scholarly journals Integrin αvβ3 on human endothelial cells binds von Willebrand factor strings under fluid shear stress

Blood ◽  
2009 ◽  
Vol 113 (7) ◽  
pp. 1589-1597 ◽  
Author(s):  
Jing Huang ◽  
Robyn Roth ◽  
John E. Heuser ◽  
J. Evan Sadler
Keyword(s):  
Endothelial Cells ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Fluid Shear Stress ◽  
Immunofluorescence Microscopy ◽  
Integrin Αvβ3 ◽  
Platelet Glycoprotein ◽  
Human Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Acutely secreted von Willebrand factor (VWF) multimers adhere to endothelial cells, support platelet adhesion, and may induce microvascular thrombosis. Immunofluorescence microscopy of live human umbilical vein endothelial cells showed that VWF multimers rapidly formed strings several hundred micrometers long on the cell surface after stimulation with histamine. Unexpectedly, only a subset of VWF strings supported platelet binding, which depended on platelet glycoprotein Ib. Electron microscopy showed that VWF strings often consisted of bundles and networks of VWF multimers, and each string was tethered to the cell surface by a limited number of sites. Several approaches implicated P-selectin and integrin αvβ3 in anchoring VWF strings. An RGDS peptide or a function-blocking antibody to integrin αvβ3 reduced the number of VWF strings formed. In addition, integrin αv decorated the VWF strings by immunofluorescence microscopy. Furthermore, lentiviral transduction of shRNA against the αv subunit reduced the expression of cell-surface integrin αvβ3 and impaired the ability of endothelial cells to retain VWF strings. Soluble P-selectin reduced the number of platelet-decorated VWF strings in the absence of Ca2+ and Mg2+ but had no effect in the presence of these cations. These results indicate that VWF strings bind specifically to integrin αvβ3 on human endothelial cells.

scholarly journals Gamma-interferon modulates von Willebrand factor release by cultured human endothelial cells

Blood ◽  
1990 ◽  
Vol 75 (11) ◽  
pp. 2177-2184 ◽  
Author(s):  
SH Tannenbaum ◽  
HR Gralnick
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
High Molecular Weight ◽  
Von Willebrand Factor ◽  
Inflammatory Mediators ◽  
Interleukin 1 ◽  
Gamma Interferon ◽  
Human Endothelial Cells ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Endothelial cells (EC) synthesize and secrete von Willebrand factor (vWF), a multimeric glycoprotein required for normal hemostasis. Within human endothelial cells, vWF multimers of extremely high molecular weight are stored in rod-shaped organelles known as Weibel-Palade bodies. Inflammatory mediators, such as interleukin-1, induce in vitro a variety of procoagulant responses by EC, including the secretion of stored vWF. We postulated that other inflammatory mediators might act to balance this procoagulant reaction, thereby assisting in the maintenance of blood fluidity during immune activation. Both gamma- interferon (gamma-IFN) and tumor necrosis factor (TNF) were found to act independently and cooperatively to depress the stimulated release of vWF from EC. Analysis of stored vWF in either gamma-IFN and/or TNF- treated EC demonstrated a loss of high molecular weight multimers while immunofluorescent studies documented a loss of visible Weibel-Palade bodies. This suggests that gamma-IFN and TNF interfere with normal vWF storage. gamma-IFN acted in a dose-, time-, and RNA-dependent fashion, and its inhibition of vWF release was reversible with time. No effect of gamma-IFN on EC was noted when anti-serum to gamma-IFN was added. Unlike gamma-IFN, alpha-interferon did not effect EC vWF. Therefore, gamma-IFN and TNF may be important in decreasing vWF release during inflammatory or immunologic episodes.

scholarly journals Studies of Multimerin in Human Endothelial Cells

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1304-1317 ◽  
Author(s):  
Catherine P. M. Hayward ◽  
Elisabeth M. Cramer ◽  
Zhili Song ◽  
Shilun Zheng ◽  
Roxanna Fung ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Von Willebrand Factor ◽  
Culture Media ◽  
Cellular Membrane ◽  
Human Endothelial Cells ◽  
Early Passage ◽  
Endothelial Cell Cultures ◽  
Dense Core Granules ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Multimerin is a novel, massive, soluble protein that resembles von Willebrand factor in its repeating, homomultimeric structure. Both proteins are expressed by megakaryocytes and endothelial cells and are stored in the region of platelet α-granules resembling Weibel-Palade bodies. These findings led us to study the distribution of multimerin within human endothelial cells. Multimerin was identified in vascular endothelium in situ. In cultured endothelial cells, multimerin was identified within round to rod-shaped, dense-core granules, some of which contained intragranular, longitudinally arranged tubules and resembled Weibel-Palade bodies. However, multimerin was found primarily in different structures than the Weibel-Palade body proteins von Willebrand factor and P-selectin. After stimulation with secretagogues, multimerin was observed to redistribute from intracellular structures to the external cellular membrane, without detectable accompanied secretion of multimerin into the culture media. In early passage endothelial cell cultures, multimerin was associated with extensive, fibrillary, extracellular matrix structures, in a different distribution than fibronectin. Although multimerin and von Willebrand factor are stored together in platelets, they are mainly found within different structures in endothelial cells, indicating that there are tissue-specific differences in the sorting of these soluble, multimeric proteins.

