scholarly journals Factor IXa Inhibition Contributes to the Heparin Effect

1991 ◽  
Vol 66 (03) ◽  
pp. 306-309 ◽  
Author(s):  
Suzette Béguin ◽  
Frédérique Dol ◽  
H Coenraad Hemker
Keyword(s):  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heparin Cofactor Ii ◽  
Factor Ixa ◽  
Factor Viiia ◽  
At Iii ◽  
Inhibition Of Thrombin ◽  
Thrombin Formation

SummaryWe investigated whether the inactivation of factor IXa contributes to the partial inhibition of thrombin formation that is observed at therapeutic concentrations of heparin. The action of standard unfractionated heparin (0.05 U/ml) on thrombin formation in the intrinsic system was compared to that of a mixture of dermatan sulfate (DS) and a synthetic pentasaccharide (PS). DS enhances the action of heparin cofactor II which inhibits thrombin only. PS specifically enhances the anti-factor Xa activity of antithrombin III (AT III). The concentrations of DS and PS were chosen so as to obtain equal anti-thrombin and anti-factor Xa activities as in 0.05 U/ml heparin. An extra inhibitory effect of heparin over the mixture is observed in situations where free factor IXa, not bound to factor VIIIa and phospholipid, limits the rate of thrombin formation, notably in contact activated plasma. We conclude that the inactivation of free factor IXa by heparin contributes importantly to the inhibition of thrombin formation in the intrinsic system such as e.g. measured in the activated partial thromboplastin time.

Homozygous Variant of Antithrombin III that Lacks Affinity for Heparin, AT III Kumamoto

1989 ◽  
Vol 61 (01) ◽  
pp. 020-024 ◽  
Author(s):  
Kenji Okajima ◽  
Hidetsugu Ueyama ◽  
Youichiro Hashimoto ◽  
Yasuto Sasaki ◽  
Keiko Matsumoto ◽  
...  
Keyword(s):  
Affinity Chromatography ◽  
Autosomal Dominant ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Heparin Cofactor Ii ◽  
At Iii ◽  
Antifactor Xa ◽  
Cofactor Activity ◽  
Inhibition Of Thrombin ◽  
Plasma Heparin

SummaryAbnormal antithrombin III (AT III) was found in the plasma of a 31-year-old female who suffered from recurrent thrombotic episodes. Heparin cofactor activity was 28% of normal and undetectable when measured by inhibition of thrombin and factor Xa (F. Xa), while both progressive antithrombin and antifactor Xa activities were normal. The concentration of plasma AT III antigen was 37 mg/dl. Analysis by crossed-immunoelec- trophoresis (CIE) in the presence of heparin and affinity chromatography on heparin-Sepharose revealed that the propositus’ AT III did not bind to heparin. When heparin cofactor II (HC II) was removed from propositus’ plasma, heparin cofactor activity of AT III was not detected. Thus, HC II seemed to account for the plasma heparin cofactor activity found in the presence of thrombin. The patient’s parents and three of her brothers demonstrated qualitative abnormality of AT III; heparin cofactor activity was 30-50% of normal levels in the presence of both thrombin and F. Xa. These findings indicate that the propositus’ AT III lacks affinity for heparin and the mode of its inheritance seems to be autosomal dominant and, hence, the propositus would be a homozygote. For this variant, the name of AT III Kumamoto is proposed.

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.

Modulation of Heparin Cofactor II Function by S Protein (Vitronectin) and Formation of a Ternary S Protein-Thrombin-Heparin Cofactor II Complex

1988 ◽  
Vol 60 (03) ◽  
pp. 399-406 ◽  
Author(s):  
Klaus T Preissner ◽  
Pierre Sié
Keyword(s):  
Dermatan Sulfate ◽  
Enzyme Inhibitor ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Binding Domain ◽  
Coagulation System ◽  
Pentosan Polysulfate ◽  
Heparin Cofactor Ii ◽  
S Protein ◽  
Inhibition Of Thrombin

