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.

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.

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.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

NEUTRALIZING EFFECT OF PLATELET FACTOR 4 OR HISTIGINE-RICH GLYCOPROTEIN AGAINST LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN

1987 ◽  
Author(s):  
K Takahashi ◽  
M Niwa ◽  
N Sakuragawa
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Bleeding Tendency ◽  
Human Origin ◽  
Platelet Factor ◽  
At Iii ◽  
Antifactor Xa

Purpose: Low molecular weight(LMW) heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. Platelet factor 4(Pf-4) and histidine-rich glycoprotein(HRG) neutralize heparin. We investigated on the heparin neutralizing effects of them to both kinds of heparinMaterials and methods: LMW heparin(Kabi and Pharmuka) and UF heparin(Novo) were used. Antithrombin III(AT-III), HRG(human origin ) and pf-4( bovine origin ) were purified by our methodsTH(Green-Cross) and F-Xa(Sigma) were used. Reaction mixtures for anti-TH or anti-F-Xa were as follows: 1 vol of AT-III( 0.1 U/ml)+ 1 vol of heparin( 10 ug/ml)+l vol of pf-4 or HRG(varied)→incubated for 5 min→+l vol of TH(5 U/ml) or F-Xa( 7 nKat/ml)→incubated for 5 min→ + S-2238 or S-2222→ recorded at 405 nm.Results: (1) Pf-4 showed the equivalent anti-TH effect on both kinds of heparin, and 3 ug of pf-4 neutralized 1 ug of heparinOn F-Xa neutralizing effect, 13 ug of pf-4 neutralized 1 ug of UF heparin, but could not neutralize LMW heparin. (2) HRG showed the same results on anti-TH effect of both kinds of heparin, but could not neutralize the anti-F-Xa effect of LMW heparin on the same amount of HRG which neutralized that of UF heparin. Conclusion: Anti-F-Xa effect of. LMW heparin could not be easily neutralized by pf-4 or HRG compared with that of UF heparin.

Isolation and Partial Characterization of Equine Antithrombin III.

1979 ◽  
Author(s):  
D.W. Estry ◽  
T.G. Bell ◽  
G.H. Tishkoff ◽  
J.C. Mattson ◽  
S.C. Estry
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Stable Complex ◽  
Sds Page ◽  
At Iii ◽  
Human Thrombin ◽  
Horse Plasma ◽  
Stoichiometric Complex

A protein analogous to human antithrombin III was isolated from fresh horse plasma. The procedure for purification was a modification of Thaler and Schmer’s two-step isolation procedure. The horse protein was homogeneous on 7.5% SDS-PAGE gels and had a molecular weight of 62,000 to 64,000 daltons in both reducing and non-reducing systems (human; 62,300). Rabbit anti-human antithrombin III was used to demonstrate a line of partial identity by Immunoelectrophoresis between the horse and human protein. The horse protein rapidly neutralizes human thrombin (34,000 daltons) and the reaction appears to be greatly potentiated by heparin. In order to establish the formation of 1:1 covalent stoichiometric complex between horse AT III and thrombin (IIa), time studies were run in the presence and absence of heparin. AT III (62,000) at 15 seconds, 2, 5, 10 and 60 minutes formed a stable complex with thrombin (32,000) having a molecular weight of 86,000 daltons. Additional bands developing with time are due to the autolytic capabilities of the uncomplexed IIa. The major autolytic band had a molecular weight of 70,000 daltons. Addition of heparin potentiated the interaction although it did not change the stoichio-metry of the complexes formed. The data accumulated to date demonstrates the similarities between the human and horse protein and the possibilities of using the horse as a model system for the evaluation of AT III replacement therapy in vivo.

scholarly journals Effect of heparin on the inactivation rate of human factor XIa by antithrombin-III

Blood ◽  
1982 ◽  
Vol 60 (4) ◽  
pp. 940-947 ◽  
Author(s):  
CF Scott ◽  
M Schapira ◽  
RW Colman
Keyword(s):  
Clinical Practice ◽  
Human Factor ◽  
Antithrombin Iii ◽  
Fold Increase ◽  
Factor Ix ◽  
Inactivation Rate ◽  
Amidolytic Activity ◽  
Factor Ixa ◽  
Factor Xia ◽  
Plasma Heparin

