A DEFINITION OF HEPARIN ANTICOAGULANT POTENCY APPLICABLE TO ALL HEPARINS AND HEPARIN-LIKE SUBSTANCES AND ITS PRACTICAL APPLICATION IN ASSAYING HEPARIN

1987 ◽  
Author(s):  
Craig M Jackson
Keyword(s):  
Rate Constant ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Order Rate Constant ◽  
Red Cross ◽  
Order Kinetic ◽  
High Affinity ◽  
General Terms ◽  
The Individual ◽  
Heparin Preparation

Heparins increase the rate of inactivation of proteinases by antithrombin without being consumed in the inactivation reaction. The anticoagulant activity of any heparin or heparin preparation is thus determined by the increase in the inactivaton rate which it produces. This rate increase is dependent on the concentration of the heparin in the sample and on some now well known structural properties of the individual heparin molecules that produce high affinity for antithrombin . All proteinases are not inactivated by antithrombin equally rapidly in the absence of heparin, nor are heparins and heparin derivatives of different molecular weight equally effective in the inactivation of the same proteinase. Under appropriate conditions, the observed rate constant (kObs) for the heparin catalyzed proteinase inactivation reaction is simply related to the intrinsic potencies and concentrations of the individual high affinity heparin molecules in the sample. The intrinsic potency of a high affinity heparin molecule is the efficiency with which it catalyzes the inactivation of the particular proteinase, e.g. Factor Xa or thrombin, i.e., it is a second order rate constant, (designated k*) . After k* has been determined from kobs for a known heparin or heparin preparation and a particular proteinase, the concentration of heparin in an unknown sample can be calculated from the equation[H] = [HAT] = kobs/k* In general terms, the appropriate conditions, i.e.,the antithrombin and proteinase concentrations, the pH, and ionic strength, required for this equation to be used are those conditions for which all of the high affinity heparin is bound to the antithrombin and pseudo first order kinetic behavior occurs. At very low heparin concentrations, a correction for the inactivation of the proteinase by antithrombin alone is necessary, but is easily made.Supported by Organon Teknika Corporation and an Established Investigator Award from the American National Red Cross

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 Effect of a pentosan polysulphate upon thrombin and factor Xa inactivation by antithrombin III.

1984 ◽  
Vol 222 (3) ◽  
pp. 571-578 ◽  
Author(s):  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Charge Density ◽  
Dissociation Constant ◽  
Rate Constant ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Order Rate Constant ◽  
First Order ◽  
High Affinity ◽  
High Charge Density ◽  
Pentosan Polysulphate

The kinetics of inhibition of human and bovine alpha-thrombin and human factor Xa by antithrombin III were examined under pseudo-first-order conditions as a function of the concentration of pentosan polysulphate [a fully sulphated (beta 1-4)-linked D-xylopyranose with a single laterally positioned 4-O-methyl-alpha-D-glucuronic acid]. Double-reciprocal plots of the observed first-order rate constant against concentration of pentosan polysulphate gave straight lines, intercepts on the axes giving values for maximum increase in second-order rate constant (by calculation) and apparent dissociation constant. These values were: for human alpha-thrombin 1.52×10(7) M-1 . min-1 and 3.6 microM respectively, for bovine alpha-thrombin 6.56×10(6) M-1 . min-1 and 0.16 microM and for factor Xa 6.86×106 M-1 . min-1 and 20 microM. In the presence of pentosan polysulphate the dissociation constant for the initial complex of antithrombin III and thrombin was shown to be reduced from approx. 2×10(-3) M to 61×10(-6) M without apparent change in the limiting rate constant of 750 min-1. An oligosaccharide (primarily 8-10 saccharide units) prepared from heparin and with high affinity for antithrombin III but low potency in the thrombin-antithrombin III interaction did not diminish the rate of interaction catalysed by pentosan polysulphate. The catalysis was shown to be due to a weak electrostatic interaction, since it was completely reversed by concentrations of NaCl greater than 0.3 M. It is concluded that the mechanism is independent of the heparin high-affinity binding site on antithrombin III and is probably due to binding of the high-charge-density polysaccharide to the proteinase. It is calculated that the acceleration in rate achieved, although lower than that of heparin, approaches that required to be of physiological significance and may be of importance in the anticoagulation role of antithrombin III at sites of high charge density which may occur in vivo.

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.

