Antithrombin III Binding to Surface Immobilized Heparin and Its Relation to F Xa Inhibition

1987 ◽  
Vol 58 (04) ◽  
pp. 1064-1067 ◽  
Author(s):  
K Kodama ◽  
B Pasche ◽  
P Olsson ◽  
J Swedenborg ◽  
L Adolfsson ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Surface Coating ◽  
Antithrombin Iii ◽  
Lower Class ◽  
Biologically Active ◽  
High Affinity ◽  
Heparin Coating ◽  
Continuous Surface ◽  
Approximate Molecular Weight

SummaryThe mode of F Xa inhibition was investigated on a thromboresistant surface with end-point attached partially depoly-merized heparin of an approximate molecular weight of 8000. Affinity chromatography revealed that one fourth of the heparin used in surface coating had high affinity for antithrombin III (AT). The heparin surface adsorbed AT from both human plasma and solutions of purified AT. By increasing the ionic strength in the AT solution the existence of high and low affinity sites could be shown. The uptake of AT was measured and the density of available high and low affinity sites was found to be in the range of 5 HTid 11 pic.omoles/cmf, respectively Thus the estimated density of biologically active high and low ailmity heparm respectively would be 40 and 90 ng/cm2 The heparin coating did not take up or exert F Xa inhibition by itself. With AT adsorbed on both high and low affinity heparin the surface had the capacity to inhibit several consecutive aliquots of F Xa exposed to the surface. When mainly high affinity sites were saturated with AT the inhibition capacity was considerably lower. Tt was demonstrated that the density of AT on both high and low affinity heparin determines the F Xa inhibition capacity whereas the amount of AT on high affinity sites limits the rate of the reaction. This implies that during the inhibition of F Xa there is a continuous surface-diffusion of AT from sites of a lower class to the high affinity sites where the F Xa/AT complex is formed and leaves the surface. The ability of the immobilized heparin to catalyze inhibition of F Xa is likely to be an important component for the thromboresistant properties of a heparin coating with non-compromized AT binding sequences.

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).

Characterisation of Persistent Anti-Xa Activity Following Administration of the Low Molecular Weight Heparin Enoxaparin Sodium (Clexane)

1994 ◽  
Vol 72 (02) ◽  
pp. 275-280 ◽  
Author(s):  
David Brieger ◽  
Joan Dawes
Keyword(s):  
Molecular Weight ◽  
Enoxaparin Sodium ◽  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Active Material ◽  
Biologically Active ◽  
Size Exclusion ◽  
Low Molecular Weight ◽  
Exclusion Chromatography ◽  
Liver And Kidney

SummaryIt is widely reported that persistent anti-Xa activity follows administration of low molecular weight heparins. To identify the effectors of this activity we have injected 125I-labelled Enoxaparin sodium into rabbits and subsequently analysed the circulating radiolabelled material and anti-Xa activity by affinity and size exclusion chromatography. Antithrombin III-binding material derived from the injected drug was responsible for all the anti-Xa amidolytic activity. At early times after injection additional anticoagulant activity which was largely attributable to tissue factor pathway inhibitor was measured by the Heptest clotting assay after removal of glycosaminoglycans from plasma samples. Small radiolabelled fragments, including penta/hexasaccharide with affinity for antithrombin III, were detectable in the circulation 1 week later, and sulphated oligosaccharides persisted for 3-4 weeks. Significant quantities of radiolabel remained in the liver and kidney several weeks post-injection; these organs may sequester some of the injected drug and give rise to circulating biologically active material by degradation and secretion of catabolic products into the plasma.

scholarly journals Factors influencing the acceleration of human factor XIa inactivation by antithrombin III

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1873-1879
Author(s):  
CF Scott ◽  
RW Colman
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Antithrombin Iii ◽  
Progressive Increase ◽  
Inactivation Rate ◽  
At Iii ◽  
Factor Xia ◽  
Heparin Complexes ◽  
High Concentrations ◽  
Heparin Preparation

Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa in plasma.

