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.

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.

EFFECT OF VARIOUS HEPARIN(OID)S ON HEPARIN COFACTOR II MEDIATED ANTI-THROMBIN ACTIVITY AND INHIBITION OF THROMBIN GENERATION IN VITRO

1987 ◽  
Author(s):  
T G van Dinther ◽  
F Hol ◽  
D G Meuleman
Keyword(s):  
Molecular Weight ◽  
Thrombin Generation ◽  
Factor Xa ◽  
Weight Fraction ◽  
Blood Platelet ◽  
Low Molecular Weight ◽  
Heparin Cofactor Ii ◽  
Thrombin Activity ◽  
At Iii

The effects of various heparin(oid)s, standard heparin VII (SH), dermatan sulphate (DS), a low molecular weight fraction of heparin (UMW-H), FragminR (FRA), Org 10172 = low molecular weight heparinoid, the fraction of Org 10172 with high affinity for AT-III (HA-10172) and the low affinity fraction (LA-10172) respectively were examined on in vitro thrombin generation and inactivation.Thrombin inactivation in the presence of either heparin cofactor II (HC-II) or anti-thrombin III (AT-III) was assessed with two newly developed assays using the purified cofactors, thrombin and chromogenic substrate S2238 on microtiterplates. Thrombin generation in the presence of HC-II and AT-III was studied using purified factor Xa, prothrombin and blood platelet lysate and the residual thrombin activity was assessed amidolytically.The inhibition of the compounds on thrombin activity are summarized in the tableThe following conclusions can be drawn:- SH, LMW-H, HA-10172 and FRA potentiate the AT-III mediated inactivation of Ha more strongly than the HC-II mediated inactivation.- DS and LA-10172 show the reverse pattern of inactivation, while Org 10172 potentiates both inactivaton pathways to a similar extent.Thrombin generation in the presence of HC-II is inhibited by mw-heparin(oid)s at approx. 2-5 times lower concentrations than the HC-II mediated thrombin inactivation, while the inhibiting effect of SH in both assays is comparable.AT-III mediated thrombin generation inhibition and AT-III mediated thrombin inactivation is comparable as well for SH, LMW-H and FRA. In contrast, Org 10172 and its subfractions are approx. 10 times more potent on AT-III mediated thrombin generation inhibition than on AT-III mediated thrombin inactivation.Org 10172 shows low anti-thrombin activity and this activity is mainly mediated via FC-II.

The Additive Effect of Low Molecular Weight Heparins on Thrombin Inhibition by Dermatan Sulfate

1993 ◽  
Vol 70 (03) ◽  
pp. 443-447 ◽  
Author(s):  
Benilde Cosmi ◽  
Giancarlo Agnelli ◽  
Edward Young ◽  
Jack Hirsh ◽  
Jeffrey Weitz
Keyword(s):  
Molecular Weight ◽  
Antithrombin Iii ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Additive Effect ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
Heparin Cofactor Ii ◽  
Thrombin Inhibition ◽  
Sds Page

SummaryThe aim of this study was to investigate the mechanism by which the anticoagulant activity of dermatan sulfate (DS) is increased by low molecular weight heparin (LMWH). In platelet poor plasma, LMWH enhances the effect of DS on thrombin (IIa) inhibition as determined by thrombin clotting times and with a chromogenic substrate assay. Analysis of the results of the chromogenic assays using either the algebraic fractional or the graphic isobole method suggests that LMWH has an additive effect on the anti-IIa activity of DS. This additive effect was lost when the experiments were repeated in plasma immunodepleted of antithrombin III (ATIII), indicating that the anti-IIa activity of LMWH is ATIII-dependent. To further explore the mechanism of the interaction between LMWH and DS, 125I-labeled IIa was added to plasma in the presence or absence of DS and/or LMWH and the formation of IIa-inhibitor complexes was assessed using SDS-PAGE followed by autoradiography. DS addition selectively increases the formation of heparin cofactor II (HCII)-IIa complexes, whereas LMWH enhances ATIII-IIa complex generation. Compared to plasma containing DS alone, the formation of ATIII-IIa complexes also is increased when the combination of DS and LMWH is added. These findings suggest that the additive effect of LMWH on the anti-IIa activity of DS reflects their different modes of IIa inhibition; DS potentiates IIa inhibition by HCII, while LMWH catalyses ATIII-dependent IIa inactivation. The potential clinical significance of these findings requires further investigation.

