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.

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.

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

On the Interaction Between Human Plasma Kallikrein and C1-Esterase Inhibitor

1983 ◽  
Vol 49 (03) ◽  
pp. 193-195 ◽  
Author(s):  
Torbjörn Nilsson
Keyword(s):  
Dissociation Constant ◽  
Human Plasma ◽  
Rate Constant ◽  
Enzyme Inhibitor ◽  
Order Rate Constant ◽  
Second Order ◽  
Plasma Kallikrein ◽  
First Order ◽  
Order Rate ◽  
Esterase Inhibitor

SummaryThe kinetics of the reaction between human plasma kallikrein and CĪ-esterase inhibitor was studied in a purified system. By monitoring the inhibition reaction for extended periods of time, it was found to proceed in two consecutive steps, a fast reversible second-order binding step followed by a slower, irreversible first-order transition. The rate constants in this reaction model were determined, as well as the dissociation constant of the initial, reversible enzyme-inhibitor complex. Thus, at 37° C the second-order rate constant was found to be 5 · 104 M -1 · s-1, the first order rate constant was 5 · 10-4 s-1 and the dissociation constant K was 1.5 · 10-8 M. Heparin (28 U/ml) and 6-aminohexanoic acid (10 mM) had no effect on the k1 of the of the reaction.

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.

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

scholarly journals The acceleration of the inhibition of platelet prothrombinase complex by heparin

1986 ◽  
Vol 233 (1) ◽  
pp. 161-165 ◽  
Author(s):  
V Ellis ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Unfractionated Heparin ◽  
Rate Constant ◽  
Nitrous Acid ◽  
Maximum Rate ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Binding ◽  
Prothrombinase Complex ◽  
High Affinity ◽  
Heparin Fractions

The influence of heparin on the inhibition of factor Xa has been studied under conditions where factor Xa is bound to collagen-thrombin-stimulated platelets to form the prothrombinase complex. Unfractionated heparin was found to cause a concentration-dependent acceleration of the inhibition of the platelet prothrombinase complex up to a maximum rate constant of 4.1 × 10(7) M−1 × min−1 at heparin concentrations of 0.2 microM and above. This is equivalent to a 4800-fold acceleration over the rate constant for the inhibition in the absence of heparin, and is 6.8-fold lower than the rate constant for the inhibition of uncomplexed factor Xa in the presence of saturating concentrations of heparin which was determined as 2.8 × 10(8) M−1 × min−1. The effects of three Mr fractions of heparin were also studied. These were a gel-filtered heparin of Mr 15000, a gel-filtered heparin of Mr 6000 and a heparin oligosaccharide (primarily 8-10 monosaccharide units) prepared by nitrous acid depolymerization, each with high affinity for antithrombin III. These fractions all accelerated the rate of the antithrombin III inhibition of the platelet prothrombinase complex, with maximum rate constants of 6.8 × 10(7), 1.4 × 10(7) and 9.8 × 10(6) M−1 × min−1, respectively. On comparison with the effect of these heparin fractions on the rate of inhibition of uncomplexed factor Xa a progressively increasing disparity between the rate of inhibition of uncomplexed and complexed factor Xa was observed, rising from 1.7-fold with the oligosaccharide to 6.8-fold with the unfractionated heparin. A possible mechanism for this differential activity between uncomplexed and complexed factor Xa with the various heparin fractions is discussed in terms of an involvement of heparin binding to factor Xa.

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.

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.

Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

1983 ◽  
Vol 49 (02) ◽  
pp. 109-115 ◽  
Author(s):  
M Hoylaerts ◽  
E Holmer ◽  
M de Mol ◽  
D Collen
Keyword(s):  
Molecular Weight ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Life Time ◽  
Low Molecular Weight ◽  
High Affinity ◽  
Plasma Half Life ◽  
Covalent Complex

SummaryTwo high affinity heparin fragments (A/r 4,300 and M, 3,200) were covalently coupled to antithrombin III (J. Biol. Chem. 1982; 257: 3401-3408) with an apparent 1:1 stoichiometry and a 30-35% yield.The purified covalent complexes inhibited factor Xa with second order rate constants very similar to those obtained for antithrombin III saturated with these heparin fragments and to that obtained for the covalent complex between antithrombin III and native high affinity heparin.The disappearance rates from plasma in rabbits of both low molecular weight heparin fragments and their complexes could adequately be represented by two-compartment mammillary models. The plasma half-life (t'/j) of both low Afr-heparin fragments was approximately 2.4 hr. Covalent coupling of the fragments to antithrombin III increased this half-life about 3.5 fold (t1/2 ≃ 7.7 hr), approaching that of free antithrombin III (t1/2 ≃ 11 ± 0.4 hr) and resulting in a 30fold longer life time of factor Xa inhibitory activity in plasma as compared to that of free intact heparin (t1/2 ≃ 0.25 ± 0.04 hr).

Development of Sustained Release Multi-Component Microparticulate System of Diclofenac Sodium and Tizanidine Hydrochloride

1970 ◽  
Vol 1 (1) ◽  
pp. 97-105
Author(s):  
Kamlesh Dashora ◽  
Shailendra Saraf ◽  
Swarnalata Saraf
Keyword(s):  
Particle Size ◽  
Sustained Release ◽  
Cellulose Acetate ◽  
Rate Constant ◽  
Diclofenac Sodium ◽  
Order Rate Constant ◽  
Anomalous Transport ◽  
First Order ◽  
Sem Study ◽  
Transport Type

Sustained released tablets of diclofenac sodium (DIC) and tizanidine hydrochloride (TIZ) were prepared by using different proportions of cellulose acetate (CA) as the retardant material. Nine formulations of tablets having different proportion of microparticles developed by varied proportions of polymer: drug ratio ‘’i.e.’’; 1:9 -1:3 for DIC and 1:1 – 3:1 for TIZ. Each tablet contained equivalent to 100 mg of DIC and 6mg of TIZ. The prepared microparticles were white, free flowing and spherical in shape (SEM study), with  the particle size varying from 78.8±1.94 to 103.33±1.28 µm and 175.92± 9.82 to 194.94±14.28µm for DIC  and TIZ, respectively.  The first order rate constant K1 of formulations were found to be in the range of  K1 = 0.117-0.272 and 0.083- 0.189 %hr-1for DIC and TIZ, respectively. The value of exponent coefficient (n) was found to be in the range of 0.6328-0.9412  and 0.8589-1.1954 for DIC and TIZ respectively indicates anomalous  to  non anomalous transport type of diffusions among different formulations

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