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.

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 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 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.

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.

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 Effect of a heparan sulphate with high affinity for antithrombin III upon inactivation of thrombin and coagulation factor Xa

1989 ◽  
Vol 262 (2) ◽  
pp. 651-658 ◽  
Author(s):  
M F Scully ◽  
V Ellis ◽  
N Shah ◽  
V Kakkar
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Heparan Sulphate ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Order Rate Constant ◽  
Pseudo First Order Reaction ◽  
High Affinity ◽  
Kinetics Of ◽  
Kinetics Of Inhibition

The kinetics of inhibition of human alpha-thrombin and coagulation Factor Xa by antithrombin III were examined under pseudo-first-order reaction conditions as a function of the concentration of heparan sulphate with high affinity for antithrombin III. The maximum observed second-order rate constant was, for the antithrombin III-thrombin reaction, 1.2 x 10(9) M-1.min-1 compared with 2.4 x 10(9) M-1.min-1 in the presence of high-affinity heparin. However, the maximum rate was catalysed by much higher concentrations of heparan sulphate (1.3 microM) than of heparin (0.025 microM). Differences were also observed in the maximal acceleration of the antithrombin III-Factor Xa interaction: 1.2 x 10(9) M-1.min-1 at 0.2 microM-heparin sulphate compared with 2.2 x 10(9) M-1.min-1 at 0.04 microM-heparin. The differences in properties of heparan sulphate and heparin were analysed by using the random bi-reactant model of heparin action [Griffith (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5460-5464]. It was observed that the apparent binding affinity for thrombin was higher for heparan sulphate (180 nM) than for heparin (14 nM). The rate constant for transformation of the antithrombin III-Factor Xa complex into irreversible product differed between heparan sulphate (96 min-1) and heparin (429 min-1). These properties of the high-affinity heparan sulphate may be of importance in consideration of a putative role in the control of intravascular haemostasis.

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.

High Affinity Binding of Heparin by Necrotic Tumour Cells Neutralises Anticoagulant Activity

2001 ◽  
Vol 86 (08) ◽  
pp. 616-622 ◽  
Author(s):  
Sumihito Morita ◽  
Milena Gebska ◽  
Ajay Kakkar ◽  
Michael Scully
Keyword(s):  
Ribosomal Proteins ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Live Cells ◽  
Affinity Binding ◽  
Heparin Binding ◽  
Pancreatic Carcinoma Cell Line ◽  
High Affinity Binding ◽  
High Affinity ◽  
Heparin Activity

SummaryWe have observed a striking neutralisation of the anticoagulant activity of unfractionated heparin in the presence of a pancreatic carcinoma cell line (MIA PaCa-2) due to binding of around 9 g of heparin per 107 cells (apparent Kd, 30 nM). The loss of anticoagulant activity was less marked in the presence of low molecular weight forms of heparin. Binding to the cell blocked acceleration of the thrombin:anti-thrombin interaction by heparin. Neutralisation of heparin activity was also shown to occur in the presence of a number of other tumour cell lines. FACS analysis demonstrated that live cells did not bind heparin and high affinity binding only occurred to dead MIA PaCa-2 cells. Heparin binding proteins accumulating in cell medium were identified as histone and ribosomal proteins that will become exposed during necrosis. The release of these proteins from cells within the necrotic core of a tumour or from cells killed during chemotherapy may abrogate the heparan sulphate/antithrombin system and possibly contribute to the idiopathic thromboembolism often associated with cancer (Trousseau’s syndrome). The findings also suggest a reason for the reported advantage of LMWH over UFH in treating venous thromboembolism in cancer patients and in improving patient survival.

Potentiation of Antithrombin III Activity by Sulphated Polysaccharides

1979 ◽  
Author(s):  
G. Kindness ◽  
W.F. Long ◽  
F.B. Williamson
Keyword(s):  
Antithrombin Iii ◽  
Ex Vivo ◽  
Anticoagulant Activity ◽  
Heparan Sulphate ◽  
Relative Effectiveness ◽  
Coagulation Factor ◽  
Factor X ◽  
Sulphated Polysaccharides ◽  
Molecular Rigidity ◽  
Cellulose Sulphate

Because of current interest in the mechanisms of antithrombin III-mediated anticoagulation by heparin, we examined the anticoagulant effectiveness of a number of other sulphated polysaccharides of relatively well-defined chemical structure using ex vivo assay systems. Our results suggest that heparan sulphate, cellulose sulphate, dextran sulphate, xylan sulphate, and a number of sulphated galactans derived from marine algae are able, like heparin, to potentiate antithrombin III inhibition of thrombin and activated coagulation factor X. Dermatan sulphate exerts anticoagulant activity which appears to be independent of antithrombin III. The relative effectiveness of these polymers as activators of antithrombin III may be related to their molecular rigidity.

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