scholarly journals Extracellular Histones Bind Vascular Glycosaminoglycans and Inhibit the Anti-Inflammatory Function of Antithrombin.

2021 ◽  
Vol 55 (5) ◽  
pp. 605-617
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Heparin Binding ◽  
Protease Inhibition ◽  
Proinflammatory Responses ◽  
Extracellular Histones ◽  
Anti Inflammatory ◽  
Anticoagulant Function ◽  
Cellular Phenotypes ◽  
Angiopoietin 2

Binding of histones to molecular pattern recognition receptors on endothelial cells and leukocytes provokes proinflammatory responses and promotes activation of coagulation. Histones also bind therapeutic heparins, thereby neutralizing their anticoagulant functions. The aim of this study was to test the hypothesis that histones can interact with the antithrombin (AT)-binding vascular glycosaminoglycans (GAGs) to induce inflammation and inhibit the anti-inflammatory function of AT. METHODS: We evaluated the heparin-binding function of histones by an AT-dependent protease-inhibition assay. Furthermore, we treated endothelial cells with histones in the absence and presence of AT and monitored cellular phenotypes employing established signaling assays. RESULTS: Histones neutralized AT-dependent anticoagulant function of heparin in both purified protease-inhibition and plasma-based assays. Histones also disrupted endothelial cell barrier-permeability function by a GAG-dependent mechanism as evidenced by the GAG-antagonist, surfen, abrogating their disruptive effects. Further studies revealed histones and AT compete for overlapping binding-sites on GAGs, thus increasing concentrations of one protein abrogated effects of the other. Histones elicited proapoptotic effects by inducing nuclear localization of PKC-δ in endothelial cells and barrier-disruptive effects by destabilizing VE-cadherin, which were inhibited by AT, but not by a D-helix mutant of AT incapable of interacting with GAGs. Finally, histones induced release of Weibel-Palade body contents, VWF and angiopoietin-2, and promoted expression of cell adhesion molecules on endothelial cells, which were all downregulated by AT but not by D-helix mutant of AT. CONCLUSION: We conclude that histones and AT compete for overlapping binding sites on vascular GAGs to modulate coagulation and inflammation.

scholarly journals Mechanism of thrombin binding to endothelial cells

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 368-372 ◽  
Author(s):  
PT Bauer ◽  
R Machovich ◽  
P Aranyi ◽  
KG Buki ◽  
E Csonka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Antithrombin Iii ◽  
Affinity Binding ◽  
Heparin Binding ◽  
High Affinity Binding ◽  
Aortic Endothelial Cells ◽  
High Affinity ◽  
Lysine Residues ◽  
Two Populations

Abstract The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

scholarly journals Mechanism of thrombin binding to endothelial cells

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 368-372
Author(s):  
PT Bauer ◽  
R Machovich ◽  
P Aranyi ◽  
KG Buki ◽  
E Csonka ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Antithrombin Iii ◽  
Affinity Binding ◽  
Heparin Binding ◽  
High Affinity Binding ◽  
Aortic Endothelial Cells ◽  
High Affinity ◽  
Lysine Residues ◽  
Two Populations

The interaction of human alpha-thrombin with mini-pig aortic endothelial cells was studied using 125I-labeled enzyme. Equilibrium between bound and free thrombin was attained within 1 min, and the Klotz-Hunston equations indicated two populations of binding sites. Approximately 30,000 sites/cell belonged to the high-affinity class with a Kd of about 3 x 10(-8) M. Modification of two lysine residues of thrombin with pyridoxal 5′-phosphate (PLP2-thrombin) destroyed the high- affinity binding and about three-fourths of the low-affinity bindings. When the lysine residue of thrombin involved in heparin binding was protected with heparin against chemical modification (PLP-thrombin), the modified enzyme remained similar to the native one with respect to cellular binding, with some loss of low-affinity binding only. Heparin, in a tenfold molar excess to enzyme, inhibited the binding of the native as well as the PLP-thrombin, whereas it did not influence the interaction between PLP2-thrombin and the cell. Since heparin might interfere with both the enzyme and the cell, the binding of heparin to endothelial cells was also examined. The results revealed that 3H- heparin also bound to cells. This binding was characterized by a Kd of 3 x 10(-7) M, approximately 10(6) sites/cell. Furthermore, thrombin bound to endothelial cells was released by antithrombin III. On the basis of these and other data in the literature, a model is proposed for the mechanism of the binding of thrombin to endothelial cells.