Abstract Factor XIa catalyzes an important reaction in the early phase of blood coagulation by converting factor IX to an active enzyme (factor IXa). Although antithrombin-III, an inhibitor of factor XIa, normally accounts for only one-sixth of the plasma inhibitory activity against factor XIa, its effectiveness has been reported to be enhanced by heparin. We have reinvestigated the ability of heparin to potentiate factor XIa inhibition by both purified antithrombin-III and plasma using synthetic tripeptide amide substrates as well as a coagulant assay. No increase in the inactivation rate of factor XIa amidolytic activity by purified antithrombin-III was observed in the presence of therapeutic heparin concentrations (1 U/ml), although inhibition of the amidolytic activity of thrombin by purified antithrombin-III was enhanced at least 20-fold by the same concentration of heparin. Furthermore, despite the ability of heparin (1 U/ml) to increase the inactivation rate of thrombin by plasma, no acceleration of the rate of inhibition of factor XIa by plasma was observed. Similar results were found when the inhibition of factor XIa was monitored with a coagulant assay after first removing the heparin. Only at heparin concentrations of 5 and 10 U/ml, was a 2- and 4-fold increase in the inactivation rate of factor XIa by purified antithrombin III observed. Therefore, in both purified systems as well as plasma, heparin, at concentrations observed in clinical practice, does not accelerate the inactivation rate of human factor XIa by antithrombin-III.

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.

Modification of an Amidolytic Heparin Assay to Express Protein-Bound Heparin and to Correct for the Effect of Antithrombin III Concentration

1987 ◽  
Vol 58 (03) ◽  
pp. 884-887 ◽  
Author(s):  
Sandra G Lyon ◽  
Elliott C Lasser ◽  
Rosalyn Stein
Keyword(s):  
Molecular Weight ◽  
Dextran Sulfate ◽  
Antithrombin Iii ◽  
Low Molecular Weight ◽  
Accurate Assessment ◽  
Blood Samples ◽  
Concurrent Control ◽  
At Iii ◽  
Antithrombin Iii Concentration ◽  
Plasma Heparin

SummaryA modification of an anti-Xa assay for plasma heparin has been devised using a low molecular weight dextran sulfate that competitively binds protein heparin neutralizers and displaces masked heparin. The addition of 0.12 mg dextran sulfate per ml of plasma permits heparin, neutralized by the products of platelet aggregation, to recover full functional activity against Xa. The assay will permit a more accurate assessment of both exogenous plasma heparin and endogenous liepaiin-like activity in blood samples collected with varying techniques. A further modification is proposed employing polybrene to neutralize the plasma heparin-like material providing a concurrent control for each sample that increases accuracy by eliminating the effect of varying AT-III levels which have anti-Xa activity.

scholarly journals The inhibition of high and low molecular weight urokinase in plasma

Blood ◽  
1980 ◽  
Vol 55 (3) ◽  
pp. 430-436
Author(s):  
G Murano ◽  
D Aronson ◽  
L Williams ◽  
L Brown
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Antithrombin Iii ◽  
Polyacrylamide Gel Electrophoresis ◽  
Low Molecular Weight ◽  
Biologic Activity ◽  
At Iii ◽  
Neutralization Process ◽  
Alpha 1 Antitrypsin ◽  
Formation Of Complexes

The rates of inhibition of high molecular weight (HMW) and low molecular weight (LMW) urokinase (UK) incubated in plasma or with purified antithrombin III (AT-III) were compared. Using a fibrinolytic assay system to determine residual biologic activity, polyacrylamide gel electrophoresis to demonstrate the formation of complexes, and selective immunoprecipitation techniques to identify the plasma inhibitors participating in the neutralization process, it was established that: (A) HMW-UK is inhibited more rapidly than LMW-UK, both in plasma and with purified AT-III; (B) heparin (3--10 U/ml accelerates the neutralization process in both systems, but only slightly; and (C) in plasma, several inhibitors, alpha 2-macroglobulin, alpha 1-antitrypsin, and antithrombin III, neutralize the activity of HMW-UK and LMW-UK.

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