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.

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.

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.

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

Abstract 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.

scholarly journals Optimization of Heparin Monitoring with Anti-FXa Assays and Impact of Dextran Sulfate for Measuring All In-Vivo Drug Activity

2021 ◽  
Author(s):  
JEAN AMIRAL ◽  
Cedric AMIRAL ◽  
Claire DUNOIS
Keyword(s):  
Molecular Weight ◽  
Blood Circulation ◽  
Dextran Sulfate ◽  
Drug Dosage ◽  
Low Molecular Weight ◽  
Key Factors ◽  
The One ◽  
Assay Conditions ◽  
Plasma Heparin

Heparins, Unfractionated or Low Molecular Weight, are permanently at the spotlight of both clinical indications and laboratory monitoring. An accurate drug dosage is necessary for an effi-cient and safe therapy. The one-stage anti-FXa kinetics’ assays are the most widely and universally used with full automation for large series, without needing exogenous Antithrombin. WHO in-ternational standards are available for UFH and LMWH, but external quality assessment surveys still report a high inter-assay variability. This heterogeneity results from: assay formulation, designed without or with dextran sulfate to measure all heparin in blood circulation; calibrators for testing UFH or LMWH with the same curve; and automation parameters. The various factors which impact heparin measurements are reviewed, and we share our experience to optimize assays for completely testing plasma heparin. Evidence is provided on the usefulness of low molecular weight dextran sulfate to mobilize all drug present in blood circulation. Other key factors concern adjustment of assay conditions to obtain fully superimposable calibration curves for UFH and LMWH, and automation parameters. The study is illustrated by the performances of the various anti-FXa assays used for testing heparin on UFH or LMWH treated patients’ plasmas and obtained using citrate or CTAD anticoagulants. Comparable results are obtained only when CTAD anti-coagulant is used. Using citrate UFH is underestimated in the absence of dextran sulfate. Heparin calibrators, adjustment of automation parameters and data treatment contribute to other smaller differences.

COMPARATIVE STUDIES ON ANTITHROMBIN III AFFINITY OF LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN

1987 ◽  
Author(s):  
N Sakuragawa ◽  
T Shimotori ◽  
K Takahashi
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Affinity Column ◽  
Low Molecular Weight ◽  
Bleeding Tendency ◽  
Heparin Cofactor Ii ◽  
Linear Gradient ◽  
Affinity Column Chromatography ◽  
At Iii ◽  
Different Characteristics

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. These characteristics were surmised to be depend on antithrombin III(AT-III) affinity of the heparin. Materials and methods: LMW heparin(Kabi and Pharmuka), UF heparin (Novo) and heparin cofactor II(HC-II) purified by our method were used. AT-III affinity column chromatography with 0.1 M Tris-buffer (pH 7.4)-NaCl 0.02 to 2.5 M linear gradient was performed. From the point of AT-III affinity strength, non-affinity(Na), low affinity (La) and high affinity(Ha) were separated, and aPTT, anti-F-Xa and anti-TH activities were assayed on each fractions. HC-II was assayed by biological activity.Results: (1) Kabi-LMW heparin; Na 34.5%, La 39.3%, Ha 26.2%, Pharmuka-LMW heparin; Na 58.0%, La 24.1%, Ha 17.3%. Novo; Na 0%, La 50%, Ha 50%. (2) APTT; Na showed no effect, but Ha showed the strongest prolonging effects on aPTTs even having less amount of uronic acid, and more prominent effects were observed in UF(Novo)-heparin than LMW heparins. (3) La showed higher activity of anti-F-Xa and anti-TH activities than Ha. (4) Anti-TH activity of AT-III was observed in both fractions of La and Ha, but that of HC-II was observed in each fractions including Na.Conclusion: It was surmised that the differences of the characteristics between LMW heparin and UF heparin were depend on to the strength of AT-III. The different characteristics of HC-II from AT-III to anti-TH were observed and surmised to be depend on the binding ability to the fractions.