SummaryThe complement inhibitor S protein, which is identical to the adhesive protein vitronectin, functions as heparin-neutralizing factor by protecting thrombin as well as factor Xa against fast inactivation by antithrombin III. The interference of S protein with glycosaminoglycan-catalyzed inhibition of thrombin by heparin cofactor II was investigated in these studies. S protein significantly counteracted the anticoagulant activity of heparin and pentosan polysulfate but not of dermatan sulfate. In the presence of 0.3 μg/ml heparin, 0.5 μg/ml pentosan polysulfate, or 2 μg/ml dermatan sulfate, S protein induced a concentrationdependent reduction of the inhibition rate of thrombin by heparin cofactor II. This resulted in a decrease of the apparent pseudo first-order rate constants by about 17-fold (heparin), or about 7-fold (pentosan polysulfate), whereas no neutralization of dermatan sulfate was demonstrable at a physiological ratio of S protein to heparin cofactor II. Exposure of the glycosaminoglycan-binding region of S protein by reduction and carboxymethylation of the protein increased the neutralizing activity of S protein towards heparin and pentosan polysulfate. The results of these functional experiments correlated well with the demonstration of direct binding of S protein to both polysaccharides but not to dermatan sulfate. While reduced/carboxymethylated S protein remained also ineffective in neutralizing other dermatan sulfate compounds with varying degree of sulfation, a synthetic highly basic tridecapeptide, representing a portion of the glycosaminoglycan-binding domain of S protein, counteracted their anticoagulant activity. Independent on the polysaccharide used, S protein was found incorporated within a ternary complex with thrombin and heparin cofactor II during the inhibition reaction as judged by crossed immunoelectrophoresis, ultracentrifugation as well as ELISA analysis, emphazising the function of S protein as scavenger protein for enzyme-inhibitor complexes of the coagulation system. These findings demonstrate the role of S protein as effective neutralising plasma protein of the anticoagulant activity of various glycosaminoglycans also with respect to heparin cofactor II. Although the glycosaminoglycan-binding domain of S protein readily neutralized different dermatan sulfate compounds, physiological modulation of heparin cofactor-II-dependent inhibition of thrombin by native S protein appears to be restricted to the vascular compartments, where other glycosaminoglycans than dermatan sulfate appear to be operative.

scholarly journals Effect of oversulphated chondroitin and dermatan sulphate upon thrombin and factor Xa inactivation by antithrombin III or heparin cofactor II

1988 ◽  
Vol 254 (2) ◽  
pp. 547-551 ◽  
Author(s):  
M F Scully ◽  
V Ellis ◽  
N Seno ◽  
V V Kakkar
Keyword(s):  
Antithrombin Iii ◽  
Chondroitin Sulphate ◽  
Factor Xa ◽  
Dermatan Sulphate ◽  
Physiological Control ◽  
Heparin Cofactor Ii ◽  
High Affinity ◽  
Human Thrombin ◽  
Sulphated Glycosaminoglycans ◽  
Inhibition Of Thrombin

The kinetics of inhibition of human thrombin and Factor Xa by antithrombin III or heparin cofactor II were examined under pseudo-first-order conditions as a function of the concentration of naturally occurring oversulphated chondroitin and dermatan sulphates. The sulphated glycosaminoglycans (GAGs) studied were chondroitin sulphate D (CSD) (GlcA-2-SO4-GalNAc-6-SO4), chondroitin sulphate K (CSK) (GlcA-3-SO4-GalNAc-4-SO4), chondroitin sulphate H (CSH) (IdA-GalNAc-4,6-diSO4) and polysulphated dermatan sulphate (DPS) (IdA-2-SO4 or -3-SO4-GalNAc-4,6-diSO4). The data for the antithrombin III inhibition of thrombin showed a low degree of maximal potentiation of this interaction (congruent to 10-fold), which would appear to be characteristic of GAGs devoid of the high-affinity antithrombin III binding site. In contrast there was a greater potentiation of the inhibition of thrombin by heparin cofactor II with DPS showing an activity comparable to heparin in this interaction at a concentration two orders of magnitude lower than dermatan sulphate. DPS potentiated antithrombin III-Factor Xa interaction by 1200-fold, similar to that shown by high-affinity heparin of 6 kDa. The antithrombin III-Factor Xa interaction was potentiated by all other GAGs studied to a degree similar to that of heparin pentasaccharide with high affinity for antithrombin III. The findings suggest more stringent structural requirements for GAG stimulation of antithrombin-thrombin interaction than for antithrombin-Factor Xa or heparin cofactor-thrombin interaction, which may also be of significance in physiological control of haemostasis.