Shear-Dependent Rolling on von Willebrand Factor of Mammalian Cells Expressing the Platelet Glycoprotein Ib-IX-V Complex

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3684-3693 ◽  
Author(s):  
Becky J. Fredrickson ◽  
Jing-Fei Dong ◽  
Larry V. McIntire ◽  
José A. López
Keyword(s):  
Von Willebrand Factor ◽  
Mammalian Cells ◽  
Platelet Adhesion ◽  
Fluid Shear Stress ◽  
Platelet Glycoprotein ◽  
Shear Stresses ◽  
Mean Velocity ◽  
The Mean ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.

Integrin αvβ3 Is Involved in the Stabilization of Ultra Long von Willebrand Factor Strings under Fluid Shear Stress on Human Endothelial Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 289-289
Author(s):  
Jing Huang ◽  
John E. Heuser ◽  
Rodger P. McEver ◽  
J. Evan Sadler
Keyword(s):  
Endothelial Cells ◽  
Shear Stress ◽  
Fluid Shear Stress ◽  
Integrin Αvβ3 ◽  
Fluid Shear ◽  
Blocking Antibody ◽  
Integrin Αv ◽  
String Formation ◽  
Von Willebrand ◽  
Willebrand Factor

Abstract Von Willebrand factor (VWF) multimers attached to endothelial cells can provide a platform for thrombosis, especially when accompanied by ADAMTS13 deficiency. We characterized the structural features of ultralarge VWF (ULVWF) molecules acutely secreted from Weibel-Palade bodies and identified a receptor responsible for their binding to the surface of cultured human umbilical vein endothelial cells (HUVECs). Using fluorescence microscopy on live cells, VWF multimers formed extended strings within minutes after stimulation. String formation did not require exogenous platelets and occurred over a range of shear stress from 2.5 dyn/cm2 to 40 dyn/cm2. A subset of ULVWF strings spontaneously bound formalin-fixed platelets via platelet GPIb. Quick-freeze, deep-etch electron microscopy showed that ULVWF strings often merged to form bundles and networks. Each string was tethered to the endothelial membrane by a limited number of anchorage sites, many of them located on small membrane projections, suggesting a specific mode of interaction. Several independent approaches implicated integrin αvβ3 in anchoring ULVWF strings to the HUVEC surface. Either “RGDS” peptide or function blocking antibody (LM609) to integrin αvβ3 specifically and dose dependently inhibited ULVWF string formation 62 ± 0.7% and 53 ± 4%, respectively. Furthermore, integrin αv was seen decorating the extending ULVWF strings using a non-functional blocking antibody (LM142) in live-cell immunofluorescence. In addition, a lentiviral vector encoding shRNA against integrin αv resulted in approximately 70% reduction in cell surface αv expression by FACS analysis with antibody LM609. HUVEC infected with this lentivirus were significantly impaired in their ability to form ULVWF strings compared to cells infected with a control virus. In multiple experiments, shRNA knockdown of αv expression reduced ULVWF strings 75.3 ± 4.4% at 2.5dyn/cm2 and 81.6 ± 2% at 7.5dyn/cm2. These results indicated that ULVWF strings bind tightly to endothelial cells via very few anchorage sites and are relatively resistant to fluid shear stress. Although integrin αvβ3 does not appear to be required to stabilize ULVWF strings on mouse endothelium, these data suggest that integrin αvβ3 may participate in the stabilization of ULVWF strings on human endothelial cell surfaces.

