Thrombin Interaction with Platelet GPIB: Role of the Heparin Binding Domain

1997 ◽  
Vol 77 (04) ◽  
pp. 735-740 ◽  
Author(s):  
Erica De Candia ◽  
Raimondo De Cristofaro ◽  
Lulgi De Marco ◽  
Moreno Mazzucato ◽  
Matilde Picozzi ◽  
...  
Keyword(s):  
Binding Site ◽  
Recognition Site ◽  
Thrombin Receptor ◽  
Membrane Glycoprotein ◽  
Heparin Binding ◽  
Chemical Modifications ◽  
Heparin Binding Site ◽  
High Affinity Binding Site ◽  
Heparin Binding Domain

SummaryThe platelet membrane glycoprotein lb (Gplb) has a high affinity binding site for α-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as “heparin binding site” (HBS) and “fibrino#gen recognition site” (FRS) were investigated as the possible domains involved in Gplb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-α-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of Gplb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of α-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A α-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Kallistatin: double-edged role in angiogenesis, apoptosis and oxidative stress

2017 ◽  
Vol 398 (12) ◽  
pp. 1309-1317 ◽  
Author(s):  
Julie Chao ◽  
Pengfei Li ◽  
Lee Chao
Keyword(s):  
Oxidative Stress ◽  
Endothelial Cells ◽  
Animal Models ◽  
Binding Site ◽  
Active Site ◽  
Heparin Binding ◽  
Heparin Binding Site ◽  
Tnf Α ◽  
Heparin Binding Domain ◽  
And Oxidative Stress

AbstractKallistatin, via its two structural elements – an active site and a heparin-binding domain – displays a double-edged function in angiogenesis, apoptosis and oxidative stress. First, kallistatin has both anti-angiogenic and pro-angiogenic effects. Kallistatin treatment attenuates angiogenesis and tumor growth in cancer-bearing mice. Kallistatin via its heparin-binding site inhibits angiogenesis by blocking vascular endothelial growth factor (VEGF)-induced growth, migration and adhesion of endothelial cells. Conversely, kallistatin via the active site promotes neovascularization by stimulating VEGF levels in endothelial progenitor cells. Second, kallistatin inhibits or induces apoptosis depending on cell types. Kallistatin attenuates organ injury and apoptosis in animal models, and its heparin-binding site is essential for blocking tumor necrosis factor (TNF)-α-induced apoptosis in endothelial cells. However, kallistatin via its active site induces apoptosis in breast cancer cells by up-regulating miR-34a and down-regulating miR-21 and miR-203 synthesis. Third, kallistatin can act as an antioxidant or pro-oxidant. Kallistatin treatment inhibits oxidative stress and tissue damage in animal models and cultured cells. Kallistatin via the heparin-binding domain antagonizes TNF-α-induced oxidative stress, whereas its active site is crucial for stimulating antioxidant enzyme expression. In contrast, kallistatin provokes oxidant formation, leading to blood pressure reduction and bacterial killing. Kallistatin-mediated vasodilation is partly mediated by H2O2, as the effect is abolished by the antioxidant enzyme catalase. Moreover, kallistatin exerts a bactericidal effect by stimulating superoxide production in neutrophils of mice with microbial infection as well as in cultured immune cells. Thus, kallistatin’s dual roles in angiogenesis, apoptosis and oxidative stress contribute to its beneficial effects in various diseases.

scholarly journals Important role of arginine 129 in heparin-binding site of antithrombin III. Identification of a novel mutation arginine 129 to glutamine.

1990 ◽  
Vol 265 (31) ◽  
pp. 18997-19001
Author(s):  
S Gandrille ◽  
M Aiach ◽  
D.A. Lane ◽  
D Vidaud ◽  
P Molho-Sabatier ◽  
...  
Keyword(s):  
Binding Site ◽  
Antithrombin Iii ◽  
Novel Mutation ◽  
Heparin Binding ◽  
Heparin Binding Site

scholarly journals Role of Glycoprotein V in the Formation of the Platelet High-Affinity Thrombin-Binding Site

Blood ◽  
1997 ◽  
Vol 89 (12) ◽  
pp. 4355-4363 ◽  
Author(s):  
Jing-Fei Dong ◽  
Garunee Sae-Tung ◽  
José A. López
Keyword(s):  
Binding Site ◽  
Surface Expression ◽  
Thrombin Receptor ◽  
Platelet Surface ◽  
High Affinity ◽  
L Cells ◽  
High Affinity Binding Site ◽  
Physiologic Function ◽  
Von Willebrand

