GAGs-Potentiated Inhibition of Thrombin, Factor Xa and Plasmin in Plasma and in a Purified System Containing Antithrombin III – Correlation with Total Charge Density

1981 ◽  
Vol 46 (04) ◽  
pp. 749-751 ◽  
Author(s):  
E Cofrancesco ◽  
A Vigo ◽  
E M Pogliani
Keyword(s):  
Charge Density ◽  
Chemical Structure ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Total Charge ◽  
Pure System ◽  
At Iii ◽  
Total Charge Density ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryThe ability of heparin and related glycosaminoglycans (GAGs) to accelerate the inhibition of thrombin, factor Xa and plasmin in plasma and in a purified system containing antithrombin III (At III) was studied using chromogenic peptide substrate assaysThere was a good correlation between the charge density of the mucopolysaccharides and the activities investigated. While the difference between potentiation of the antithrombin activity by GAGs in plasma and in the purified system was slight, the inhibition of factor Xa in plasma was more pronounced than in the presence of purified At III, indicating the mechanisms for GAGs-potentiated inhibition of thrombin and factor Xa are not identical.For the antiplasmin activity, there was a good correlation between the chemical structure and biological activity only in the pure system, confirming that the antithrombin-GAG complex plays a very limited role in the inactivation of plasmin in plasma.

Reactivity of a Hereditary Abnormal Antithrombin III Fraction in the Inhibition of Thrombin and Factor Xa

1980 ◽  
Vol 44 (02) ◽  
pp. 092-095 ◽  
Author(s):  
T H Tran ◽  
C Bondeli ◽  
G A Marbet ◽  
F Duckert
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Thrombin Inhibition ◽  
Superficial Thrombophlebitis ◽  
Heparin Concentration ◽  
Heparin Binding Site ◽  
At Iii ◽  
The Difference ◽  
Inhibition Of Thrombin

SummaryTwo different AT-III fractions were purified from the plasma of a patient with recurrent superficial thrombophlebitis. The abnormal AT-III fraction (A-AT) was compared to the normal AT-III fraction (N-AT) in the inhibition of thrombin and factor Xa. Without heparin, both inactivate proteases in a similar manner and at the same rate. However, at low heparin concentration the thrombin inhibition proceeds more slowly with A-AT than with N-AT. At high heparin concentration the difference between A-AT and N-AT becomes very small. The inhibition of factor Xa follows a similar pattern. It is suggested that the heparin binding site of A-AT differs from that of N-AT resulting in a decreased heparin cofactor activity.

HEPARIN COFACTOR II: A SIMPLE ASSAY METHOD

1987 ◽  
Author(s):  
H Vinazzer ◽  
U Pangraz
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Tris Buffer ◽  
Assay Method ◽  
Healthy Individuals ◽  
Heparin Cofactor Ii ◽  
Complete Inactivation ◽  
Thrombin Activity ◽  
At Iii ◽  
The Difference

A photometric assay method for heparin cofactor II (HC II) is described. In a first step antithrombin III (AT III) in plasma is blocked by an anti human AT III immunoglobuline from goats. After dilution of this plasma with Tris buffer pH 8.4 containing 3 IU/ml heparin and addition of thrombin the remaining thrombin activity is measured by use of the chromogenic substrate S-2238 Kabi. The following preliminary experiments were carried out: Variation of the amount of anti-AT III added to plasma resulted in complete inactivation of 1.25 units AT III by 1.0 ml of the inhibitor. Incubation of 1 ml anti AT III with 1 ml purified AT III ( 1 U/ml} or with 1 ml normal plasma completely abolished AT III activity within 60 sec. Incubation of the reaction mixture with thrombin resulted in maximum inactivation after 180 sec. This is in contrast to AT III activated by heparin which immediately inactivates thrombin. Anti-Xa activity after depletion of AT III was assayed in a similar way by addition of factor Xa to the reaction mixture and measuring the remaining Xa activity by the substrate S-2222. In these tests no anti Xa-activity was found after AT III depletion. From these experiments there was assumed that the anti thrombin activity measured under the following conditions was due to the action of HC II:Plasma ( 50 μl) was mixed with anti AT III (50 μl) and was incubated for 60 sec. Tris buffer with heparin pH 8.4 (900 μl) was added. From this mixture 200 μl was pipetted into a cuvette at 37°C followed by 200 μl thrombin ( 2 IU/ml). After an incubation time of 180 sec 200 μl S-2238 ( 2 mmol/1) was added and the difference in OD/min was determined at 405 nm. A calibration curve was made by series of dilutions of normal AT III depleted plasma from 20 healthy individuals. The following preliminary results ofrHC II activity as a percentage were obtained:

scholarly journals Factor IXa Inhibition Contributes to the Heparin Effect

1991 ◽  
Vol 66 (03) ◽  
pp. 306-309 ◽  
Author(s):  
Suzette Béguin ◽  
Frédérique Dol ◽  
H Coenraad Hemker
Keyword(s):  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heparin Cofactor Ii ◽  
Factor Ixa ◽  
Factor Viiia ◽  
At Iii ◽  
Inhibition Of Thrombin ◽  
Thrombin Formation