Influence of Tryptophan Modification upon Digestion of Antithrombin III by Elastase

1991 ◽  
Vol 65 (04) ◽  
pp. 351-354 ◽  
Author(s):  
M F Scully ◽  
N Shah ◽  
V Ellis ◽  
V V Kakkar
Keyword(s):  
Molecular Weight ◽  
Chemical Modification ◽  
Neutrophil Elastase ◽  
Antithrombin Iii ◽  
High Affinity ◽  
Tryptophan Residues ◽  
Tryptophan Modification ◽  
Modified Forms ◽  
Molecular Weight Form

SummaryChemical modification of tryptophan residues in antithrombin III by dimethyl (2-hydroxy-5-nitrobenzyl) sulfonium bromide (HNBSB) generates products with similar levels of modification (equivalent to 0.9 mole 2-hydroxy-5-nitrobenzyl [HNB] incorporated/mole of antithrombin III) but with high or low affinity for heparin-Sepharose. Upon digestion with pancreatic or neutrophil elastase the low affinity forms generate a product of molecular weight form (55 kDa) not seen in digests of native antithrombin III or modified forms with high affinity for heparin. When measured as loss of activity the obserued rate of digestion of the latter in the absence of heparin was more rapid than that of native antithrombin III. The differences in digestion are considered to be related to conformation at differences between the various forms.

Properties and characteristics of a bacteriocin-like substance produced by Propionibacterium acnes isolated from dental plaque

1988 ◽  
Vol 34 (12) ◽  
pp. 1344-1347 ◽  
Author(s):  
Greg E. Paul ◽  
S. James Booth
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Dental Plaque ◽  
Propionibacterium Acnes ◽  
Gram Positive ◽  
Gram Negative ◽  
A Cell ◽  
Approximate Molecular Weight ◽  
Low Ionic Strength

A cell-associated bacteriocinlike substance with an approximate molecular weight of 78 000 was isolated from an oral isolate of Propionibacterium acnes. The substance was bacteriostatic and was active against both gram-positive and gram-negative anaerobes. Lysozyme inhibited the activity of the bacteriocinlike substance at low ionic strength.

scholarly journals The Mode of Action of CY216 and CY222 in Plasma

1992 ◽  
Vol 67 (01) ◽  
pp. 033-041 ◽  
Author(s):  
Suzette Béguin ◽  
Simone Wielders ◽  
J C Lormeau ◽  
H Coenraad Hemker
Keyword(s):  
Molecular Weight ◽  
High Performance ◽  
Time Course ◽  
Antithrombin Iii ◽  
Platelet Rich Plasma ◽  
Gel Permeation Chromatography ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Material ◽  
At Iii

SummaryThree fractions of the low molecular weight heparin CY216 (fraxiparin, mean molecular weight [MMW] 5,090), with MMWs of respectively, 3,090, 4,400 and 7,910 were prepared by gel permeation chromatography. From CY222 (MMW 3,770) as well as from CY216 and its three fractions the material with high affinity to antithrombin III (AT III) was obtained by chromatography on immobilised AT III. The molecular weight distribution of each of the ten preparations thus obtained was determined by high performance liquid chromatography, while the content of AT III binding material was determined by stoichiometric titration of AT III, monitored by intrinsic fluorescence enhancement.We measured the effect of all heparins on the decay of endogenous thrombin in plasma and on the overall generation of thrombin in plasma, triggered via the extrinsic or via the intrinsic pathway. From these data we calculated the time course of prothrombin conversion, i. e. the course of factor Xa activity as expressed by prothrombinase activity.It was found that in platelet-poor plasma the anticoagulant properties of the heparins are largely dependent on their antithrombin action, which is determined by their content of high affinity material with a MW of 5,400 or higher. The specific antithrombin activity of all heparins, when expressed in terms of material with high affinity to antithrombin III (HAM) with a MW >5,400 is 13.0 min-1/(μg/ml) (range 10.5-15.9). The anticoagulant potency is not influenced by the presence of low-affinity material and hardly by material with MW <5,400.In platelet-rich plasma, however, the presence of non-AT III binding material enhances the inhibition, presumably by neutralising heparin binding material originating from activated platelets. The ultra low MW fractions (<3,400) show a similar activity in PPP and in PRP.

scholarly journals RABBIT MACROPHAGE INTERFERONS

1967 ◽  
Vol 125 (4) ◽  
pp. 579-593 ◽  
Author(s):  
Thomas J. Smith ◽  
Robert R. Wagner
Keyword(s):  
Molecular Weight ◽  
Peritoneal Macrophages ◽  
Biologically Active ◽  
Rabbit Kidney ◽  
Rabbit Serum ◽  
Molecular Component ◽  
Molecular Weights ◽  
Marker Proteins ◽  
Approximate Molecular Weight ◽  
Interferon Activity