Abstract The glycoprotein (GP) Ib-IX-V complex contains a high-affinity binding site for thrombin on the platelet surface with a poorly defined role in platelet activation by this agonist. Four polypeptides comprise the complex: GP Ibα, GP Ibβ, GP IX, and GP V. The site within the complex that binds thrombin has been localized to a 45-kD region at the amino terminus of GP Ibα, which also contains the site through which the complex interacts with von Willebrand factor. A GP Ib-IX complex that lacks GP V can be efficiently expressed on the surface of transfected cells. We examined the ability of L cells expressing the GP Ib-IX complex (L2H cells) to bind thrombin at high affinity, and found no increase over the level of thrombin binding to control L cells. Because it is one of the few substrates for thrombin on the platelet surface, GP V has also been implicated as possibly participating in thrombin's actions on the platelet. To examine the role of GP V in forming the high-affinity thrombin-binding site, we compared the binding of thrombin to L2H cells versus cells that express the entire GP Ib-IX-V complex (L2H/V cells). Surface expression of GP Ibα was equivalent in these two stable cell lines. Thrombin binding to L2H/V cells was detectable at 0.25 nmol/L thrombin and reached a plateau at 1 nmol/L. No binding to L2H cells was detectable at these concentrations. Comparable results were obtained when thrombin binding to L2H cells transiently expressing GP V was compared with its binding to sham-transfected L2H cells. Again, only cells transiently expressing GP V bound thrombin specifically. As with the platelet polypeptide, thrombin cleaved GP V from the surface of L2H/V cells. To test whether GP V cleavage was required for enhancing thrombin binding to the complex, we tested the binding of enzymatically inactive D-phenylalanyl-Lprolyl-L-arginine chloromethylketone (PPACK)-thrombin to L2H and L2H/V cells. Like native thrombin, PPACK-thrombin at 1 nmol/L bound only to L2H/V cells, indicating that GP V cleavage is not a prerequisite for the formation of the high-affinity thrombin receptor. These data provide the first indication of a physiologic function for GP V, and suggest that formation of the high-affinity thrombin receptor on the platelet surface has complex allosteric requirements.

Antithrombin-mediated Inhibition of Factor Vlla-Tissue Factor Complex by the Synthetic Pentasaccharide Representing the Heparin Binding Site to Antithrombin

1996 ◽  
Vol 76 (01) ◽  
pp. 005-008 ◽  
Author(s):  
Jean Claude Lormeau ◽  
Jean Pascal Herault ◽  
Jean Marc Herbert
Keyword(s):  
Binding Site ◽  
Tissue Factor ◽  
Residual Activity ◽  
Coagulation Factors ◽  
Heparin Binding ◽  
Experimental Conditions ◽  
First Order ◽  
Heparin Binding Site ◽  
Coagulant Activity

SummaryWe examined the effect of the synthetic pentasaccharide representing the minimal binding site of heparin to antithrombin on the antithrombin-mediated inactivation of factor Vila bound to tissue factor. This effect was compared to the effect of unfractionated heparin. Using purified recombinant human coagulation factors and either a clotting or an amidolytic assay for the determination of the residual activity of factor Vila, we showed that the pentasaccharide was an efficient antithrombin-dependent inhibitor of the coagulant activity of tissue factor-factor Vila complex. In our experimental conditions, assuming a mean MW of 14,000 for heparin, the molar pseudo-first order rate constants for ATIII-mediated FVIIa inhibition by ATIII-binding heparin and by the synthetic pentasaccharide were found to be similar with respective values of 104,000 ± 10,500 min-1 and 112,000 ± 12,000 min-1 (mean ± s.e.m., n = 3)

scholarly journals Localization of the major heparin-binding site in fibronectin.

1991 ◽  
Vol 266 (12) ◽  
pp. 7812-7818 ◽  
Author(s):  
F J Barkalow ◽  
J E Schwarzbauer
Keyword(s):  
Binding Site ◽  
Heparin Binding ◽  
Heparin Binding Site

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5

2000 ◽  
Vol 24 (1) ◽  
pp. 43-51 ◽  
Author(s):  
H Song ◽  
J Beattie ◽  
IW Campbell ◽  
GJ Allan
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Protein ◽  
Binding Activity ◽  
Binding Domain ◽  
Site Directed Mutagenesis ◽  
Heparin Binding ◽  
Igf I ◽  
Heparin Binding Domain ◽  
Igf Binding

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

scholarly journals Role of the heparin-binding domain in intracellular trafficking of sHB-EGF

2019 ◽  
Vol 91 (4) ◽  
pp. 26-32
Author(s):  
O. I. Krynina ◽  
◽  
K. Yu. Manoilov ◽  
D. V. Kolybo ◽  
S. V. Komisarenko ◽  
...  
Keyword(s):  
Intracellular Trafficking ◽  
Binding Domain ◽  
Heparin Binding ◽  
Heparin Binding Domain
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