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.

scholarly journals The relative molecular mass dependence of the anti-factor Xa properties of heparin

1986 ◽  
Vol 238 (2) ◽  
pp. 329-333 ◽  
Author(s):  
V Ellis ◽  
M F Scully ◽  
V V Kakkar
Keyword(s):  
Antithrombin Iii ◽  
Factor Xa ◽  
Relative Molecular Mass ◽  
Heparin Binding ◽  
High Affinity ◽  
Maximum Inhibition ◽  
Human Proteins ◽  
Heparin Fractions ◽  
Kinetics Of ◽  
Inhibition Rate Constant

The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 × 10(7) M-1 × min-1, compared with 6.3 × 10(4) M-1 × min-1 in the absence of heparin, and this rose progressively to 4.2 × 10(8) M-1 × min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III.

Effects of heparin fractions of different affinities to antithrombin III and thrombin on the inactivation of thrombin and factor Xa by antithrombin III

1984 ◽  
Vol 62 (10) ◽  
pp. 975-983 ◽  
Author(s):  
Andrew L. Cerskus ◽  
Kathy J. Birchall ◽  
Frederick A. Ofosu ◽  
Jack Hirsh ◽  
Morris A. Blajchman
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Similar Rate ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Relative Contribution ◽  
Heparin Fractions

To investigate the relative contribution of heparin-binding thrombin and antithrombin III to the enhancement of the rate of inactivation of thrombin by antithrombin III, standard heparin was fractionated on matrix-linked thrombin and (or) antithrombin III. There was a good correlation between heparin affinity for antithrombin III and its ability to enhance the inactivation of thrombin and factor Xa. In addition, there was a good correlation between affinity of heparin for thrombin and its catalytic activity on the inactivation of thrombin by antithrombin III. Thus fractions with high affinity to thrombin had similar rate-enhancing activity for thrombin inactivation to that of fractions with high affinity to antithrombin III. Fractions with high affinity to both proteins were more potent than fractions with high affinity to either protein alone. No significant differences in mean molecular weight were observed among the various heparin fractions. A heparin fraction with very low affinity to thrombin and high affinity to antithrombin III was prepared by repeated fractionation of a low molecular weight heparin on the two affinity columns. This fraction had very weak rate-enhancing activity for the inactivation of thrombin by antithrombin III, but retained substantial activity for the inactivation of factor Xa. The results of these studies support the concept that, for both standard and low molecular weight heparin, the enhancement of the inactivation of thrombin by antithrombin III requires the interaction of the heparin with both thrombin and antithrombin III.

scholarly journals The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides

1987 ◽  
Vol 243 (1) ◽  
pp. 31-37 ◽  
Author(s):  
T W Barrowcliffe ◽  
S J Havercroft ◽  
G Kemball-Cook ◽  
U Lindahl
Keyword(s):  
Protective Effect ◽  
Unfractionated Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Free Enzyme ◽  
Factor V ◽  
High Affinity ◽  
Heparin Fractions ◽  
Heparin Oligosaccharides ◽  
Bovine Factor

The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results.

The Heparin Binding Site Of Antithrombin: Identification Of A Critical Tryptophan Residue Within The Amino Acid Sequence

1981 ◽  
Author(s):  
M Blackburn
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Tryptophan Fluorescence ◽  
Tryptophan Residue ◽  
Affinity Column ◽  
Heparin Binding ◽  
High Affinity ◽  
Heparin Binding Site

Chemical modification of antithrombin III with the tryptophan reagent, dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide, results in the incorporation of one hydroxynitrobenzyl (HNB) moiety per molecule of antithrombin III. Heparin protects against tryptophan modification, particularly at low reagent concentrations. Unlike native antithrombin, which has high affinity for heparin, HNB-anti- thrombin does not bind to a heparin-agarose affinity column. Furthermore, the heparin-induced increase in tryptophan fluorescence, obtained with native antithrombin, is not observed with the singly modified inhibitor. HNB-anti- thrombin does not exhibit heparin-promoted rate enhancement in the inactivation of thrombin and Factor Xa. However, in the absence of heparin, HNB-antithrombin and native antithrombin possess progressive antithrombin activity, inactivating these proteases at identical rates. These results indicate that the integrity of a specific tryptophan residue is required for the binding of heparin to antithrombin III. Chemical and enzymatic cleavage techniques have been used to isolate peptides containing this tryptophan from both HNB-labeled and native antithrombin and to identify this critical tryptophan residue within the amino acid sequence of the antithrombin molecule.

