Venous Occlusion Does Not Release von Willebrand Factor, Factor VIII or PAI-1 from Endothelial Cells – The Importance of Consensus on the Use of Correction Factors for Haemoconcentration

SummaryCultured human endothelial cells synthesize and secrete a protein(s) which has factor VIII antigen and von Willebrand factor activity. Subcellular membrane and granule fractions derived from human platelets also contain the factor VIII antigen and von Willebrand factor activity. Circulating platelets constitute a significant reservoir of VIII antigen containing approximately 15 % of the amount present in platelet-poor plasma. Thus normal platelets contain surface bound as well as intracellularly stored von Willebrand factor, a protein synthesized by endothelial cells which is required for normal platelet function.

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Immunohistochemical Detection of Factor VIII/von Willebrand Factor in Hyperplastic Endothelial Cells in Glioblastoma Multiforme and Mixed Glioma-Sarcoma

Journal of Neuropathology & Experimental Neurology ◽

10.1097/00005072-198209000-00001 ◽

1982 ◽

Vol 41 (5) ◽

pp. 497 ◽

Cited By ~ 34

Author(s):

RODNEY D. MCCOMB ◽

TREVOR R. JONES ◽

SALVATORE V. PIZZO ◽

DARELL D. BIGNER

Keyword(s):

Endothelial Cells ◽

Glioblastoma Multiforme ◽

Factor Viii ◽

Von Willebrand Factor ◽

Immunohistochemical Detection ◽

Mixed Glioma ◽

Von Willebrand ◽

Willebrand Factor

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The Effect of von Willebrand Factor (VWF) on Site-Specific Factor VIII (FVIII) Expression in Hemophilia A Mice.

Blood ◽

10.1182/blood.v110.11.764.764 ◽

2007 ◽

Vol 110 (11) ◽

pp. 764-764

Author(s):

Qizhen Shi ◽

Scot A. Fahs ◽

Erin L. Kuether ◽

Robert R. Montgomery

Keyword(s):

Endothelial Cells ◽

Endothelial Cell ◽

Factor Viii ◽

Von Willebrand Factor ◽

Double Knockout ◽

Site Specific ◽

Von Willebrand ◽

Willebrand Factor

Abstract von Willebrand factor (VWF) is a carrier protein for factor VIII (FVIII) and protects plasma FVIII from protease degradation. Our laboratory has had a longstanding interest in the association of FVIII with VWF both in vitro and in vivo. Our in vitro studies have demonstrated that FVIII stores together with VWF in both endothelial cells and megakaryocytes if FVIII is made in these cells. Furthermore, we demonstrated that FVIII and VWF are both releasable by agonist stimulation. To investigate the association of VWF and FVIII in vivo, we generated two lines of transgenic mice that express FVIII either in endothelial cells or in platelets using either the endothelial cell-specific Tie2 promoter or the platelet-specific αIIb promoter, respectively. When the platelet-specific FVIII (2bF8) transgene is bred into the FVIIInull mouse, FVIII can only be detected in platelets, with a level of 0.76 ± 0.27 mU/108 platelets in heterozygous and 1.53 ± 0.14 mU/108 platelets in homozygous 2bF8 mice. When the endothelial cell-specific FVIII (Tie2F8) transgene is bred into the FVIIInull mouse, homozygous Tie2F8 mice maintained normal plasma FVIII levels (1.15 ± 0.16 U/ml) and 50% levels in heterozygous mice (0.56 ± 0.16 U/ml). Both 2bF8trans and Tie2F8trans phenotypes effectively abrogate the bleeding diathesis in hemophilic mice. When 2bF8 transgene was bred into a FVIII and VWF double knockout background, the level of platelet-FVIII significantly decreased, but this platelet-derived FVIII was still stored in a-granules and still maintained clinical efficacy. In contrast, when the Tie2F8 transgene was bred into the double knockout background, plasma FVIII dropped to undetectable levels. This is in contrast to the situation in VWFnull mice in which normal endogenous murine FVIII is synthesized with about 10% of normal FVIII activity persisting in plasma. This could be due to a difference in survival between human FVIII and murine FVIII. All Tie2F8trans/FVIIInullVWFnull mice (n=15) survived tail clipping even though there is no FVIII:C detected in the plasma. To investigate the effect of murine VWF on the levels of plasma FVIII, plasma from FVIIInull mice was infused into Tie2F8trans/FVIIInullVWFnull mice to restore VWF levels to 25% of normal. As expected, the endothelial cell-derived plasma FVIII was stabilized by the infused VWF and was detected within 1 hour after infusion, with a peak (25% level) at 4 hours. The level of plasma FVIII at 24 hours was still about 20% of normal while the level of remaining VWF was only 5% of normal. These results demonstrate that VWF is important for site-specific FVIII expression. Co-expression with VWF in platelets is important for optimal platelet-specific FVIII expression and endothelial cell-derived plasma FVIII is VWF-dependent.

