scholarly journals Anticoagulant activities of heparin oligosaccharides and their neutralization by platelet factor 4

1984 ◽  
Vol 218 (3) ◽  
pp. 725-732 ◽  
Author(s):  
D A Lane ◽  
J Denton ◽  
A M Flynn ◽  
L Thunberg ◽  
U Lindahl
Keyword(s):  
Nitrous Acid ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Molecular Size ◽  
Protein Molecule ◽  
Platelet Factor 4 ◽  
Gel Chromatography ◽  
Platelet Factor ◽  
Thrombin Inhibition ◽  
Heparin Oligosaccharides

Oligosaccharides of well-defined molecular size were prepared from heparin by nitrous acid depolymerization, affinity chromatography on immobilized antithrombin III (see footnote on Nomenclature) and gel chromatography on Sephadex G-50. High affinity (for antithrombin III) octa-, deca-, dodeca-, tetradeca-, hexadeca- and octadeca-saccharides were prepared, as well as oligosaccharides of larger size than octadecasaccharide. The inhibition of Factor Xa by antithrombin III was greatly accelerated by all of these oligosaccharides, the specific anti-Factor Xa activity being invariably greater than 1300 units/mumol. The anti-Factor Xa activity of the decasaccharide was not significantly decreased in the presence of platelet factor 4, even at high platelet factor 4/oligosaccharide ratios. Measurable but incomplete neutralization of the anti-Factor Xa activities of the tetradeca- and hexadeca-saccharides was observed, and complete neutralization of octadeca- and larger oligo-saccharides was achieved with excess platelet factor 4. The octa-, deca-, dodeca-, tetradeca- and hexadeca-saccharides had negligible effect on the inhibition of thrombin by antithrombin III, whereas specific anti-thrombin activity was expressed by the octadeca-saccharide and by the larger oligosaccharides. An octadecasaccharide is therefore the smallest heparin fragment (prepared by nitrous acid depolymerization) that can accelerate thrombin inhibition by antithrombin III. The anti-thrombin activities of the octadecasaccharide and larger oligosaccharides were more readily neutralized by platelet factor 4 than were their anti-Factor Xa activities. These findings are compatible with two alternative mechanisms for the action of platelet factor 4, both involving the binding of the protein molecule adjacent to the antithrombin III-binding site. Such binding results in either steric interference with the formation of antithrombin III-proteinase complexes or in displacement of the antithrombin III molecule from the heparin chain.

scholarly journals Binding of platelet factor 4 to heparin oligosaccharides

1983 ◽  
Vol 209 (2) ◽  
pp. 455-460 ◽  
Author(s):  
J Denton ◽  
D A Lane ◽  
L Thunberg ◽  
A M Slater ◽  
U Lindahl
Keyword(s):  
Specific Activity ◽  
Gel Filtration ◽  
Factor Xa ◽  
Platelet Factor 4 ◽  
Platelet Factor ◽  
Physiological Conditions ◽  
Heparin Fractions ◽  
Specific Activities ◽  
Binding Sequence ◽  
Heparin Oligosaccharides

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.

scholarly journals Low Molecular Weight Heparin-Catalyzed Inactivation of Factor Xa and Thrombin by Antithrombin III – Effect of Platelet Factor 4

1991 ◽  
Vol 66 (04) ◽  
pp. 435-441 ◽  
Author(s):  
Pieter Schoen ◽  
Theo Lindhout ◽  
Jo Franssen ◽  
H Coenraad Hemker
Keyword(s):  
Molecular Weight ◽  
Rate Constants ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Factor Xa ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Platelet Factor ◽  
International Standard ◽  
Molecular Weights

SummaryLow molecular weight (LMW) heparin preparations have unknown distributions of ATIII-binding material, so mean molecular weights as such might bear little information on their anti-factor Xa and anti-thrombin activities, and on the neutralization of these activities by platelet factor 4 (PF4). These properties were investigated in pure systems with proteins of human origin. Pseudo-first order rate constants of inactivation of factor Xa and thrombin by antithrombin III were determined as function of heparin concentration, in the presence of 4.0 mM CaCl2. Despite a large variation in the mean molecular weights, the ratios of the anti-factor Xa over the anti-thrombin activities were essentially the same for the 4th International Standard for heparin (0.46), the 1st International Standard for LMW heparin (0.32), CY216 (0.42) and enoxaparin (0.50). The ultra LMW heparin CY222 had only a 2-times higher ratio (0.98). Analysis of CY216 subfractions, obtained by gel filtration, showed that the heparin molecules of the upper region of the molecular weight distribution are responsible for the anti-thrombin, but also to a large extent for the anti-factor Xa activities. The results indicate that depolymerization of unfractionated heparin does not result in an increased anti-factor Xa/anti-thrombin ratio, because in the presence of Ca2+-ions the rate constants of inactivation of factor Xa are lowered as compared to those of native heparin. PF4-dependent neutralization of anti-factor Xa and anti-thrombin activities of fixed concentrations of the LMW heparins was studied by measuring rate constants as function of PF4 concentration. All anti-thrombin and 50% of the anti-factor Xa activities were readily neutralized. Excess PF4 was required to neutralize another 35-50% of the anti-factor Xa activities. At PF4 levels obtained at maximal release of the content of platelet α-granules, all anti-thrombin and most (≥85%) of the anti-factor Xa activities can be neutralized.