Shear-Dependent Rolling on von Willebrand Factor of Mammalian Cells Expressing the Platelet Glycoprotein Ib-IX-V Complex

Blood ◽  
1998 ◽  
Vol 92 (10) ◽  
pp. 3684-3693 ◽  
Author(s):  
Becky J. Fredrickson ◽  
Jing-Fei Dong ◽  
Larry V. McIntire ◽  
José A. López
Keyword(s):  
Von Willebrand Factor ◽  
Mammalian Cells ◽  
Platelet Adhesion ◽  
Fluid Shear Stress ◽  
Platelet Glycoprotein ◽  
Shear Stresses ◽  
Mean Velocity ◽  
The Mean ◽  
Von Willebrand ◽  
Willebrand Factor

Mural thrombi form on exposed arterial subendothelium by a two-step process of platelet adhesion and aggregation. At high shear stresses such as are found in stenotic arteries, both steps are mediated by von Willebrand factor (vWF). Platelets initially adhere on vWF affixed to the subendothelial matrix through the glycoprotein (GP) Ib-IX-V complex. To examine the role of the GP Ib-IX-V complex under dynamic conditions, we modeled initial platelet adhesion at shear stresses ranging from 2 to 40 dyn/cm2 using vWF-coated glass slides, mammalian cells expressing full or partial GP Ib-IX-V complexes, and a parallel plate flow chamber with phase contrast video microscopy and digital image processing. Mammalian cells expressing the full complex tethered and rolled on the vWF substrate, whereas control cells did not. The rolling was completely inhibited by the monoclonal GP Ib antibody, AK2, or the vWF antibody, 5D2, both shown previously to block vWF-dependent platelet aggregation. Other GP Ib antibodies, WM23 and SZ2, did not significantly change the number or mean velocity of rolling cells. At low levels of GP Ib surface expression, cells expressing the full complex rolled slower than cells expressing the complex without GP V, indicating that GP V strengthens the interactions with the vWF surface under these conditions. Preshearing vWF for 5 minutes at 40 dyn/cm2 immediately before introducing cells into the chamber did not significantly change the number or the mean velocity of rolling cells. Inhibiting sulfation of the tyrosine residues within the GP Ib subunit reduced the number but did not change the mean velocity of the rolling cells. Our results indicate that, under the conditions of these experiments, bonds between vWF and GP Ib constantly form and break under fluid shear stress. Additionally, our results suggest that GP Ib-IX-V complexes behave like selectin receptors in their ability to mediate smooth rolling while cells maintain continuous surface contact. Such a mechanism, in vivo, would allow platelets to slow down and eventually arrest on the blood vessel wall. The system described provides a valuable approach for investigating the structure-function relationship of individual receptors and ligands in the process of platelet adhesion and thrombosis.

Cytotoxicity of silica nanoparticles through exocytosis of von Willebrand factor and necrotic cell death in primary human endothelial cells

Biomaterials ◽  
2011 ◽  
Vol 32 (33) ◽  
pp. 8385-8393 ◽  
Author(s):  
Alexander T. Bauer ◽  
Elwira A. Strozyk ◽  
Christian Gorzelanny ◽  
Christoph Westerhausen ◽  
Anna Desch ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Death ◽  
Silica Nanoparticles ◽  
Von Willebrand Factor ◽  
Necrotic Cell Death ◽  
Human Endothelial Cells ◽  
Necrotic Cell ◽  
Von Willebrand ◽  
Willebrand Factor

P-selectin binds to the D′-D3 domains of von Willebrand factor in Weibel-Palade bodies

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3922-3924 ◽  
Author(s):  
Grégoire Michaux ◽  
Timothy J. Pullen ◽  
Sandra L. Haberichter ◽  
Daniel F. Cutler
Keyword(s):  
Endothelial Cells ◽  
Membrane Protein ◽  
Cell Surface ◽  
Von Willebrand Factor ◽  
Integral Membrane Protein ◽  
Hek293 Cells ◽  
Binding Partner ◽  
Lumenal Domain ◽  
Von Willebrand ◽  
Willebrand Factor

It has recently been shown that the ultralarge platelet–recruiting von Willebrand factor (VWF) strings formed immediately at exocytosis from endothelial cells may be anchored to the cell surface by interaction with the integral membrane protein P-selectin. This finding of a new binding partner for VWF immediately prompts the question which domains of VWF bind to P-selectin. We have exploited the fact that VWF expression in HEK293 cells triggers the formation of Weibel-Palade body–like structures that can recruit P-selectin. A suitably modified version of this assay using coexpressed truncations of VWF, together with P-selectin variants in HEK293 cells, allowed us to determine which domains of VWF would recruit P-selectin within a physiologically appropriate intracellular environment. Confirming the results of such a cellular assay by conventional coimmunoprecipitation, we concluded that the lumenal domain of P-selectin interacts with the D′-D3 domains of VWF.

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