Structure of the Antithrombin III-Binding site in Heparin

1979 ◽  
Author(s):  
U. Lindahl ◽  
G. Bäckström ◽  
N. Höök ◽  
J. Riesenfeld ◽  
L. Thunberg ◽  
...  
Keyword(s):  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Periodate Oxidation ◽  
Variable Structure ◽  
Variable Regions ◽  
High Affinity ◽  
Swedish Medical ◽  
A Minor ◽  
Binding Sequence ◽  
Partial Chemical

Fragments with high affinity for antithrombin III (AT), composed of 12 to 16 monosaccharide units, were isolated from heparin after partial chemical or enzymatic depolymerization of the polysaccharide. Analysis of such fragments based on identification ot deamination products suggested that nonsulfated L-iduronic acid (a minor constituent) is essential for the anticoagulant activity of heparin. The location of this unit in the AT-binding sequence was determined by periodate oxidation. Furthermore, an N-sulfate group essential for activity was located by structural analysis of partially N-desulfated fragments retaining high affinity for AT. It is proposed that the AT-hinding sequence in heparin has a variable structure containing certain nonvanable regions. A tentative structure for this sequence is presented, with indication of identified constant and variable regions.(Supported by grant No. 2309 from the Swedish Medical Research Council).

scholarly journals Biological implications of the structural, antithrombin affinity and anticoagulant activity relationships among vertebrate heparins and heparan sulphates

1986 ◽  
Vol 237 (2) ◽  
pp. 573-581 ◽  
Author(s):  
P Hovingh ◽  
M Piepkorn ◽  
A Linker
Keyword(s):  
Antithrombin Iii ◽  
Structural Characteristics ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Variable Structure ◽  
Cellular Level ◽  
High Affinity ◽  
Highly Active ◽  
Wide Range ◽  
Species Specific

We analysed the distribution, structural characteristics, antithrombin-III-binding properties and anticoagulant activities of heparins and heparan sulphates isolated from the tissues of a wide range of vertebrates. Heparin has a curiously limited distribution, since it was absent from lower aquatic vertebrate species, present in only certain organs such as intestine in many higher vertebrates, and completely absent from the rabbit among mammals examined. The heparins were structurally diverse, and they exhibited a broad range of anticoagulant activities, from approx. 50% to 150% of average commercial heparins. Although there was a rough correlation between the anticoagulant potency of the starting isolate and the proportional content of material exhibiting high-affinity binding to the proteinase inhibitor antithrombin III, activities of high-affinity fractions from heparins low in activity overlapped those of low-affinity fractions from highly active heparins. Heparan sulphates, which in contrast were isolated from nearly all vertebrate organs, contained high-affinity subfractions constituting up to 5% of the starting material and possessing anticoagulant potencies of 2-30 units/mg. In consideration of the heparin data, we infer that its biological function is either species-specific or may be served by other molecular elements, and that there exists considerable diversity in the antithrombin-III-binding sequence of heparin. The more-generally distributed glycosaminoglycan heparan sulphate possesses within its variable structure a small high-affinity subfraction with low anticoagulant potency, whether isolated from aorta or other tissues. Although heparan sulphate appears to have an essential function at the cellular level, we suggest that this is probably not that of providing heparin-like antithrombotic effects on vascular surfaces.

scholarly journals The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

1981 ◽  
Vol 197 (3) ◽  
pp. 599-609 ◽  
Author(s):  
B Casu ◽  
P Oreste ◽  
G Torri ◽  
G Zoppetti ◽  
J Choay ◽  
...  
Keyword(s):  
Uronic Acid ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Amino Sugar ◽  
Active Species ◽  
Model Compounds ◽  
High Affinity ◽  
The Common ◽  
Binding Sequence ◽  
Heparin Oligosaccharides

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].