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.

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.

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.

Circulating Activities During Constant Infusion of Heparin or a Low Molecular Weight Derivative (Enoxaparine): Failure to Demonstrate any Circadian Variations

1987 ◽  
Vol 58 (04) ◽  
pp. 1068-1072 ◽  
Author(s):  
P Toulon ◽  
J F Vitoux ◽  
C Leroy ◽  
T Lecomte ◽  
M Roncato ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Circadian Rhythm ◽  
Biological Activities ◽  
Constant Infusion ◽  
Low Molecular Weight ◽  
Circadian Variations ◽  
Heparin Cofactor Ii ◽  
Heparin Infusion ◽  
Chromogenic Assay ◽  
Heparin Affinity

SummaryWe compared in six patients successively treated with an unfractionated heparin (UFH) and a low molecular weight heparin (LMWH) the variations in plasma anti-Xa activity, measured in a chromogenic assay, during a 36 h constant infusion. The values varied in a wider range during UHF infusion, but remained in the therapeutic range except once in one patient. No circadian rhythm could be demonstrated in our six patients. LMWH infusion yielded very constant anti-Xa circulating activities. In both cases, there were no significant modifications of three proteins with high heparin affinity (antithrombin III, heparin cofactor II, histidine-rich glycoprotein).Our results suggest that the circadian rhythm of the biological activities previously observed in patients treated with constant heparin infusion using clotting method is due to other factors than heparin itself.

Antithrombin III and Heparin Cofactor II in Patients with Chronic Renal Failure Undergoing Regular Hemodialysis

1987 ◽  
Vol 57 (03) ◽  
pp. 263-268 ◽  
Author(s):  
P Toulon ◽  
C Jacquot ◽  
L Capron ◽  
M -O Frydman ◽  
D Vignon ◽  
...  
Keyword(s):  
Renal Failure ◽  
Chronic Renal Failure ◽  
Plasma Proteins ◽  
Antithrombin Iii ◽  
Vascular Wall ◽  
Relative Increase ◽  
Heparin Cofactor Ii ◽  
Normal Ranges ◽  
At Iii ◽  
Regular Hemodialysis

SummaryHeparin enhances the inhibition rate of thrombin by both antithrombin III (AT III) and heparin cofactor II (HC II). We studied the activity of these two plasma proteins in patients with chronic renal failure (CRF) undergoing regular hemodialysis as their heparin requirements varied widely. In 77 normal blood donors, normal ranges (mean ± 2 SD) were 82-122% for AT III and 65-145% for HC II. When compared with these controls 82 dialyzed CRF patients had a subnormal AT III activity and a significantly (p <0.001) lower HC II activity. To evaluate the effect of hemodialysis we compared AT III, HC II and total proteins in plasma before and after dialysis in. 24 patients (12 with normal and 12 with low basal HC II activity). AT III and HC II activities significantly (p <0.001) increased in absolute value. When related to total plasma proteins, in order to suppress the influence of hemoconcentration induced by dialysis, AT III decreased significantly (p <0.01) whereas HC II increased slightly but significantly (p <0.01) in the 12 patients with low initial HC II activity. The decrease of AT III induced by heparin administrated during dialysis is likely to account for this relative decrease of AT III activity. A modification of the distribution of both HC II and heparin between the vascular wall and the circulating blood is evoked to explain the relative increase in HC II activity and the need for higher heparin dosage in patients with low HC II levels.

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.

Sign in / Sign up

Export Citation Format

Share Document