SummaryWe investigated whether the inactivation of factor IXa contributes to the partial inhibition of thrombin formation that is observed at therapeutic concentrations of heparin. The action of standard unfractionated heparin (0.05 U/ml) on thrombin formation in the intrinsic system was compared to that of a mixture of dermatan sulfate (DS) and a synthetic pentasaccharide (PS). DS enhances the action of heparin cofactor II which inhibits thrombin only. PS specifically enhances the anti-factor Xa activity of antithrombin III (AT III). The concentrations of DS and PS were chosen so as to obtain equal anti-thrombin and anti-factor Xa activities as in 0.05 U/ml heparin. An extra inhibitory effect of heparin over the mixture is observed in situations where free factor IXa, not bound to factor VIIIa and phospholipid, limits the rate of thrombin formation, notably in contact activated plasma. We conclude that the inactivation of free factor IXa by heparin contributes importantly to the inhibition of thrombin formation in the intrinsic system such as e.g. measured in the activated partial thromboplastin time.

Homozygous Variant of Antithrombin III that Lacks Affinity for Heparin, AT III Kumamoto

1989 ◽  
Vol 61 (01) ◽  
pp. 020-024 ◽  
Author(s):  
Kenji Okajima ◽  
Hidetsugu Ueyama ◽  
Youichiro Hashimoto ◽  
Yasuto Sasaki ◽  
Keiko Matsumoto ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Autosomal Dominant ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Cofactor Ii ◽  
At Iii ◽  
Antifactor Xa ◽  
Cofactor Activity ◽  
Inhibition Of Thrombin ◽  
Plasma Heparin

SummaryAbnormal antithrombin III (AT III) was found in the plasma of a 31-year-old female who suffered from recurrent thrombotic episodes. Heparin cofactor activity was 28% of normal and undetectable when measured by inhibition of thrombin and factor Xa (F. Xa), while both progressive antithrombin and antifactor Xa activities were normal. The concentration of plasma AT III antigen was 37 mg/dl. Analysis by crossed-immunoelec- trophoresis (CIE) in the presence of heparin and affinity chromatography on heparin-Sepharose revealed that the propositus’ AT III did not bind to heparin. When heparin cofactor II (HC II) was removed from propositus’ plasma, heparin cofactor activity of AT III was not detected. Thus, HC II seemed to account for the plasma heparin cofactor activity found in the presence of thrombin. The patient’s parents and three of her brothers demonstrated qualitative abnormality of AT III; heparin cofactor activity was 30-50% of normal levels in the presence of both thrombin and F. Xa. These findings indicate that the propositus’ AT III lacks affinity for heparin and the mode of its inheritance seems to be autosomal dominant and, hence, the propositus would be a homozygote. For this variant, the name of AT III Kumamoto is proposed.

An Antithrombin III Assay Based on Factor Xa Inhibition Provides a More Reliable Test to Identify Congenital Antithrombin III Deficiency Than an Assay Based on Thrombin Inhibition

1993 ◽  
Vol 69 (03) ◽  
pp. 231-235 ◽  
Author(s):  
Christine Demers ◽  
Penny Henderson ◽  
Morris A Blajchman ◽  
Michael J Wells ◽  
Lesley Mitchell ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Factor Xa ◽  
Chromogenic Substrate ◽  
Cross Sectional ◽  
Thrombin Inhibition ◽  
Factor Xa Inhibition ◽  
At Iii ◽  
The Difference