Interaction of Heparin with Histidine-Rich Glycoprotein and with Antithrombin III

1983 ◽  
Vol 50 (02) ◽  
pp. 560-562 ◽  
Author(s):  
H R Lijnen ◽  
B van Hoef ◽  
D Collen
Keyword(s):  
Metal Ions ◽  
Antithrombin Iii ◽  
Potent Inhibitor ◽  
Anticoagulant Activity ◽  
Divalent Metal ◽  
Clinical Grade ◽  
Divalent Metal Ions ◽  
Apparent Increase ◽  
High Affinity

SummaryThe interaction between heparin, histidine-rich glycoprotein and antithrombin III was studied in purified systems. Histidine- rich glycoprotein binds heparin and thereby interferes with its interaction with antithrombin III, resulting in neutralization of the anticoagulant activity. This interaction occurs with clinical grade heparin as well as with high affinity (for antithrombin III) heparin and with a high affinity heparin fragment with Mr. 4,300.Low affinity heparin competes with high affinity heparin for the binding to histidine-rich glycoprotein which results in an apparent increase of the anticoagulant activity of high affinity heparin.The interaction between heparin and histidine-rich glycoprotein is counteracted by Ca2+-binding anticoagulants, indicating that it is dependent on the presence of divalent metal ions. Ethylenediaminetetraacetate is a much more potent inhibitor of the interaction between heparin and histidine-rich glycoprotein than citrate.

ANTITHROMBIN III BINDING TO IMMOBILIZED HEPARIN FRAGMENTS AND ITS RELATION TO F XA INHIBITION

1987 ◽  
Author(s):  
K Kodama ◽  
O Larm ◽  
P Olsson ◽  
B Pasehe ◽  
J Risenfeldt ◽  
...  
Keyword(s):  
Binding Sites ◽  
Antithrombin Iii ◽  
Cellular Membrane ◽  
Tris Buffer ◽  
Buffer Solution ◽  
Thrombin Inhibition ◽  
High Affinity ◽  
Inhibitory Capacity ◽  
Nacl Concentration ◽  
Binding Sequence

Covalent end-point attachment of heparin fragments (8 000 daltons) to artitifical materials results as published before in a highly thromboresistant surface. Approximately one out of six bonded fragments carry the antithrombin III (AT) binding sequence. i.e. the high affinity site. With regard to thrombin inhibition the heparin surface resembles the endothelium. The present work deals with the uptake on the immobilized heparin and its significance for F Xa inhibition. The uptake of AT was studied at 0.15 M and 0.35 M NaCl concentration (TRIS buffer, pH 7.4) respectively and determined as disappearance of AT activity in the exposed solutions. Large amounts were adsorbed at 0.15 M and the uptake was both concentration and time dependent. At 0.35 M the uptake was the same at all the tested AT concentrations: 5 pico-moles/square cm. It was deduced that mainly high affinity sites had taken up AT at 0.35 M and that both high and low affinity sites had taken up AT at 0.15 M.The non-AT-adsorbed heparin surface did not induce inhibition of F Xa (in TRIS buffer solution) after exposure. The surface AT-adsorbed at 0.15 M induced F Xa inhibition with the same rate in several consecutive exposed aliquots. The surface AT-adsorbed at 0.35 M had a lower inhibitory capacity and only the first F Xa aliquot was inhibited at the same rate as on the surface AT-adsorbed at 0.15 M. The inhibition rate for a second aliquot was slowlier due to the facts that AT had been consumed and that the density of AT on high affinity sites had decreased. It is concluded that AT on high affinity sites determines the rate at which F Xa is inhibited whereas the amount of AT on low affinity sites determines the inhibitory capacity by continously providing the high affinity sites with AT. The migration of AT must take place horizontally in the 100-200 A thick surface layer and may mimic events happening on a cellular membrane with binding sites of different classes for a substance.

