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.

Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

1982 ◽  
Vol 47 (03) ◽  
pp. 244-248 ◽  
Author(s):  
D P Thomas ◽  
Rosemary E Merton ◽  
T W Barrowcliffe ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Xa ◽  
Blood Levels ◽  
Thrombin Time ◽  
High Affinity ◽  
Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

scholarly journals Studies on Fractionated Heparin

1977 ◽  
Author(s):  
P. A. Lane ◽  
I. Macgregor ◽  
R. Michalski ◽  
V. V. Kakkar
Keyword(s):  
Molecular Weight ◽  
Specific Activity ◽  
Gel Filtration ◽  
Factor Xa ◽  
Assay Method ◽  
Clotting Time ◽  
Reactive Material ◽  
Heparin Fractions ◽  
Specific Activities ◽  
Low Activity

Commercial porcine heparin has been divided into five different molecular weight components following gel filtration upon a polyacrylamide-agarose gel matrix. Rechromatography has shown that the two extreme fractions, which contain very low and very high molecular weight material, were totally separable upon gel filtration, while the intermediate fractions contained material in common with other fractions. Four of the five fractions contained almost equivalent specific activity when measured by an anti-Factor Xa clotting assay (l) and yet had very different specific activities if assayed by either thrombin clotting time or kaolin-cephalin clotting time methods. The highest MW fraction had low specific activity in all of the clotting assays, suggesting that it contained the greatest percentage of a non-reactive material demonstrated by Rosenberg et al, (2) while the lowest MW fraction had low activity only in thrombin clotting time and KCCT assays. The lowest MW fraction produced a smaller acceleration of purified fibrin monomer polymerisation rate and also smaller inhibition of thrombin induced platelet aggregation in plasma. The demonstration of different results for specific activities of the heparin fractions, depending upon the assay method used is in agreement with a recent study (3) and has important implications in regard to the pharmacopeal assays for heparin. It also suggests that heparins with higher specific anti-Factor Xa (or antithrombotic) effect can be prepared from commercial heparins, although these may have a relatively low ability to neutralise thrombin in plasma.

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:

The Measurement of Heparin

1973 ◽  
Vol 30 (03) ◽  
pp. 471-479 ◽  
Author(s):  
K. W. E Denson ◽  
John Bonnar
Keyword(s):  
Degradation Products ◽  
Factor Xa ◽  
Assay Method ◽  
Thrombin Time ◽  
Fibrinogen Degradation Products ◽  
Low Levels

SummaryA method for the measurement of heparin utilising the potentiating effect of heparin on the action of anti-factor Xa is described. The effect on the assay of platelet contamination of plasma, the presence of fibrinogen degradation products and low levels of anti-factor Xa have been studied. The assay method has been compared with the calcium thrombin time method and a group of obstetrical patients have been studied using both methods.

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.

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.

A Comparison of Anticoagulation Activity of a Potent Heparin Preparation in Different Test

1979 ◽  
Author(s):  
A.S. Bhargava ◽  
J. Heinick ◽  
Chr. Schöbel ◽  
P. Günzel
Keyword(s):  
Blood Clotting ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Thrombin Time ◽  
Beagle Dogs ◽  
Clotting Time ◽  
Relative Potencies ◽  
Heparin Preparation

The anticoagulant effect of a new potent heparin preparation was compared with a commercially available heparin in vivo after intravenous application in beagle dogs. The anticoagulant activity was determined using thrombin time, activated partial thromboplastin time and whole blood clotting time after 5, 10 and 30 minutes of application. The relative potency of the new heparin preparation (Scherinq) was found to be 1.62 to 2.52 times higher than heparin used for comparison (150 USP units/mg, Dio-synth). The anticoagulant properties of both preparations were also studied in vitro using dog and human plasma. The relative potencies in vitro correlated well with those obtained in vivo. Further characterization with amidolytic method using chromogenic substrate for factor Xa and thrombin (S-2222 and S-2238 from KABI, Stockholm) showed that heparin (Schering) contains 243 to 378 USP units/raq depending upon the test systems used to assay the anticoagulation activity and in addition, proves the validity of the amidolytic method.