A Comparison of Pentosan Polysulphate (SP54) and Heparin I: Mechanism of Action on Blood Coagulation

1982 ◽  
Vol 47 (02) ◽  
pp. 104-108 ◽  
Author(s):  
A-M Fischer ◽  
T W Barrowcliffe ◽  
D P Thomas
Keyword(s):  
Factor Xa ◽  
Factor X ◽  
Factor Ixa ◽  
Crossed Immunoelectrophoresis ◽  
Low Concentrations ◽  
At Iii ◽  
Pentosan Polysulphate ◽  
High Concentrations ◽  
Marked Suppression ◽  
Inhibition Of Thrombin

SummaryThe effects of SP54 on inhibition of thrombin, factor Xa and factor IXa, in the presence and absence of antithrombin III (At III), have been examined and compared to those of heparin. SP54 potentiated inhibition of thrombin and Xa by purified At III, but crossed immunoelectrophoresis data indicated that these effects were mediated by binding to the enzyme, rather than to At III. Relatively high concentrations of SP54 were required for inhibition of thrombin and Xa in plasma, but at concentrations less than 2 μg/ml there was a marked suppression of the intrinsic activation of factor X. This effect was shown to be independent of At III, and to be due largely to inhibition of factor IXa. Prothrombin activation by factor Xa and phospholipid was also suppressed by SP54 in the absence of At III, and its effect on the APTT was also shown to be independent of At III. It is concluded that at relatively low concentrations the anticoagulant actions of SP54 are mainly due to these At III-independent pathways.

Antithrombotic and Anticoagulant Activity of Depolymerized Fragment of the Glycosaminoglycan Extracted from Stichopus japonicus Selenka

1991 ◽  
Vol 65 (04) ◽  
pp. 369-373 ◽  
Author(s):  
Norihiko Suzuki ◽  
Kenji Kitazato ◽  
Junki Takamatsu ◽  
Hidehiko Saito
Keyword(s):  
Antithrombin Iii ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Activated Partial Thromboplastin Time ◽  
Low Molecular Weight ◽  
Clotting Time ◽  
Antithrombotic Agents ◽  
Chromogenic Assay ◽  
Factor Ixa ◽  
At Iii

SummaryThe antithrombotic and anticoagulant activities of depolymerized fragment (DHG-l) of glycosaminoglycan extracted from Stichopw japonicus Selenka (FGAG) were compared with those of unfractionated heparin (UFH) or low molecular weight heparin (LMWH). DHG-1 at more than 0.3 mg/kg i. v. significantly prevented death of mice treated with thrombin (800 U/kg i.u.). Under the same conditions, FGAG, UFH and LMWH significantly prevented death of mice at more than 0.3, 0.3 and 0.6 mg/kg i.v., respectively. In normal plasma, the concentration required to double the activated partial thromboplastin time (doubling APTT) of DHG-I, FGAG, LMWH and UFH were 12.0, 2.4, 5.8, and 1.2 μg/ml, respectively. In antithrombin III (AT Ill)-depleted plasma, doubling APTT of DHG-1, FGAG, and UFH were 11.3, 2.1, and 18.5 μg/ml, respectively. Prothrombin activation in contact-activated plasma was inhibited completely for 60 s at doubling APTT by all glycosaminoglycans used in this study. DHG-I, however, showed much less antithrombin activity than UFH as tested by thrombin clotting time in plasma and chromogenic assay in the presence of AT III. Moreover, DHG-1 showed much less inhibitory activity on factor Xa, factor IXa, and glass surface-induced factor IXa generation than UFHThese results suggested that DHG-1 is one of the promising antithrombotic agents with quite different anticoagulant property from UFH or LMWH.