Relative Contributions Of Thrombin And Antithrombin III Affinities Of Heparin Fractions To The Rate Of Inactivation Of Thrombin By Antithrombin III

1981 ◽  
Author(s):  
A L Cerskus ◽  
K J Birchall ◽  
F Ofosu ◽  
M A Blajchman ◽  
J Hirsh
Keyword(s):  
Antithrombin Iii ◽  
Volume Fraction ◽  
Anticoagulant Activity ◽  
Order Rate Constant ◽  
Void Volume Fraction ◽  
Significant Activity ◽  
High Affinity ◽  
At Iii ◽  
Heparin Fractions ◽  
Heparin Affinity

Heparin enhances the rate of inactivation of thrombin (IIa) and other coagulant proteases by antithrombin III (AT III). The anticoagulant activity of heparin is associated with heparin moieties which have high affinity to AT III. However, the contribution of the IIa affinity to the anticoagulant activity has not been as clear. Standard porcine mucosal heparin was fractionated on affinity columns consisting of purified human AT III and IIa immobilized on agarose to determine the effect of heparins of various affinities on the second order rate constant (K") for the inactivation of IIa by AT III. Results are expressed as the rate enhancement factor (REF) which is defined as the ratio of the K" in the presence of 50 ng/ml heparin to the K" in the absence of heparin. The REF for unfractionated heparin was 9.5. Chromatography on either AT III-agarose or IIa-agarose resulted in elution of three heparin fractions corresponding to a void volume fraction, a low affinity fraction and a high affinity fraction. The REF’s of the fractions eluted from AT III-agarose were 1.3, 1.4 and 18.8, respectively while the REF’s for the corresponding IIa fractions were 5.6, 14.9 and 18.1, respectively. Rechromatography of the high affinity fraction from AT III-agarose on the IIa-agarose column resulted in the elution of three fractions with REF’s of 17.9, 21.1 and 27.0, respectively. Conclusions: 1. Heparin affinity to AT III is critical for anticoagulant activity as all of the activity is found in the high affinity fraction. 2. Heparin affinity to IIa also contributes to anticoagulant activity but is not critical as significant activity is observed in low affinity fractions. 3. High affinity to both IIa and AT III results in greater activity than that seen with fractions of high affinity to either protein alone.

scholarly journals Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 252-254
Author(s):  
A Tefferi ◽  
BA Owen ◽  
WL Nichols ◽  
TE Witzig ◽  
WG Owen
Keyword(s):  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Transitional Cell ◽  
Chemical Properties ◽  
Thrombin Time ◽  
High Affinity ◽  
Carcinoma Of The Bladder ◽  
D Dimer ◽  
And Chemical Properties

A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.

Turnover And Anticoagulant Properties Of Heparin-Antithrombin III Complexes, Stabilized By Covalent Bonds

1981 ◽  
Author(s):  
D Collen ◽  
R Ceustermans ◽  
M De Mol ◽  
M Hoylaerts
Keyword(s):  
Intravenous Injection ◽  
Half Life ◽  
High Performance ◽  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Gel Filtration ◽  
Water Soluble ◽  
Amino Groups ◽  
Covalent Bonds ◽  
High Affinity

High affinity heparin obtained by chromatography of clinical grade heparin on antithrombin III-Sepharose was covalently coupled to antithrombin III using a three step procedure: 1) introduction of free amino groups in the heparin molecule; 2) reaction of these amino groups with the difunctional reagent tolylene-2,4-diisothiocyanate; and 3) reaction of the remaining isothiocyanate group with amino groups of antithrombin III. Amino groups were introduced in the heparin molecule by limited N-desulfation or by reaction of carboxyl groups with hexamethylenediamine with the use of a water soluble carbodiimide. Between 1 and 4 moles of NH2 groups were introduced per 15,000 g heparin and the yield of the coupling procedures was 25 to 30 percent. The complexes could be separated from free active heparin by chromatography on antithrombin III-Sepharose and from unreacted antithrombin III by gel filtration on a high performance liquid chromatography column. Coupling occurred for 80 percent with an apparent 1:1 stoichiometry. The specific anticoagulant activity of the complexes (expressed per mg heparin) was approximately 75 percent of that of modified heparin and approximately 50 percent of the original high affinity heparin. The half-life of these complexes in blood following intravenous injection of about 100 heparin equivalent units in rabbits was 0.68 ± 0.08 hours for the N-desulfated heparin-antithrombin III complex and 0.99 ± 0.27 hours for the hexamethylenediamine substituted heparin-antithrombin III complex which is 2.4 and 3.5 times longer than the half-life of free heparin in the blood. This finding indicates that the main mechanism of disappearance of the anticoagulant activity following intravenous injection of heparin is by removal of free heparin and dissociation of the heparin-antithrombin III complex and not by clearing of the intact complex.

