scholarly journals High-resolution probing heparan sulfate–antithrombin interaction on a single endothelial cell surface: single-molecule AFM studies

2015 ◽  
Vol 17 (20) ◽  
pp. 13301-13306 ◽  
Author(s):  
Cunlan Guo ◽  
Xian Fan ◽  
Hong Qiu ◽  
Wenyuan Xiao ◽  
Lianchun Wang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
High Resolution ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Single Molecule ◽  
Binding Sites ◽  
Specific Interactions ◽  
Endothelial Cell Surface ◽  
Afm Probe

Using an antithrombin functionalized AFM probe, the specific interactions between antithrombin and heparan sulfate on endothelial cell surface were determined, revealing previously unexpected diverse binding sites as well as critical roles of N-, 2-O- and 6-O-sulfation in the interactions.

CULTURED HUMAN ENDOTHELIAL CELLS BIND AND INTERNALIZE HIGH MOLECULAR WEIGHT KININOGEN

1987 ◽  
Author(s):  
Freek van Iwaarden ◽  
G Philip ◽  
de Groot ◽  
Bonno N Bouma
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
High Molecular Weight ◽  
Binding Sites ◽  
High Molecular Weight Kininogen ◽  
Human Endothelial Cells ◽  
Endothelial Cell Surface ◽  
Coagulation Pathway

The presence of High Molecular Weight kininogen (HMWK) was demonstrated in cultured human endothelial cells (EC) by immunofluorescence techniques. Using an enzyme linked immunosorbent assay a concentration of 58 ng HMWK/10 cells was determined. Immunoprecipitation studies performed with lysed metabolically labelled endothelial cells and mono-specific antisera directed against HMWK suggested that HMWK is not synthesized by the endothelial cells. Endothelial cells cultured in the presence of HMWK-depleted serum did not contain HMWK. This, suggests that endothelial cells can internalize HMWK. Using 125I-HMWK it was demonstrated that cultured endothelial cells bind HMWK in a time-dependent, specific and saturable.way. The cells were found to internalize 125I-HMWK, since I-HMWK was detected in solubilized endothelial cells after the cell bound 125I-HMWK had been eluted with dextran sulphate.The binding of I-HMWK required the presence of zinc ions. Optimal binding of 125I-HMWK was observed at 50 μM Zn++ . Calcium ions inhibited the Zn++ dependent binding of 125I-HMWK |25EC. In the presence of 3 mM CaCl2 the total binding of 125I-HMWK was significantly decreased, and a .concentration of 200 μM Zn++ was Required for the binding of 125I-HMWK to thecells. Higher,. Ca concentrations did not further decrease the binding of 125I-HMWK. Analysis of tl^e binding data by the ligand computer program indicated 3.2 x 10 binding sites per cell for HMWK with a Kd of 35 nM at 50 μM ZnCl2 and 1 mM CaCl2. Specify binding of HMWK did also occur at physiological plasma Zn++ concentrations. Half maximal binding was observed at HMWK concentrations of ± 105 nM at 10 μM ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 pM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway. M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 10 molecules of HMWK bound per cell and at 80 nM with 2.8 x 10 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.M ZnCl2 and 45 nM at 25 μM ZnCl2. The HMWK binding sites were saturatecT at HMWK concentrations of 130 nM with 1.6 x 16 molecules of HMWK bound per cell and at 80 nM with 2.8 x 106 molecules of HMWK bound per cell at 10 and 25 μM ZnCl2 respectively. These results suggest that at physiological zinc, calcium and HMWK concentrations the HMWK binding sites on the endothelial cell are saturated. The presence of HMWK on the endothelial cell surface may play a role in the initiation of the intrinsic coagulation pathway.

Increased responsiveness of hypoxic endothelial cells to FGF2 is mediated by HIF-1α-dependent regulation of enzymes involved in synthesis of heparan sulfate FGF2-binding sites

2002 ◽  
Vol 115 (9) ◽  
pp. 1951-1959
Author(s):  
Jian Li ◽  
Nicholas W. Shworak ◽  
Michael Simons
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Cell Surface ◽  
Heparan Sulfate ◽  
Binding Sites ◽  
Cell Mass ◽  
Growth Stimulation ◽  
Receptor Expression ◽  
Hypoxic Conditions

Binding of basic fibroblast growth factor (FGF2) to its high affinity receptors requires the presence of specific heparan sulfate (HS) moieties on the cell surface that act as coreceptors. To determine the contribution of cell-surface HS to modulation of FGF2-dependent cell growth, we studied the changes in the cell mass and FGF2 binding of endothelial cell HS under normoxic and hypoxic conditions in vitro. Both large vein and cardiac microvascular endothelial cells cultured under hypoxic conditions demonstrated an increase in the ratio of cell-surface HS to chondroitin sulfate (CS), as well as an increase in the number of low affinity (HS-associated) binding sites for FGF2 with no change in the apparent Kd. This increase in the number of HS-FGF2 binding sites, in the absence of a significant change in FGF receptor expression, resulted in enhanced responsiveness of hypoxic,compared with normoxic, endothelial cells to FGF2 stimulation. Gene expression studies demonstrated increased expression of the key regulatory enzyme responsible for HS chain synthesis, 1,4 GlcNAc transferase(GlcNAcT-I), as well as increased expression of 2-O sulfotransferase (HS2ST),the enzyme responsible for sulfation of IdoA, a crucial part of the HS-FGF2 binding site. Transduction of cells with an adenovirus encoding a HIF-1αexpression construct resulted in a similar increase in GlcNAcT-I and HS2ST expression. We conclude that hypoxia increases endothelial cell responsiveness to FGF2 by promoting preferential synthesis of HS rather than CS chains and increasing the number of FGF2-binding sites on HS chains. Both of these events are mediated by a HIF-1α-dependent increase in expression of the enzymes GlnNAcT-I and HS2ST. This shift in cell-surface HS composition results in enhanced cell sensitivity to FGF2-induced growth stimulation.

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