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.

Investigations of the Immunoglobulin Subtype Transformation of Anti–Heparin-Platelet Factor 4 Antibodies During Treatment With a Low-Molecular-Weight Heparin (Clivarin) in Orthopedic Patients

2003 ◽  
Vol 127 (5) ◽  
pp. 584-588
Author(s):  
Sarfraz Ahmad ◽  
H. Peter Bacher ◽  
Michael R. Lassen ◽  
Debra A. Hoppensteadt ◽  
Helen Leitz ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Serotonin Release ◽  
Platelet Factor 4 ◽  
Enzyme Linked Immunosorbent Assay ◽  
Low Molecular Weight ◽  
Platelet Factor ◽  
Significant Difference ◽  
Antibody Titers ◽  
Orthopedic Patients

Abstract Context.—It is now widely accepted that the pathophysiology of heparin-induced thrombocytopenia (HIT) syndrome is mediated by the generation of a wide array of functional and molecularly heterogeneous anti–heparin-platelet factor 4 (AHPF4) antibodies that may mediate platelet and/or endothelial cell activation/destruction. Objective.—We investigated the differential prevalence and functionality of AHPF4 immunoglobulin subtypes (IgA, IgG, and IgM) in plasmas obtained from orthopedic patients immobilized with Plaster-Cast and treated with clivarin (a low-molecular-weight heparin) in comparison to a placebo for the prophylaxis of deep-vein thrombosis. Design and Methods.—Clivarin was administered subcutaneously at a fixed daily dosage of 1750 U without any adjustment or loading dosage. Citrated plasmas were obtained at baseline, at 10 to 14 days, and at postbrace procedure (5–12 weeks). An enzyme-linked immunosorbent assay (ELISA) was used to quantitate the AHPF4 antibody titers. The functionality of the ELISA-positive samples was determined by a 14C-serotonin release assay (SRA). Results.—In the ELISA test, 16 of 1073 samples (1.5%; 6 in clivarin and 10 in placebo groups) were positive for AHPF4 antibodies (mean optical density [OD] = 0.46 ± 0.02). None of the ELISA-positive samples for AHPF4 antibodies could mediate platelet activation responses as determined by the SRA (0%–3% serotonin release, P > .10, n = 16). Through differential immunoglobulin subtype analysis of the samples positive for (cumulative) AHPF4 antibodies, we determined that their relative prevalence in plasma were as follows: IgM (mean OD = 0.71 ± 0.13) > IgG (0.31 ± 0.08) > IgA (0.14 ± 0.02). Although there was no significant difference in the total antibody titers between clivarin and placebo groups, the antibody subtyping data showed conversion trends (ie, IgA [clivarin to placebo], IgG [placebo to clivarin], and IgM [clivarin to placebo]). Conclusion.—These observations indicate that even at reduced dosages, clivarin can shift the immunogenic up-regulation toward the IgG subpopulation; however, the IgG subtype is of a nonfunctional type of AHPF4 antibody and thus may not cause any HIT-related pathogenic responses.

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 Heparin-Mobilisable Pool of Platelet Factor 4: A Comparison of Intravenous and Subcutaneous Heparin and Kabi Heparin Fragment 2165

1985 ◽  
Vol 54 (04) ◽  
pp. 735-738 ◽  
Author(s):  
J R O’Brien ◽  
M D Etherington ◽  
Michelle A Pashley
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
Platelet Factor ◽  
Once Daily ◽  
Subcutaneous Heparin

SummarySome clinical advantages are claimed for low molecular weight heparin so the mobilisation of platelet factor 4 (PF 4) from the endothelial pool by the heparins may be relevant. Unfractionated (UF) heparin has been compared with Kabi heparin fragment 2165. A single intravenous (i. v.) injection of 60 iu/kg heparin was compared with 5000 anti-Xa units of Kabi-2165. Less PF 4 was mobilised by Kabi-2165 and some apparently remained in the pool and was released when the pool was subsequently challenged by giving i.v. heparin. Subcutaneous (s. c.) injections of 5000 iu heparin twice daily were compared with 5000 anti-Xa units of Kabi-2165 once daily, each given for a week. The plasma PF 4 was never raised yet when finally challenged with i.v. heparin the pool was “empty” or refractory after the s.c. heparin but some PF 4 remained after the s.c. Kabi-2165. The two glycosaminogly-cans (GAGs) had widely differing half-lives but the t/2 of the PF 4 mobilised by the two GAGs was similar even though the PF 4 is apparently bound to the GAG.

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.

scholarly journals The Mode of Action of Low Molecular Weight Heparin Preparation (PK10169) and Two of its Major Components on Thrombin Generation in Plasma

1989 ◽  
Vol 61 (01) ◽  
pp. 030-034 ◽  
Author(s):  
S Béguin ◽  
J Mardiguian ◽  
T Lindhout ◽  
H C Hemker
Keyword(s):  
Molecular Weight ◽  
Mode Of Action ◽  
Low Molecular Weight Heparin ◽  
Thrombin Generation ◽  
Platelet Rich Plasma ◽  
Weight Fraction ◽  
Platelet Factor 4 ◽  
Low Molecular Weight ◽  
High Molecular Weight Fraction ◽  
Platelet Factor

SummaryWe studied the mode of action of the low molecular weight heparin PK10169 and two of its constituent fractions: EMT 966 High Molecular Weight Fraction and EMT 967 Low Molecular Weight Fraction.EMT 966 like standard heparin, acts primarily on thrombin formed and not on prothrombinase (S type heparin). In contrast EMT 967 has no direct effect on thrombin. At high concentrations, it inhibits the prothrombinase complex (P type heparin). PK10169, that contains the two EMTs shows both activities: anti thrombin and antiprothrombinase (mixed type heparin).The addition of increasing amounts of EMT 967 to a constant amount of EMT 966 does not influence the breakdown constant of endogenous thrombin which is determined by the concentration of EMT 966 only. This demonstrates the absence of competition for AT III between the two components of PK10169.In platelet rich plasma, EMT 966 inhibits and postpones thrombin generation more efficiently than unfractionated heparin, probably because it is less sensitive to neutralization by platelet components (platelet factor 4). Amounts of EMT 967 that hardly inhibit thrombin generation in platelet rich plasma enhance the effect of EMT 966 probably by neutralizing platelet factor 4.

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