The Influence Of Heparin, Phospholipid And Platelets On The Thrombin Or Factor Xa Inactivation By Antithrombin III

1981 ◽  
Author(s):  
F Ofosu ◽  
M Blajchman ◽  
A Cerskus ◽  
J Hirsh
Keyword(s):  
Vitamin K ◽  
Rate Constants ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Platelet Factor 4 ◽  
Detectable Effect ◽  
Platelet Phospholipid ◽  
Clotting Factors ◽  
Platelet Factor ◽  
Calcium Dependent

It has been known for many years that the effects of heparin are antagonized by platelets. This effect has generally been attributed to the release of platelet factor 4 (PF4). Heparin has recently been shown to disrupt the interactions of vitamin K-dependent clotting factors with phospholipid. This is due in part to the binding of heparin to phospholipid. The present investigations therefore explored the possible effects of phospholipid (cephalin) and degranulated platelets on the interaction of thrombin or factor Xa with antithrombin-III in the presence of heparin. These interactions were determined by the measurement of the second order rate constants for the inactivation of purified factor Xa or thrombin by purified antithrombin-III. The rates of inactivation of thrombin or factor Xa by antithrombin-III increased with increasing concentrations of heparin. Cephalin reduced the magnitude of the heparin effect on factor Xa but had no additional effect on thrombin inactivation. Relative to cephalin, gel filtered platelets reduced further the ability of heparin to enhance the inactivation of factor Xa by antithrombin-III. When the platelets were depleted of PF4 by degranulation with thrombin (2u/ml), the effects of platelets on the protease- antithrombin-III-heparin reactions approached those obtained with cephalin. However, no detectable effect on the inactivation of factor Xa or thrombin by antithrombin- III was demonstrable by gel filtered platelets, degranulated platelets, or cephalin in the absence of heparin. These results indicate that in addition to the contribution made by the release of PF4 by platelets, the calcium dependent binding of factor Xa to cephalin and platelet phospholipid is capable of protecting factor Xa from inactivation by antithrombin-III and heparin.

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.

Neutralization of Antithrombin VI (Fibrinogen Breakdown Products) with Platelet Antiheparin Factor (Platelet Factor 4)*

1965 ◽  
Vol 14 (03/04) ◽  
pp. 490-499 ◽  
Author(s):  
S Niewiarowski ◽  
R Farbiszewski ◽  
A Popławski
Keyword(s):  
Zinc Acetate ◽  
Antithrombin Iii ◽  
Platelet Factor 4 ◽  
Breakdown Product ◽  
Platelet Factor ◽  
Chromatography Column ◽  
Antiheparin Activity

SummaryIt has been found that fibrinogen breakdown product – antithrombin VI – is neutralized by the purified preparation of platelet factor 4, obtained by means of zinc acetate precipitation and DEAE chromatography column. It has been suggested that antiheparin activity of platelet factor 4 and its ability to neutralize antithrombin VI may be related to the same protein.The purified preparation of platelet factor 4 does not influence the fibrinogen – fibrin conversion by thrombin. This means that platelet factor 2 and platelet factor 4 are not the same substance.Crude platelet extracts neutralize antithrombin III and V. However, the purified product did not interferes with the action of these antithrombins.

scholarly journals The structure of heparin oligosaccharide fragments with high anti-(factor Xa) activity containing the minimal antithrombin III-binding sequence Chemical and13C nuclear-magnetic-resonance studies

1981 ◽  
Vol 197 (3) ◽  
pp. 599-609 ◽  
Author(s):  
B Casu ◽  
P Oreste ◽  
G Torri ◽  
G Zoppetti ◽  
J Choay ◽  
...  
Keyword(s):  
Uronic Acid ◽  
Antithrombin Iii ◽  
Factor Xa ◽  
Amino Sugar ◽  
Active Species ◽  
Model Compounds ◽  
High Affinity ◽  
The Common ◽  
Binding Sequence ◽  
Heparin Oligosaccharides