SummaryObjectives: To determine whether functional antithrombin III (AT-III) levels measured by a factor Xa inhibition (AT-III-Xa) assay identifies AT-III deficient individuals more reliably than functional AT-III levels measured by a thrombin inhibition (AT-III-IIa) assay.Study design: Cross-sectional study.Patient population: Sixty-seven members of a large family with type 2 AT-III deficiency.Intervention: DNA analysis was used as the reference diagnostic standard for AT-III status and subjects were classified as AT-III deficient or non deficient according to these results. Functional AT-III levels were measured in all subjects using: 1) a chromogenic substrate for thrombin and added human thrombin (AT-III-IIa), and 2) a chromogenic substrate for factor Xa and added bovine factor Xa (AT-III-Xa). Functional heparin cofactor II (HC-II) levels were measured using a commercially available kit. The proportions of 125I-α-thrombin complexed to AT-III and HC-II were measured by polyacrylamide gel electrophoresis and autoradiography.Results: Thirty-one (46%) individuals were classified as AT-III deficient and 36 (54%) as AT-III non deficient. AT-III-Xa assay measured a significantly lower mean AT-III value and a narrower range for individuals classified as AT-III deficient than the AT-III-IIa assay. Using the AT-III-IIa assay, six subjects had borderline AT-III levels compared to none with the AT-III-Xa assay. Thrombin inhibition by HC-II likely accounts for the AT-III-IIa assay giving higher values than the AT-III-Xa assay since 1) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and HC-II levels, 2) the mean level of HC-II was significantly higher for individuals who had a positive difference between AT-III-IIa and AT-III-Xa levels compared to those who had a negative difference and 3) there was a significant correlation between the difference in AT-III-IIa and AT-III-Xa levels and the percentage of 125I-α-thrombin complexed to HC-II.Conclusion: The AT-III-Xa assay is a better discriminant between AT-III deficient and AT-III non deficient individuals than the AT-III-IIa assay.

High precision measurement of Debye-Waller factors for NiAl

1995 ◽  
Vol 53 ◽  
pp. 140-141
Author(s):  
W. Niichter ◽  
A.L. Weickenmeier ◽  
J. Mayer
Keyword(s):  
Charge Density ◽  
Atomic Displacement ◽  
Waller Factor ◽  
Total Charge ◽  
High Precision Measurement ◽  
Atomic Vibrations ◽  
Total Charge Density ◽  
The Difference ◽  
Mott Formula ◽  
Image Position

Motivation Quantitative convergent beam electron diffraction (CBED) is increasingly appreciated as a tool to determine bonding charge densities of crystalline materials. Simulated CBED patterns are fitted to experimental ones to derive the structure factors. These are converted by means of the Mott formula to yield the total charge density. Finally a neutral atom total charge density is subtracted and the difference is interpreted as the bonding charge density. Accounting for the temperature by a Debye-Waller factor (DWF) the g-th Fourier coefficient of the bonding charge density is then given by where u denotes the thermal root mean square atomic displacement. Here we have assumed thesimplest case of identical isotropic atomic vibrations for all atoms in the crystal. In order to estimate the error in due to the uncertainty in u we insert the results obtained by Fox and Tabbernor for NiAl at room temperature. They found differences between atomic and measured values of in the order of 2 percent.

Coagulation Inhibitors in Plasma and Serum

1975 ◽  
Author(s):  
O. R. Ødegård ◽  
U. Abildgaard
Keyword(s):  
Antithrombin Iii ◽  
Close Correlation ◽  
The Other ◽  
At Iii ◽  
Coagulation Inhibitors ◽  
Patient Groups ◽  
Cofactor Activity ◽  
The Difference ◽  
Immuno Assay ◽  
Activity Method

Heparin cofactor activity and antithrombin III (At-III) activity measured with amidolytic methods; antifactor Xa by a clotting method (Biggs et al., Brit. J. Haemat. 19, 287, 1970) and immunoassay of At-III (Fagerhol & Abildgaard, Scand. J. Haemat. 7, 10, 1970) in plasma and serum showed:1) There was a close correlation between the plasma values as measured by all these methods (r = 0.84–0.93).2) The difference between plasma and serum values (“consumption”) was lower in warfarin treated and in haemophiliacs than in the other groups.3) The difference between plasma and serum was greater when measured by the heparin cofactor activity method than by the other methods. The reason for this discrepancy will be discussed. The results in different patient groups will be reported.4) As the heparin cofactor activity assay can be completed within 10 minutes after blood sampling, and has a higher precision than clotting assay and immuno assay, it is preferable for clinical use.