Influence Of Variations In The Chemical Structure Of Heparin On Its Anticoagulant And Anti Xa Activities

1981 ◽  
Author(s):  
A S Perlin ◽  
L Ayotte ◽  
J C Lormeau
Keyword(s):  
Molecular Weight ◽  
Chemical Composition ◽  
Chemical Structure ◽  
Reaction Rate ◽  
Glucuronic Acid ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Iduronic Acid ◽  
Catalytic Effects ◽  
Heparin Preparation

Fractions of hog mucosal heparin, prepared by graded sedimentation as barium salts, are distinguished by large differences in chemical composition and relatively small differences in molecular weight. These fractions were found to vary widely in anticoagulant activity and in their ability to potentiate the inhibition of Factor Xa. Anticoagulant activity was highest for fractions that consisted mainly of L-iduronic acid 2-sulfate and 2-deoxy-2-sulfamino-D-glucose 6-sulfate, and decreased overall by a factor of 3 (from 145 to 56 USP units) as the proportion of residues of 2- acetamido-2-deoxy-D-glucose, D-glucuronic acid and L- iduronic acid became progressively more prominent. Conversely, a 10-fold increment in anti Xa activity (from 59 to 639 units/mg) accompanied the increase in the relative proportions of the minor, non-sulfated, heparin constituents. These findings suggest that individual steps of the coagulation process are subject to selective catalytic effects on reaction rate by differently constituted molecules within a heparin preparation

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 On the molecular-weight-dependence of the anticoagulant activity of heparin

1979 ◽  
Vol 181 (1) ◽  
pp. 241-243 ◽  
Author(s):  
L Thunberg ◽  
U Lindahl ◽  
A Tengblad ◽  
T C Laurent ◽  
C M Jackson
Keyword(s):  
Molecular Weight ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Coagulation Factor ◽  
Molecular Weights ◽  
High Affinity ◽  
Polysaccharide Chain ◽  
Molecular Weight Dependence ◽  
Heparin Fractions

The inactivation of thrombin and factor Xa by antithrombin was determined in the presence of heparin fractions of different molecular weights and with high affinity for antithrombin. The ability to potentiate the inactivation of either coagulation factor increased with increasing length of the polysaccharide chain.

EFFECT OF ANTITHROMBIN III AND HEPARIN ON FACTOR X ACTIVATION BY FACTOR IXa

1987 ◽  
Author(s):  
J Pieters ◽  
G willems ◽  
H C Hemker ◽  
T Lindout
Keyword(s):  
Rate Constant ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Order Rate Constant ◽  
Free Solution ◽  
Factor X ◽  
Order Rate ◽  
Factor Ixa ◽  
At Iii ◽  
Factor X Activation

The heparin-catalyzed inactivation of activated coagulation factors by antithrombin III (AT III) has mostly been studied for isolated serine proteases. However, we decided to study the action of heparin and AT III under more physiological conditions, i.e. during the activation of factor X by factor IXa in the presence of phospholipid and calcium. Thereby we made use of a mathematical model which describes the generation of factor Xa by factor IXa, phospholipid and calcium in the presence of AT III and heparin. Fitting the experimental factor Xa generation curve to a set of equations gave the pseudo-first-order rate constants of factor Xa and factor IXa. In a first approach we examined the effect of AT III alone on factor X activation. We found that the second order rate constant of inhibition of formed factor Xa was 2 x 10 5M-1min-1 , whereas that of factor Xa in free solution was 5 x 10 5M-1min-1 , indicating that phospholipid-bound factor X competes with AT III for factor Xa. The second order rate constant of inhibiton of factor IXa, either in the presence or absence of accessory components, was 8 x 103 M-1min-1. Unfractionated heparin (UFH; 168 USP units/mg) was found to stimulate the inhibition of generated factor Xa by AT III (200 nM) with 0.1 min-1 per nM of UFH, and a synthetic pentasaccharide (PS; 4000 anti-Xa units/mg) stimulated this inhibition with only 0.03 min-1per nM. Due to the presence of phospholipid-bound factor X this stimulation was 4-fold less when compared with factor Xa in free solution. At UFH concentrations higher than 3 nM, and PS concentrations exceeding 10 nM hardly any active factor Xa generation could be measured because of the rapid inactivation of factor Xa whereas factor IXa was not inhibited. Using a factor IXa assay we found that PS, even at relatively high concentrations, had no effect on factor IXa inactivation by AT III (200 nM), both in the presence and absence of accessory components. The inactivation of factor IXa by AT III (200 nM) during factor X activation was stimulated by UFH with 1.6 x 10 -2min-1 per nM of UFH. Surprisingly, this was 4-fold more when compared with factor IXa in the absence of accessory components. We established that calcium stimulates the heparin-dependent inhibition of factor IXa.