Inhibition of Thrombin by Antithrombin III and Heparin Cofactor II In Vivo

1995 ◽  
Vol 73 (03) ◽  
pp. 405-412 ◽  
Author(s):  
Longbin Liu ◽  
Lori Dewar ◽  
Yingqi Song ◽  
Myron Kulczycky ◽  
Blajchman A Morris ◽  
...  
Keyword(s):  
Pregnant Women ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Critical Role ◽  
Experimental Studies ◽  
Thrombin Inhibitor ◽  
Heparin Cofactor Ii ◽  
Inhibition Of Thrombin

SummaryThe critical role of thrombin in the pathogenesis of venous and arterial thrombosis, and the effectiveness of glycosaminoglycans as antithrombotic drugs are well known. Antithrombin III is a major inhibitor of thrombin and augmentation of its inhibitory actions by heparin is the basis for the clinical uses of heparin. Recent clinical and experimental studies have demonstrated that another glycosaminoglycan, dermatan sulfate, is an effective antithrombotic drug. Dermatan sulfate catalyses the inhibition of thrombin by heparin cofactor II. The concentrations of heparin cofactor II are higher in the plasmas of individuals with congenital antithrombin III deficiency and pregnant women than controls. The role of heparin cofactor II as a physiologic thrombin inhibitor is unknown. Enzyme-linked immunosorbent assays were used to quantify thrombin-heparin cofactor II and thrombin-antithrombin III endogenous to the plasmas of adult antithrombin III-Hamilton deficient subjects, their siblings with normal antithrombin III levels, pregnant women at term and 3 to 5 days after delivery. Both thrombin-antithrombin III and thrombin-heparin cofactor II complexed with vitronectin were detected in all the plasmas. Significantly, the concentrations of thrombin-heparin cofactor II-vitronectin were higher in the plasmas of congenital antithrombin III deficient subjects and in pre- and post-delivery plasmas than those of normal subjects. In addition, the concentrations of thrombin-heparin cofactor II decreased 3 to 5 days after delivery, reflecting the disappearance of the catalytically active dermatan sulfate elaborated by the placenta. Thus, heparin cofactor II normally inactivates thrombin in vivo, with its role increasing in conditions associated with high levels of heparin cofactor II and/or dermatan sulfate.

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:

EVIDENCE FOR A HOMOLOGY BETWEEN THE HEPARIN AND HEPARAN SULFATE BINDING REGIONS TO ANTITHROMBIN III

1987 ◽  
Author(s):  
A M Fischer ◽  
J Tapon-Bretaudière ◽  
M D Dautzenberg ◽  
C Sternberg ◽  
J Choay
Keyword(s):  
Heparan Sulfate ◽  
Antithrombin Iii ◽  
Competitive Inhibition ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Heparin Binding ◽  
At Iii ◽  
Binding Regions ◽  
Partial Homology ◽  
Type 3

As we have previously demonstrated, heparan sulfate, a glycosaminoglycan physiologically present on the endothelial wall, is, like heparin, unable to potentiate the inhibitory effect against Factors IIa and Xa of two abnormal type 3 antithrombin III (AT III) variants. We report here that a synthetic pentasaccharide which constitutes the sequence of the heparin binding site to AT III is also unable to potentiate these two AT III variants in an anti-Factor Xa assay. According to these data, we speculated the existence of a homology between the heparin and heparan sulfate binding regions to normal AT III. We thus studied the competitive inhibition by the pentasaccharide of heparin and heparan sulfate in their potentiation of AT III activity. Such a competitive inhibition can be observed in an AT III antiFactor IIa assay because the pentasaccharide which exhibits a high anti-Factor Xa activity is devoid of any anti-Factor IIa activity. In the absence of pentasaccharide, with both heparin and heparan sulfate, a plateau is reached in AT III potentiation for concentrations of respectively 2.5 ng and 17.2 μg (corresponding to the same anti-Factor IIa activity of 0.4 u/ml for both glycosaminoglycans). In the presence of 5.5 μg of pentasaccharide, the inhibition of heparin cofactor activity is 70%. With heparan sulfate, the inhibition by the same amount of pentasaccharide is less pronounced, being only 30%. These results strongly suggest the existence of a partial homology between heparin and heparan sulfate binding sites to AT III. For heparan sulfate, the exact sequence of this site remains to be identified .

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