Antithrombin III and Venous Thrombosis

1979 ◽  
Author(s):  
Duncan P. Thomas
Keyword(s):  
Venous Thrombosis ◽  
Antithrombin Iii ◽  
Physiological Role ◽  
Factor Xa ◽  
Original Description ◽  
Clear Cut ◽  
Naturally Occurring ◽  
At Iii ◽  
General Studies

Increasing interest in the physiological role of inhibitors of coagulation has highlighting the role of antithrombin III (AT III) as the most important naturally occurring inhibitor of venous thrombosis. Since Egeberg’s original description in 1965, it has been recognized that inherited deficiency of AT III is associated with an increased incidence of venous thromboembolism. The role of acquired deficiency of AT III in the pathogenesis of thromboembolism remainsless clear-cut, partly due to methodological differences. While low values have been reported in groups of patients with thromboembolism, estimations of AT III in individual patients are not allways abnormal. In general, studies which have measured protein concentration rather than functional activity, or cl otting assays which measure total antithrombin activity and not specific anti-Factor Xa activity have failed to demonstrate a clear relationship between AT III and thromboembolism. However, in two groups of patients, namely women on oral contraceptives and patients undergoing total hipreplacement, an acquired deficiency of AT III, particularly when measured by anti-Xa clotting assays, correlates highly with postoperative venous thrombosis. Although venous thrombosis may develop in patients despite normal AT III values, an activity below approximately 80% in an anti-Xa clotting assay has been found to be of predictive value in patients subjected to the stress of trauma or surgery.

Studies On The Dynamics Of Antithrombin III During Heparin Infusion By Radioimmunoassay

1981 ◽  
Author(s):  
S Urano ◽  
M Nakagawa ◽  
T Kitani ◽  
Y Maeda ◽  
M Watada ◽  
...  
Keyword(s):  
Plasma Level ◽  
Antithrombin Iii ◽  
Treated Group ◽  
Control Group ◽  
Heparin Infusion ◽  
Significant Difference ◽  
At Iii ◽  
Wet Weight ◽  
The Difference ◽  
Radioimmunoassay Method

A radioimmunoassay method for antithrombin III (ATIII) was developed in order to detect the AT III levels correctly in plasma and tissues and the effect of heparin infusion was investigated on rat using this method and 125I labeled ATIII. Rat AT III was purified from rat defibrinated plasma by heparin sepharose affinity chromatography and gel filtrations. This purified AT III was used for the preparation of specific AT III antiserum. Labeling of AT III with 125I was performed according to the method by Hunter and Greenwood. Plasma level of AT III were significantly decreased in the treated group with heparin for 6 hours, although significant difference was not observed in AT III contents in various organs. The behavior of i.v. injected AT III laveled with 125I in the normal control and treated groups proved the difference on the half life of AT III. Control group gave 52 hours and it was shortened in the treated group. The percent radioactivity per ml plasma after 6 hours of heparin infusion was 1.16±0.51, and 2.01±0.38 in the control group, and significant difference was observed (p < 0.05). On the contrary the percent dose radioactivity per g tissue wet weight was significantly increased in the liver, lungs, and large intestine on the heparin treated group. The decreased amount of the intravenously injected laveled AT III appears to be trapped and metabolized in the various organs mainly in the liver during heparin infusion. The decrease of plasma AT III levels on the patients treated with heparin may be explained from these experimental results.

scholarly journals The Activity of Heparin Preparations Studied by Amidolytic and Clotting Methods

1977 ◽  
Author(s):  
A.N. Teien ◽  
U. Abildgaard ◽  
M. Höök ◽  
U. Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
Factor Xa ◽  
Test System ◽  
Assay Method ◽  
Thrombin Time ◽  
Assay Methods ◽  
Specific Activities ◽  
The Difference ◽  
Heparin Preparation

Two heparin standards, heparin isolated from human mastocytoma tissue, four commercial heparins and two heparin preparations separated by affinity chromatography (“High affinity heparin”=HAH and “Low affinity heparin”=LAH) were assayed by the activated partial thromboplastin time method (APTT), the calcium thrombin time method (CaTT) and two amidolytic methods (measuring the accelerating effect of heparin on the inactivation of thrombin or factor Xa by antithrombin III), with and without plasma in the test system. The specific activities of the various heparins were expressed relative to that of the 3rd. Int. Standard (=100). Found specific activities ranged 3 - 198 (LAH and HAH, respectively). In all assay systems HAH had the highest specific activity, followed by one of the commercial preparations and the 3rd Int. Standard. LAH and human heparin had very low specific activities, except in the APTT test system, an assay method which in addition mirrors other anticoagulant effects of heparin than the acceleration of antithrombin III. Apart from the higher effect of LAH and human heparin on the APTT, the difference in specific activities found for each individual heparin preparation with these various assay methods was slight.In view of the reproducibility and simplicity of the amidolytic methods, it is suggested that they be adapted for heparin standardization.

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