance was read at nm and at nm using a Tecan Infinite M plate reader.
Subtraction of measurements at nm corrected for absorbance differences between wells. Bands for both proteins were visible on Coomassie stained gels Fig 1A. The high concentration of VWF present in Weibel-Palade bodies WPBs was used to demonstrate that images detected at the first wavelength channel nm were not cross contaminated by fluorescence generated from the second wavelength channel nm , and reciprocally, the nm channel was not affected by fluorescence from the nm channel.
The images in A and C were detected at nm and the images in B and D were detected at nm. FVIII and VWF were distinctly identified by using mono-specific primary antibodies combined with secondary detection antibodies with widely separated wavelengths for excitation and emission, as shown in Fig 2.
The cellular site of synthesis of factor VIII in the circulation has long been disputed, but 2 papers in this issue of Blood by Fahs et al 1 and Everett et al 2 finally identify the cell type that makes factor VIII in the liver and, by implication, in the rest of the body.
To understand why this quest was so prolonged, one needs to go back to the technologies of the s and s. Not surprisingly, it was the last but one of the classical blood coagulation factors to yield to the advance of molecular biology in , 2 years before tissue factor and von Willebrand factor. To purify enough protein for biochemical characterization was beyond the limits of technology available to researchers up to Then, progressively new techniques enabled even the rarest of proteins to be isolated.
Thus, factor VIII was purified and sequenced and its gene cloned. Later, we found that a hepatocyte-rich cell fraction from human donor liver contained factor VIII messenger RNA and factor VIII antigen, 5 but there remained lingering doubt about the actual cell type involved. Studies in rodents showed that liver endothelium contained factor VIII antigen, 6 and the original cross transplantation work demonstrated that cells other than hepatocytes must be able to make half the factor VIII circulating in blood.
Recently, it has been shown convincingly that factor VIII activity and antigen are confined to cells sorted individually from liver that carry markers restricted to endothelium. In the other study, Lman1 conditional knockout mice were generated to characterize the FVIII secretion profiles of endothelial cells and hepatocytes. Taken together, these studies conclusively demonstrate that hepatocytes have no role in factor VIII biosynthesis, a role that is fulfilled by endothelial cells in the liver and elsewhere.
This result must raise the question of why hepatocytes synthesize every other circulating factor necessary for clot formation yet specifically omit one crucial cofactor in the enzymatic cascade leading to thrombin formation and conversion of fibrinogen to fibrin.
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