Antiviral factors present in cultures of rabbit peritoneal macrophages or rabbit kidney (RK) cells infected with Newcastle disease virus (NDV) and those in cultures of uninfected macrophages all fulfilled the biological and physicochemical criteria for classification as interferons. Virus-induced macrophage and RK interferons were slightly more stable to heat or acid than "spontaneously produced" or endotoxin-induced macrophage interferon. Interferon activity in serum of NDV-infected rabbits was decidedly more labile than NDV-induced macrophage interferon. However, these differences in lability were too slight to serve as a useful basis for distinguishing one rabbit interferon from another. Rabbit interferons from various sources could be differentiated by filtration through Sephadex G-100 and their molecular weights estimated by comparison with elution profiles of a series of marker proteins of known molecular weight. Each of four different preparations of rabbit interferons was found to contain more than one molecular component. Elution peaks for three NDV-induced interferons were equivalent to the following molecular weights: RK ≃44,000–45,000 and &gt; 134,000 (variable and &lt; 1% when present); macrophage ≃37,000, 44,000–45,000, and &gt; 134,000 (variable and &lt;1% when present); and serum ≃50,000–52,000 and &gt; 134,000 (∼10% and heat labile). NDV-induced serum interferon may also contain another molecular component of mol wt ≃45,000 represented by a trailing shoulder from the major 51,000 mol wt peak. Endotoxin-induced macrophage interferon proved to be polydisperse. Sephadex filtration of this interferon did not reveal clear and consistent elution patterns, partially owing to its low initial titer and lability. However, variable peaks of biological activity could be detected in Sephadex fractions equivalent to approximate molecular weight values of &gt; 134,000, 72,000–78,000, 33,000–38,000, 28,000–30,000, and possibly a component of 42,000–45,000. A major component of mol wt ≃37,000 was present in all samples of endotoxin-induced macrophage interferon. The other constituents may be biologically active subunits or polymers. These data indicate that rabbit macrophages produce two primary kinds of interferon: (a) an RK-like component of mol wt ≃45,000 that is synthesized in greatest amount after viral induction, and (b) a different species of mol wt ≃ 37,000 that can also be synthesized in the absence of viral induction. The presence of major interferon constituents of mol wt ≃51,000 and &gt; 134,000 in rabbit serum after viral induction suggests that macrophages are not the principal interferon-producing cells that respond to intravenous injection of NDV.

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 Factors influencing the acceleration of human factor XIa inactivation by antithrombin III

Blood ◽  
1989 ◽  
Vol 73 (7) ◽  
pp. 1873-1879 ◽  
Author(s):  
CF Scott ◽  
RW Colman
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Antithrombin Iii ◽  
Progressive Increase ◽  
Inactivation Rate ◽  
At Iii ◽  
Factor Xia ◽  
Heparin Complexes ◽  
High Concentrations ◽  
Heparin Preparation

Abstract Controversy exists in the literature concerning the potentiating effect of heparin on the inactivation rate of factor XIa by antithrombin III (AT III) in both purified systems and in plasma. We have analyzed the factors that could influence this reaction and found that ionic strength of the medium, as well as the type and concentration of the heparin preparations accounted for the major discrepancies in the literature. At I = 0.43 N, a preparation of bovine lung heparin at 1 U/mL did not augment the inactivation rate of factor XIa by inhibitors in plasma or by purified AT III. However, when ionic strength was decreased, a progressive increase in the potentiating effect was observed, reaching 6.5-fold at I = 0.15 N. At saturating concentrations of heparin, which results in the formation of 100% AT III-heparin complex, (greater than ten-fold molar excess over AT III) in purified systems, all heparin preparations (porcine, bovine, low molecular weight [LMW], and high affinity) yielded an approximately 30-fold augmentation of the factor XIa inactivation rate. However, when heparin was less than saturating, we observed that various heparin preparations affected the AT III-induced inactivation of factor XIa to different degrees even though they exhibited the same inhibitory activity (1 U/mL) against thrombin. This variation resulted from differences in the number of AT III binding sites in each heparin preparation, despite a similar Kd for each. Addition of high molecular weight kininogen (HK) to AT III-heparin complexes did not enhance their ability to inhibit factor XIa, and high concentrations of HK decreased the inactivation rate. A high therapeutic dose of heparin only permits the formation of 2.5% to 16.5% of the AT III-heparin complexes that can be achieved at saturation. We observed that 1 U/mL heparin (bovine lung heparin) (high therapeutic concentration) in virtually undiluted plasma only accelerated the inactivation rate of factor XIa (in the absence of other active enzymes) less than two-fold. These new observations further support our previous conclusion that therapeutic levels of heparin have little to no influence on the inactivation rate of factor XIa in plasma.

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