scholarly journals Interaction of plasmin with endothelial cells

1984 ◽  
Vol 218 (1) ◽  
pp. 119-124 ◽  
Author(s):  
P I Bauer ◽  
R Machovich ◽  
K G Büki ◽  
E Csonka ◽  
S A Koch ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Binding Sites ◽  
Monolayer Culture ◽  
Scatchard Analysis ◽  
Heparin Binding ◽  
Active Centre ◽  
Aortic Endothelial Cells ◽  
Heparin Binding Site ◽  
Equilibrium Chemical

Interaction of human plasmin with a monolayer culture of mini-pig aortic endothelial cells was studied by using the 125I-labelled enzyme. The binding of plasmin was time- and concentration-dependent. Equilibrium between bound and free enzyme was obtained within 90s, and Scatchard analysis indicated a high- and a low-affinity population of binding sites of approx. 1.24 × 10(4) sites/cell having a Kd of 1.4 × 10(-9) M and 7.2 × 10(4) sites/cell with a Kd of 2 × 10(-8) M respectively. Plasmin, bound to cell, was spontaneously released within 2 min, suggesting a rapid equilibrium. Chemical modification of the enzyme with phenylmethanesulphonyl fluoride or pyridoxal 5′-phosphate revealed that neither the active centre nor the heparin-binding site of plasmin was involved in the interaction with the endothelial cell. In terms of endothelial-cell receptors, the binding sites of cells for plasmin and thrombin were different: the two enzymes did not compete with each other, and the pretreatment of cells with neuraminidase or chondroitin ABC lyase resulted in a 50% decrease of thrombin or plasmin binding respectively. Arachidonic acid incorporated into phospholipids of the cell was released by plasmin, but a change in the rate of prostacyclin formation was not measurable. The interaction of plasmin with endothelial cells seems to be specific in the fibrinolytic system, since plasminogen did not bind to these cells under similar conditions.

scholarly journals Conserved Surface-Exposed K/R-X-K/R Motifs and Net Positive Charge on Poxvirus Complement Control Proteins Serve as Putative Heparin Binding Sites and Contribute to Inhibition of Molecular Interactions with Human Endothelial Cells: a Novel Mechanism for Evasion of Host Defense

2000 ◽  
Vol 74 (12) ◽  
pp. 5659-5666 ◽  
Author(s):  
Scott A. Smith ◽  
Nick P. Mullin ◽  
John Parkinson ◽  
Sergei N. Shchelkunov ◽  
Alexei V. Totmenin ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Binding Sites ◽  
Positive Charge ◽  
Expression System ◽  
Binding Activity ◽  
Heparin Binding ◽  
Dna Encoding ◽  
Human Endothelial Cells ◽  
Charged Amino Acids ◽  
Pathway Activation

ABSTRACT Vaccinia virus complement control protein (VCP) has been shown to possess the ability to inhibit both classical and alternative complement pathway activation. The newly found ability of this protein to bind to heparin has been shown in previous studies to result in uptake by mast cells, possibly promoting tissue persistence. It has also been shown to reduce chemotactic migration of leukocytes by blocking chemokine binding. In addition, this study shows that VCP—through its ability to bind to glycosaminoglycans (heparin-like molecules) on the surface of human endothelial cells—is able to block antibody binding to surface major histocompatibility complex class I molecules. Since heparin binding is critical for many functions of this protein, we have attempted to characterize the molecular basis for this interaction. Segments of this protein, generated by genetic engineering of the DNA encoding VCP into the Pichia pastoris expression system, were used to localize the regions with heparin binding activity. These regions were then analyzed to more specifically define their properties for binding. It was found that the number of putative binding sites (K/R-X-K/R), the overall positive charge, and the percentage of positively charged amino acids within the protein were responsible for this interaction.

Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

1989 ◽  
Vol 62 (02) ◽  
pp. 699-703 ◽  
Author(s):  
Rob J Aerts ◽  
Karin Gillis ◽  
Hans Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Single Chain ◽  
Human Endothelial Cells ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

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