Proton NMR spectroscopic studies on the interactions between human plasma antithrombin III and defined low molecular weight heparin fragments

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.

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Covalent Complexes Between Low Molecular Weight Heparin Fragments and Antithrombin III – Inhibition Kinetics and Turnover Parameters

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657333 ◽

1983 ◽

Vol 49 (02) ◽

pp. 109-115 ◽

Cited By ~ 4

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).

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

10.1055/s-0038-1643499 ◽

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.

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