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 The heparin-catalysed inhibition of human factor XIa by antithrombin III is dependent on the heparin type

1988 ◽  
Vol 256 (3) ◽  
pp. 815-820 ◽  
Author(s):  
H Soons ◽  
G Tans ◽  
H C Hemker
Keyword(s):  
Unfractionated Heparin ◽  
Binding Constant ◽  
Kinetic Data ◽  
Maximum Rate ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Pseudo First Order Reaction ◽  
Rate Enhancement ◽  
Factor Xia ◽  
Heparin Fractions

The effect of various well-characterized heparin preparations on the inactivation of human Factor XIa by human antithrombin III was studied. The heparin preparations used were unfractionated heparin and four heparin fractions obtained after anion-exchange chromatography. Inactivation of Factor XIa was monitored with S2366 as chromogenic substrate and followed pseudo-first-order reaction kinetics under all reaction conditions tested. Enhancement of the rate of inhibition of Factor XIa in the presence of unfractionated heparin correlated to the binding of antithrombin III to heparin. From the kinetic data a binding constant of 0.1 microM was inferred. The maximum rate enhancement, achieved at saturating heparin concentrations, was 30-fold. The rate enhancement achieved in the presence of each of the heparin fractions could also be correlated to the binding of antithrombin III to the heparin. The binding constant inferred from the kinetic data varied from 0.10 to 0.28 microM and the number of binding sites for antithrombin III varied from 0.06 to 0.74 site per heparin molecule. The maximum rate enhancements, achieved at saturating heparin concentrations, were strongly dependent on the type of heparin used and varied from 7-fold for fraction A to 41-fold for fraction D. Therefore, although the stimulation of Factor XIa inactivation by antithrombin III could be quantitatively correlated to the binding of antithrombin III to heparin, the heparin-catalysed inhibition of Factor XIa is dependent not only upon the degree of binding of antithrombin III to heparin but also upon the type of heparin to which antithrombin III is bound.

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.

Binding of Heparin Fractions to von Willebrand Factor: Effect of Molecular Weight and Affinity for Antithrombin III

1994 ◽  
Vol 71 (01) ◽  
pp. 141-146 ◽  
Author(s):  
D Baruch ◽  
Nadine Ajzenberg ◽  
Cécile Denis ◽  
Paulette Legendre ◽  
Jean-Claude Lormeau ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Antithrombin Iii ◽  
Heparin Binding ◽  
Agarose Beads ◽  
Heparin Interaction ◽  
Factor Effect ◽  
Heparin Fractions ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryTo investigate the influence of the structure of heparin on its binding to vWF, we compared heparin fractions of different molecular weight (MW) or affinity for antithrombin III (ATIII). We studied the interaction of purified 125I-vWF or plasma vWF, labeled with a pool of 125I-monoclonal antibodies to vWF, with unfractionated heparin immobilized on agarose beads. Fractions were compared as competitors of these interactions and their effect was quantitated by their half-maximal inhibition (IC50). When the MW of the fractions decreased, especially below 7500, their IC50 increased, indicating that the affinity of the fractions for vWF decreased with their MW. Using heparin-derived oligosaccharides, we also demonstrated that a minimal chain length of 18 monosaccharides was required for heparin binding to vWF. In addition, different fractions with low affinity for ATIII were compared as competitors of 125-vWF binding to heparin-agarose. Despite a very low content of ATIII binding sites, some fractions retained a low IC50. Thus, heparin interaction with vWF is independent of the presence of the ATIII binding site and is mostly dependent on the length of the heparin chain. These data suggest that unfractionated heparin is a more potent inhibitor of vWF-dependent functions than low MW heparin fractions.

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

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.

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