Binding of Dextran Sulphate to Antithrombin

1979 ◽  
Author(s):  
B. Nordenman ◽  
I. Björk
Keyword(s):  
Anticoagulant Activity ◽  
Factor Xa ◽  
Molecular Size ◽  
Spectroscopic Properties ◽  
Dextran Sulphate ◽  
High Affinity ◽  
Fluorescence Measurements ◽  
Heparin Fractions ◽  
Difference Absorption

The interaction between dextran sulphate (DS) a synthetic sulphated polysaccharide with in vitro anticoagulant activity, and antithrombin (AT) has been studied. About 90% of a commercial, molecular-size-fractionated preparation of DS bound to an AT-Sepharose column. This material was eluted by a salt gradient at a peak ionic strength of about 0.5 and was used for further studies. Difference absorption and fluorescence measurements showed that in solution an average of about 3 molecules of AT bound to each molecule of DS of the size used (~18000 daltons) with an average binding constant of 3 x 106 M-1. The changes of the spectroscopic properties of AT induced by DS were intermediate to those caused by the low-affinity and high-affinity heparin fractions. This was parallelled by the ability of DS to accelerate-the AT-thrombin reaction to a greater extent than low-affinity heparin, but not as pronounced as high-affinity heparin. DS had relatively little effect on the reaction between AT and Factor Xa, while the influence of high-affinity heparin on this reaction was similar to that on the AT-thrombin reaction. These results suggest that DS may induce a conformational change of AT which is less extensive than that caused by high-affinity heparin. This change may be sufficient to accelerate the reaction between AT and thrombin but inadequate to correspondingly affect the reaction between the inhibitor and Factor Xa.

ROLE OF THE HIGH-AFFINITY PENTASACCHARIDE IN HEPARIN ACCELERATION OF ANTITHROMBIN III INHIBITION OF THROMBIN AND FACTOR Xa

1987 ◽  
Author(s):  
Steven T Olson ◽  
Ingemar Bjork ◽  
Paul A Craig ◽  
Joseph D Shore ◽  
Jean Choay
Keyword(s):  
Ionic Strength ◽  
Conformational Change ◽  
Rate Constant ◽  
Rate Constants ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Order Conditions ◽  
Bimolecular Rate Constant ◽  
High Affinity ◽  
Inhibition Of Thrombin

The high-affinity heparin pentasaccharide (H5) and an 8000 Mr high-affinity heparin (H26) have been compared with respect to their interaction with antithrombin III (AT) and their accelerating effect on AT inhibition of thrombin (T) and factor Xa by rapid kinetic and equilibrium binding studies at pH 7.4, 25°C. Kds of .068 μM at I 0.15 and 0.57 μM at I 0.3 were determined for tne AT-H5 interaction, which were 5 and 2.5-fold weaker, respectively, than affinities determined for H26. Comparison of the kinetics of binding of H5 and H26 to AT at I 0.15 under pseudofirst order conditions ([H]o>> [AT]o) demonstrated a saturable dependence of the observed rate constant for both reaction with indistinguishable limiting rate constants of 700 +/-120 s-1 and 520 +/-90 s-1 , but somewhat different Kds for the initial binding interaction of 20 and 29 μM for H5 and H26, respectively. These results indicate that H5 induces the same conformational change in AT as the larger heparin, but that the rate of reversal of this conformational change is greater for H5 which is the basis for its weaker AT affinity. Bimolecular rate constants for neutralization of factor Xa and thrombin by AT-H5 and AT-H26 complexes were determined by p-aminobenzamidine displacement under pseudo-first order conditions([AT-H] >> [T]o or [Xa]o). I-in-dependent values of .62 μM-1 s-1 were obtained for Xa inhibition by AT-H5 at I 0.15 and 0.3, compared to I-dependent values of 1.4 and 0.91 μM-1 s-1 for AT-H26. For thrombin inhibition by AT-H5, and I-independent enhancement of 1.6-fold in the bimolecular rate constant from .0098 to .016 μM-1 s-1 was observed, in sharp contrast to the marked I-independent enhancement by AT-H26 of the bimolecular rate constant ranging from 4000 to 200-fold at I 0.15 and 0.3, respectively. These results are consistent with a primary ionic strength-independent contribution of the AT conformational change to heparin enhancement of factor Xa but not thrombin neutralization by AT, with an ionic strength-dependent component for both reactions, compatible with a differential role for a protease-heparin interaction. Supported by grant HL-30237

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