The chemical composition and the 13C n.m.r. spectra of heparin oligosaccharides (essentially octasaccharides), having high affinity for antithrombin III and high anti-(Factor Xa) activity, prepared by three independent approaches (extraction, partial deaminative cleavage with HNO2 and partial depolymerization with bacterial heparinase), leading to different terminal residues, have been studied and compared with those of the corresponding inactive species. Combined wit chemical data, the spectra of the active oligosaccharides and of their fragmentation products afforded information on composition and sequence. The three types of active oligosaccharides were shown to have the common hexasaccharide core I-Aa-G-As*-Is-As, where I and alpha-L-idopyranosyl-uronic acid, Aa = 2-acetamido-2-deoxy-alpha-D-glucopyranose, G = beta-D-glucopyranosyl-uronic acid, Is = alpha-L-idopyranosyluronic acid 2-O-sulphate, As = 2-deoxy-2-sulphamino-alpha-D-glucopyranose 6-O-sulphate. The fourth residue (As*) is an unusually substituted amino sugar resistant to mild deamination. The 13C spectra of the active species are characterized by signals from the above atypical amino sugar, the most evident of which is at 57.7 p.p.m. These signals, compared with those of appropriate synthetic model compounds, are compatible with the recently proposed 3-O-sulphation of the residue As* [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555].

scholarly journals Heparin-induced thrombocytopenia: in vitro studies on the interaction of dabigatran, rivaroxaban, and low-sulfated heparin, with platelet factor 4 and anti-PF4/heparin antibodies

Blood ◽  
2012 ◽  
Vol 119 (5) ◽  
pp. 1248-1255 ◽  
Author(s):  
Krystin Krauel ◽  
Christine Hackbarth ◽  
Birgitt Fürll ◽  
Andreas Greinacher
Keyword(s):  
Factor Xa ◽  
Platelet Factor 4 ◽  
Thrombin Inhibitors ◽  
Direct Thrombin Inhibitors ◽  
Platelet Factor ◽  
Heparin Induced Thrombocytopenia ◽  
Alternative Anticoagulation ◽  
Heparin Complexes ◽  
History Of

Abstract Heparin is a widely used anticoagulant. Because of its negative charge, it forms complexes with positively charged platelet factor 4 (PF4). This can induce anti-PF4/heparin IgG Abs. Resulting immune complexes activate platelets, leading to the prothrombotic adverse drug reaction heparin-induced thrombocytopenia (HIT). HIT requires treatment with alternative anticoagulants. Approved for HIT are 2 direct thrombin inhibitors (DTI; lepirudin, argatroban) and danaparoid. They are niche products with limitations. We assessed the effects of the DTI dabigatran, the direct factor Xa-inhibitor rivaroxaban, and of 2-O, 3-O desulfated heparin (ODSH; a partially desulfated heparin with minimal anticoagulant effects) on PF4/heparin complexes and the interaction of anti-PF4/heparin Abs with platelets. Neither dabigatran nor rivaroxaban had any effect on the interaction of PF4 or anti-PF4/heparin Abs with platelets. In contrast, ODSH inhibited PF4 binding to gel-filtered platelets, displaced PF4 from a PF4-transfected cell line, displaced PF4/heparin complexes from platelet surfaces, and inhibited anti-PF4/heparin Ab binding to PF4/heparin complexes and subsequent platelet activation. Dabigatran and rivaroxaban seem to be options for alternative anticoagulation in patients with a history of HIT. ODSH prevents formation of immunogenic PF4/heparin complexes, and, when given together with heparin, may have the potential to reduce the risk for HIT during treatment with heparin.

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 Platelet Factor 4 (PF4) and Protamine Neutralisation of Heparin in Plasma

1977 ◽  
Author(s):  
R. Michalski ◽  
D.A. Lane ◽  
D. Pepper ◽  
V.V. Kakkar
Keyword(s):  
Intravenous Injection ◽  
Factor Xa ◽  
Weight Basis ◽  
Platelet Factor 4 ◽  
Assay System ◽  
Platelet Factor ◽  
Protamine Sulphate ◽  
Heparin Activity ◽  
Plasma Heparin

The ability of PF4 and protamine sulphate to neutralise heparin in plasma has been studied using a specific anti-Factor Xa assay and a KCCT assay to measure residual heparin. When heparin is added to plasma in vitro PF4 and protamine neutralise almost equivalent amounts of heparin on a weight basis, 1.0 unit of heparin being neutralised by approximately 20 μg of PF4 and 15 μg of protamine. Similar results are obtained using either of the heparin assays. However, following intravenous injection of heparin only about one half of the circulating heparin could be neutralised in vitro by PF4 or protamine when it was measured by anti-Factor Xa assay. Total neutralisation was obtained with both neutralising agents in the KCCT assay system. These results demonstrate that the choice of assay is important when a protamine titration is used to measure plasma heparin levels, and that PF4 and protamine are unable to totally neutralise circulating antithrombotic heparin activity.

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