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Title: Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody

Abstract

Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation.

Authors:
; ;  [1]
  1. (Univ. of California, Irvine (USA))
Publication Date:
OSTI Identifier:
5707724
Resource Type:
Journal Article
Resource Relation:
Journal Name: Biochemistry; (USA); Journal Volume: 29:38
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; ANTICOAGULANTS; CROSS-LINKING; HEPARIN; RADIORECEPTOR ASSAY; ANTIBODIES; IODINE 125; MONOCLONAL ANTIBODIES; PURIFICATION; SERINE PROTEINASES; THROMBIN; AMINES; BETA DECAY RADIOISOTOPES; CARBOHYDRATES; CHEMICAL REACTIONS; COAGULANTS; DAYS LIVING RADIOISOTOPES; DRUGS; ELECTRON CAPTURE RADIOISOTOPES; ENZYMES; HEMATOLOGIC AGENTS; HEMOSTATICS; HYDROLASES; INTERMEDIATE MASS NUCLEI; IODINE ISOTOPES; ISOTOPE APPLICATIONS; ISOTOPES; MUCOPOLYSACCHARIDES; NUCLEI; ODD-EVEN NUCLEI; ORGANIC COMPOUNDS; ORGANIC SULFUR COMPOUNDS; PEPTIDE HYDROLASES; POLYMERIZATION; POLYSACCHARIDES; RADIOISOTOPES; SACCHARIDES; TRACER TECHNIQUES; 550201* - Biochemistry- Tracer Techniques