scholarly journals Differential role of fractionated heparin in antithrombin-III proteolysis

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 576-581 ◽  
Author(s):  
E Marciniak
Keyword(s):  
Affinity Chromatography ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Lower Affinity ◽  
Inhibitor Complex ◽  
High Affinity ◽  
Highly Active ◽  
At Iii

Abstract Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

scholarly journals Isolation of a heparin-like anticoagulant from the plasma of a patient with metastatic bladder carcinoma

Blood ◽  
1989 ◽  
Vol 74 (1) ◽  
pp. 252-254 ◽  
Author(s):  
A Tefferi ◽  
BA Owen ◽  
WL Nichols ◽  
TE Witzig ◽  
WG Owen
Keyword(s):  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Transitional Cell ◽  
Chemical Properties ◽  
Thrombin Time ◽  
High Affinity ◽  
Carcinoma Of The Bladder ◽  
D Dimer ◽  
And Chemical Properties

Abstract A 73-year-old woman with metastatic transitional cell carcinoma of the bladder developed vaginal bleeding a few days after undergoing radical cystectomy. She had no other signs of mucocutaneous bleeding. Coagulation studies revealed a markedly prolonged thrombin time (greater than 600 seconds), a slightly prolonged reptilase time (20 seconds), and mildly elevated fibrinogen (4.39 g/L), and fibrin D-dimer (200 to 500 ng/mL) levels. Treatment of the patient's plasma in vitro with protamine or barium sulfate normalized the thrombin time. The anticoagulant activity corresponded to 0.15 heparin U/mL when measured by a thrombin time assay using normal plasma as substrate and standardized with porcine heparin. The anticoagulant was quantitatively bound to and subsequently eluted with 1 mol/L NaCl from quaternary aminoethyl (QAE) Sephadex, and then isolated by affinity chromatography on immobilized antithrombin III. The isolated anticoagulant was shown to be sensitive to heparinase digestion. Therefore, the inhibitor has functional and chemical properties similar to those of high-affinity heparin. Thus far, this is the only anticoagulant of this type isolated from the plasma of a patient bearing a tumor other than plasma cell myeloma.

scholarly journals Differential role of fractionated heparin in antithrombin-III proteolysis

Blood ◽  
1982 ◽  
Vol 59 (3) ◽  
pp. 576-581
Author(s):  
E Marciniak
Keyword(s):  
Affinity Chromatography ◽  
Antithrombin Iii ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Lower Affinity ◽  
Inhibitor Complex ◽  
High Affinity ◽  
Highly Active ◽  
At Iii

Commercial heparin was fractionated by affinity chromatography on immobilized antithrombin-III (AT-III) into nonbinding (NB), lower affinity (LA), and high affinity (HA) heparin, with specific anticoagulant activity of 9, 205, and 284 U/mg, respectively, Each fraction, in microgram quantities, was examined in the reaction of alpha-thrombin with a molar excess of 125I-labeled AT-III. Proteolysis of residual AT-III was assessed on the basis of distribution of radioactivity in SDS-polyacrylamide gels after electrophoresis. In the presence of HA heparin, 36% of AT-III participating in the reaction was degraded into a 50,000-dalton inactive fragment. Similarly designed proteolysis obtained in the presence of LA heparin was 21%, while in the presence of the NB fraction, or in the absence of heparin, only 8% of inhibitor was in the fragment form. When added to human plasma together with purified thrombin, both HA and LA heparin caused functional and electrophoretic changes suggestive of AT-III proteolysis. These observations support the concept that the conformational change, induced by binding of heparin, exposes specific polypeptide bonds susceptible to thrombin, except that nonproductive proteolysis may then occur even more rapidly than the formation of a stable enzyme-inhibitor complex. This, in turn, suggests that the presence of highly active heparin may contribute to reduction of the protective inhibitor in blood, if induction of proteolysis by thrombin is in effect.

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