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Factor VIII alters tubular organization and functional properties of von Willebrand factor stored in Weibel-Palade bodies

Blood ◽

10.1182/blood-2011-05-355354 ◽

2011 ◽

Vol 118 (22) ◽

pp. 5947-5956 ◽

Cited By ~ 16

Author(s):

Eveline A. M. Bouwens ◽

Marjon J. Mourik ◽

Maartje van den Biggelaar ◽

Jeroen C. J. Eikenboom ◽

Jan Voorberg ◽

...

Keyword(s):

Electron Microscopy ◽

Endothelial Cells ◽

Confocal Microscopy ◽

Factor Viii ◽

Functional Properties ◽

Von Willebrand Factor ◽

Equal Length ◽

Platelet Binding ◽

Von Willebrand ◽

Willebrand Factor

Abstract In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

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HUMAN HEPATIC ENDOTHELIAL CELLS AND HEPATOCYTES IN CULTURE: MORPHOLOGICAL FEATURES, AND PRODUCTION OF VON WILLEBRAND FACTOR AND FIBRINOGEN

10.1055/s-0038-1643350 ◽

1987 ◽

Author(s):

R Harrison

Keyword(s):

Endothelial Cells ◽

Factor Viii ◽

Von Willebrand Factor ◽

Human Liver ◽

Liver Cells ◽

Mixed Population ◽

Dependent Manner ◽

Cell Type ◽

Von Willebrand ◽

Willebrand Factor

Liver cells were derived from cadaveric organ donors. Pieces of human liver 5 to 50 grams were minced, washed, and incubated in collagenase at 37 degrees C. After washing, the cell suspension was plated into culture vessels that had been briefly pre-treated with an extract derived from human liver. A mixed population of liver cells, including endothelial cells, hepatocytes, and Kupffer cells, attached within hours. At the end of 2 to 3 weeks there developed clusters of densely packed cells of two types. The most numerous cells were initially fusiform but grew as a monolayer even when densely packed. As density increased they assumed a polygonal form; cells with this morphological appearance stained immunocytochemically for von Willebrand factor antigen. They were relatively small and resembled cells derived from human umbilical vein except that the cytoplasm was more filmy in appearance. The second prominent cell type was significantly larger and likewise replicated to form clusters. These large cells sometimes contained multiple nuclei, exhibited a relatively low nuclear to cytoplasmic ratio, and immunocytochemically stained for human fibrinogen. A more distinct nuclear membrane and prominent nucleoli were characteristics of hepatocytes that were useful light microscopically in distinguishing these cells from sinusoidal endothelial cells. Ultrastructurally, endothelial cells were characterized by small size, holes in and among the cells that probably were the in vitro analogue of fenestrae, and numerous Weibel-Palade bodies in the cytoplasm, which otherwise was relatively bland. Hepatocytes, by contrast, had an active appearing cytoplasm containing more organelles. Canaliculi and typical tight junctions formed between adjacent hepatocytes. Levels of vWF and fibrinogen increased in a time dependent manner in media overlying this mixed population of cells. Human factor VIII has not yet been detected in the media overlying these mixed cells derived from human liver, and factor VIII antigen has not yet been demonstrable immunocytochemically in either cell type.

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