Citation Formats

Smith, J.W., Dey, B., and Knauer, D.J.. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody. United States: N. p., 1990. Web. doi:10.1021/bi00490a010.
Smith, J.W., Dey, B., & Knauer, D.J.. Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody. United States. doi:10.1021/bi00490a010.
Smith, J.W., Dey, B., and Knauer, D.J.. Tue . "Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody". United States. doi:10.1021/bi00490a010.
@article{osti_5707724,
title = {Heparin binding domain of antithrombin III: Characterization using a synthetic peptide directed polyclonal antibody},
author = {Smith, J.W. and Dey, B. and Knauer, D.J.},
abstractNote = {Antithrombin III (ATIII) is a plasma-borne serine protease inhibitor that apparently forms covalent complexes with thrombin. The interaction between ATIII and thrombin is enhanced several thousandfold by the glycosaminoglycan, heparin. The authors have previously proposed that the heparin binding site of ATIII residues within a region extending from amino acid residues 114-156. Computer-assisted analysis of this region revealed the presence of a 22 amino acid domain (residues 124-145), part of which shows a strong potential for the formation of an amphipathic helix: hydrophobic on one face and highly positively charged on the other. In the presence studies, polyclonal antisera were generated against a synthetic peptide corresponding to residues 124-145 in native human ATIII. Affinity-purified IgG from these antisera, as well as monovalent Fab's derived from them, specifically blocked the binding of heparin to ATIII. Additionally, occupancy of the heparin binding site by these same monovalent and bivalent IgG's at least partially substituted for heparin, accelerating linkage formation between ATIII and thrombin. These results provide the first immunological evidence that region 124-145 is directly involved in the binding of heparin to ATIII and that an antibody-induced conformational change within this region can mediate ATIII activation.},
doi = {10.1021/bi00490a010},
journal = {Biochemistry; (USA)},
number = ,
volume = 29:38,
place = {United States},
year = {Tue Sep 25 00:00:00 EDT 1990},
month = {Tue Sep 25 00:00:00 EDT 1990}
}
  • The denaturation of human and bovine antithrombin III by guanidine hydrochloride has been followed by /sup 1/H NMR spectroscopy. The same unfolding transition seen previously from circular dichroism studies at low denaturant concentration was detected here by discontinuous changes in the chemical shifts of the C(2) protons of two of the five histidines in human antithrombin III and of three of the six histidines in bovine antithrombin III. These two histidines in human antithrombin III are assigned to residue 1 and, more tentatively, to residue 65. Two of the three histidines similarly affected in the bovine protein appear to bemore » homologous to residues in the human protein. This supports the proposal of similar structures for the two proteins. In the presence of heparin, the discontinuous titration behavior of these histidine resonances is shifted to higher denaturant concentration, reflecting the stabilization of the easily unfolded first domain of the protein by bound heparin. From the tentative assignment of one of these resonances to histidine-1, it is proposed that the heparin binding site of antithrombin III is located in the N-terminal region and that this region forms a separate domain from the rest of the protein. The pattern of disulfide linkages is such that this domain may well extend from residue 1 to at least residue 128. Thermal denaturation also leads to major perturbation of these two histidine resonances in human antithrombin III, though stable intermediates in the unfolding were not detected.« less
  • Bovine antithrombin III (AT III) interaction with the luminal surface of bovine aortic segments with a continuous layer of endothelium was examined. Incubation of /sup 125/I-AT III with vessel segments, previously washed free of endogenous AT III, demonstrated specific, time-dependent binding to the protease inhibitor to the endothelium. Half-maximal binding was observed at an added AT III concentration of 14 nM. Binding of /sup 125/I-AT III to the vessel wall was reversible (50% dissociated in 4 min), and addition of either heparin or Factor Xa accelerated displacement of /sup 125/I-AT III from the vessel segment. Dissociation of /sup 125/I-AT IIImore » from the vessel segment in the presence of factor Xa coincided with the formation of a Factor Xa-/sup 125/I-AT III complex. Inactivation of Factor IXa and Factor Xa by AT III was facilitated in the presence of vessel segments. Pretreatment of vessel segments with highly purified Flavobacterium heparinase precluded the vessel-dependent augmentation of AT III anticoagulant activity as well as specific binding of /sup 125/I-AT III to the vessel endothelium. In contrast, pretreatment of the vessel segments with chrondroitinases (ABC or AC) had no detectable effect on /sup 125/I-AT III binding or on AT III anticoagulant activity. AT III binding to vessel segments was competitively inhibited by increasing concentration of platelet factor 4. Binding of the protease inhibitor to vessel segments was inhibited by chemical modification of AT III lysyl or tryptophan residues. These AT III derivatives retained progressive inhibitory activity. These data suggest that heparin-like molecules are present on the aortic vessel wall and mediate binding of AT III to the vessel surface, as well as enhancing the anticoagulant activity of AT III at these sites.« less
  • A receptor binding region of mouse interferon {gamma} (IFN{gamma}) has previously been localized to the N-terminal 39 amino acids of the molecule by use of synthetic peptides and monoclonal antibodies. in this report, a detailed analysis of the synthetic peptide corresponding to this region, IFN{gamma}(1-39), is presented. Circular dichroism (CD) spectroscopy indicated that the peptide has stable secondary structure under aqueous conditions and adopts a combination of {alpha}-helical and random structure. A peptide lacking two N-terminal amino acids, IFN{gamma}(3-39), had similar secondary structure and equivalent ability to compete for receptor binding, while peptides lacking four or more N-terminal residues hadmore » reduced {alpha}-helical structure and did not inhibit {sup 125}I-IFN{gamma} binding. A peptide lacking eight C-terminal residues, IFN{gamma}(1-31), did not block {sup 125}I-IFN{gamma} binding and had no detectable {alpha}helical structure, suggesting a requirement of the predicted second {alpha}-helix (residues 20-34) for receptor interaction and helix stabilization. These data support the presence of a binding domain in the N-terminus of IFN{gamma} and indicate a role for residues 3, 4, 14, and at least some of residues 32-39 in IFN{gamma}(1-39) interaction with receptor. This study provides insight into the structural/functional basis for IFN{gamma} interaction with receptor that should be useful for the development of potent agonists and antagonists of IFN{gamma}action.« less
  • Previous findings indicated that binding of heparin to antithrombin III (AT III) facilitates thrombin-induced proteolysis of the inhibitor. Researchers now studied this property of heparin in regard to its molecular weight and anticoagulant activity. Commercial heparin was resolved on Sephadex G-200 into six fractions of decreasing molecular weight. From each fraction high affinity (HA) heparin was isolated by chromatography on AT III-Sepharose and examined in reaction of alpha-thrombin with a molar excess of /sup 125/I AT III. Proteolysis of the inhibitor was assessed by SDS polyacrylamide gel electrophoresis. In the presence of the HA heparin from 18% to 38% ofmore » AT III participating in reaction appeared in the form of inactive 50,000-dalton fragment, as opposed to 7% of AT III fragmented in the absence of heparin. Although the ability to potentiate proteolysis was at its peak in the medium-molecular-size heparin fraction, the amount of degraded inhibitor relative to anticoagulant activity increased with decreasing molecular weight of the polysaccharide. These findings are consistent with the possibility that the ability of bound heparin to facilitate the cleavage of AT III by thrombin is generally less contingent upon secondary characteristics of the polysaccharide than the anticoagulant activity.« less
  • Highlights: Black-Right-Pointing-Pointer HBP sequence identified from HB-EGF has cell penetration activity. Black-Right-Pointing-Pointer HBP inhibits the NF-{kappa}B dependent inflammatory responses. Black-Right-Pointing-Pointer HBP directly blocks phosphorylation and degradation of I{kappa}B{alpha}. Black-Right-Pointing-Pointer HBP inhibits nuclear translocation of NF-{kappa}B p65 subunit. -- Abstract: A heparin-binding peptide (HBP) sequence from human heparin-binding epidermal growth factor-like growth factor (HB-EGF) was identified and was shown to exhibit cell penetration activity. This cell penetration induced an anti-inflammatory reaction in lipopolysaccharide (LPS)-treated RAW 264.7 macrophages. HBP penetrated the cell membrane during the 10 min treatment and reduced the LPS-induced production of nitric oxide (NO), inducible nitric oxide synthase (iNOS),more » and cytokines (TNF-{alpha} and IL-6) in a concentration-dependent manner. Additionally, HBP inhibited the LPS-induced upregulation of cytokines, including TNF-{alpha} and IL-6, and decreased the interstitial infiltration of polymorphonuclear leukocytes in a lung inflammation model. HBP inhibited NF-{kappa}B-dependent inflammatory responses by directly blocking the phosphorylation and degradation of I{kappa}B{alpha} and by subsequently inhibiting the nuclear translocation of the p65 subunit of NF-{kappa}B. Taken together, this novel HBP may be potentially useful candidate for anti-inflammatory treatments and can be combined with other drugs of interest to transport attached molecules into cells.« less