Studies of the Anti-Xa Activity of Human Plasma and Purified Antithrombin III

1979 ◽  
Author(s):  
T.W. Barrowcliffe ◽  
Anne C. Eggleton
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Aluminium Hydroxide ◽  
Low Molecular Weight Heparin ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Inhibitory Effect ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
At Iii

When samples of purified antithrombin (At III) were compared to plasma at the same At III concentration, in the absence of heparin, the anti-Xa activity of plasma was considerably higher. In the presence of heparin the anti-Xa activity of purified At III was much greater than plasma. This was shown to be due to an inhibitory effect on the heparin. At III-Factor Xa interaction in plasma which could be removed by absorption with aluminium hydroxide [Al(OH)3]. This inhibition was dependent on the molecular weight of the heparin; low molecular weight heparin was inhibited less than high molecular weight heparin, and this probably accounts for the apparently high anti-Xa activity of low molecular weight heparin.A1(OH)3 absorption of plasma also increased its anti-Xa activity in the absence of heparin. Addition of Factor IX concentrate to the absorbed plasma reduced its anti-Xa activity to that of normal plasma, and studies with purified proteins showed that this effect was due to the prothrombin in the concentrate. The addition of Factor IX concentrate or prothrombin to purified At III did not affect its anti-Xa activity.These results suggest that, in addition to At III, there is another Xa-inhibitor in plasma which competes with prothrombin for binding of Factor Xa.

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.

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).

Antithrombin III Alger: A New Homozygous AT III Variant

1986 ◽  
Vol 55 (02) ◽  
pp. 218-221 ◽  
Author(s):  
A M Fischer ◽  
P Cornu ◽  
C Sternberg ◽  
F Mériane ◽  
M D Dautzenberg ◽  
...  
Keyword(s):  
Family History ◽  
Antithrombin Iii ◽  
Antigen Concentration ◽  
Crossed Immunoelectrophoresis ◽  
The Family ◽  
At Iii ◽  
Molecular Abnormality ◽  
Slow Moving ◽  
Cofactor Activity ◽  
Plasma Heparin

SummaryA qualitative abnormality of antithrombin III (AT III) was found in the plasma of a 41-year old patient. The plasmatic AT III antigen concentration was 130% and the progressive anti-F IIa and anti-F Xa activities were normal (105% and 137%). The plasma heparin cofactor activity was less than 10%, when measured by F Ila or F Xa inhibition. Crossed immunoelectrophoresis of AT III in the presence of heparin revealed in the plasma an abnormal slow-moving peak. When tested by affinity chromatography on heparin Sepharose, this abnormal AT III did not bind to heparin. Among the investigated relatives, 5 subjects had normal AT III levels, whatever the test used, the nine others having reduced levels of antithrombin heparin cofactor activity (45-61%) but normal levels of immunoreactive AT III (97-122%). Consanguinity was found in the family history. We therefore considered our patient as homozygous for an AT III molecular abnormality affecting the binding site for heparin.

Neutralisation of Heparan Sulphate and Low Molecular Weight Heparin by Protamine

1985 ◽  
Vol 53 (01) ◽  
pp. 086-089 ◽  
Author(s):  
A R Hubbard ◽  
C A Jennings
Keyword(s):  
Molecular Weight ◽  
Plasma Proteins ◽  
Low Molecular Weight Heparin ◽  
Heparan Sulphate ◽  
Activated Partial Thromboplastin Time ◽  
Weight Basis ◽  
Low Molecular Weight ◽  
Protamine Sulphate ◽  
At Iii ◽  
Reference Preparation

SummaryThe neutralisation by protamine sulphate (PS) of heparan sulphate (HS), a low molecular weight heparin (LMWH), and a reference preparation of unfractionated heparin (UH), was studied by activated partial thromboplastin time (APTT) and anti-Xa clotting assays. UH was most easily neutralised in the APTT assay by PS (on a weight for weight basis), followed by LMWH and HS. The neutralisation of APTT activity by PS closely followed the loss of activity in the anti-Xa clotting assay, when plasma was used as the source of At III. When the anti-Xa clotting assay was carried out using purified At III in place of plasma, HS and LMWH were neutralised by much lower amounts of PS and resembled UH neutralisation more closely. Resistance of HS anti-Xa activity to PS neutralisation decreased with increasing plasma dilution. The presence of bovine albumin with purified At III concentrate increased the resistance of HS to PS neutralisation. It is concluded that PS binding to UH, HS and LMWH is probably related more to their degree of sulphation than molecular weight and that non-specific interactions between PS and plasma proteins inhibit the binding of PS to HS and LMWH.

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