scholarly journals Activity and Concentration of Antithrombin III in Plasma and Serum

1977 ◽  
Author(s):  
L. Róka ◽  
H. Bleyl
Keyword(s):  
Antithrombin Iii ◽  
Specific Activity ◽  
Simultaneous Occurrence ◽  
Single Individual ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Specific Activities ◽  
Antithrombin Iii Activity ◽  
Heparin Affinity

The concentration and activity of antithrombin III contained in plasma and serum of a single individual were compared with each other. The concentration of antithrombin III was determined by means of the rockett technique according to Laurell, antithrombin activity was measured by thrombin neutralization, residual thrombin activity was quantified using the chromogenic substrate Chromozym TH. The patients’ plasmas examined could be divided into different groups according to their antithrombin III specific activity. Most of the samples contained about 34 U/mg, the specific activities of two other groups were about 20 u/mg and 15 U/mg respectively. Only 3 samples contained more than 45 U/mg. In plasma samples with low specific antithrombin III activity the simultaneous occurrence of free antithrombin and an antithrombin-thrombin complex formed in vivo has been demonstrated by means of the two-dimensional Immunoelectrophoresis with heparin added to the agarose-gel medium. (Sas et al., 1975). The antithrombin-thrombin complex could be separated from free antithrombin by adsorption to a heparin-agarose column and fractionated elution, using the different heparin affinity of complex-bound and free antithrombin.

Evaluation of Five Methods for the Determination of Heparin

1975 ◽  
Author(s):  
A. Teien ◽  
M. Lie
Keyword(s):  
Coefficient Of Variation ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Thrombin Time ◽  
Heparin Concentration ◽  
Normal Plasma

Heparin was added in vitro to plasma and the heparin content assayed by five clotting methods. The accuracy was lower in pathological than in normal plasma. At a heparin concentration of 0.5 U/ml plasma the coefficient of variation in eleven pathological plasmas was: Polybrene titration 3 per cent, method of Denson and Bonnar 15 per cent, method of Yin et al. 20 per cent, calcium thrombin time 40 per cent. With the APTT method the results ranged from 0.05 U/ml to above 0.6 U/ml (no clotting in five pathological plasmas).At a heparin concentration of 0.05 U/ml plasma, only the calcium thrombin time and the factor Xa methods were sensitive, but the coefficient of variation ranged from 61 to 78 per cent. The influence of the concentration of fibrinogen, antithrombin III and α1-acid glucoprotein was studied. It is concluded that of the methods tested, only polybrene titration gives accurate results in pathological plasmas. This method is, however, laborious and insensitive to heparin concentrations below 0.1 U/ml.

Biochemical and Functional Study of Antithrombin III in Newborn Infants

1982 ◽  
Vol 47 (01) ◽  
pp. 056-058 ◽  
Author(s):  
M M McDonald ◽  
W E Hathaway ◽  
E B Reeve ◽  
B D Leonard
Keyword(s):  
Protein Concentration ◽  
Antithrombin Iii ◽  
Specific Activity ◽  
Newborn Infants ◽  
Factor Xa ◽  
Functional Study ◽  
Functional Studies ◽  
Sds Page ◽  
At Iii ◽  
Heparin Affinity

SummaryAntithrombin III (AT-III) was isolated by heparin affinity chromatography from adult venous and newborn term and preterm umbilical cord blood. The purified proteins were compared by SDS-PAGE, rocket immuno-electrophoresis, protein concentration by microbiuret relative to optical density at 280 nm, heparin cofactor specific activity, progressive neutralization of thrombin and factor Xa at 37°C and pH related antithrombin kinetics. The structural evaluations revealed a fetal AT-III of molecular weight, charge and electrophoretic migration indistinguishable from adult AT-III. The functional studies showed that, on an equimolar basis, the rates of thrombin and Xa interactions with fetal AT-III were as rapid as those with adult AT-III. The catalytic rates of various concentrations of heparin were also equal. The newborn infant, therefore, displays a quantitative but not qualitative deficiency of AT-III.

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