Significant Differences in Neutralization of Heparin and Its Low Molecular Fragments by Protamine Sulfate: An In Vivo Study.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1850-1850
Author(s):  
Mark A. Crowther ◽  
Klement Petr ◽  
Liao Peng ◽  
Chen Frank ◽  
Berry B. Leslie ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Factor Xa ◽  
In Vivo Model ◽  
Low Molecular Weight ◽  
Protamine Sulphate ◽  
Heparin Plasma ◽  
Optimal Regimen

Abstract In clinical practice, patients receiving low molecular weight heparin (LMWH) occasionally suffer bleeding. Protamine sulphate (PS) is often used to reverse the anticoagulant effect of LMWH in such cases. However, the optimal regimen of PS for complete neutralization of LMWH fragments has not been established. Results from our previous in vitro studies indicate that the ability of PS to neutralize LMWHs is inversely related to the charge of the low molecular weight heparin molecule; more heavily charged LMWHs (such as Tinzaparin) are more readily neutralized than less charged LMWHs (such as Enoxaparin). The aim of the current study was to confirm these findings using an in vivo model. Twenty minutes after administration of either saline, unfractionated heparin [UFH, 100U/kg], Tinzaparin [100U/kg] or enoxaparin [100U/kg], 50% of anesthetized rabbits received either saline or PS [1 mg/100 U of heparin or LMWH]. The efficacy of PS neutralization was assessed by serial measurements of anti-factor Xa heparin plasma levels. Results are presented as mean of the anti-factor Xa heparin activities normalized to the level at 10 minutes and summarized in the table below. As expected, PS completely neutralized the anti-factor Xa effect of UFH. However, PS was significantly less effective for neutralization of Tinzaparin (about 66%) and Enoxaparin (about 44%) at the dose tested. We conclude that when tested in an in vivo model LMWHs vary in their protamine neutralizability. More highly charged LMWHs (e.g. Tinazaparin) are more neutralizable than less highly charged products (e.g. Enoxaparin). Residual Anti-Xa heparin effect Time Enoxaparin Tinzaparin UFH Saline + PS Enoxaparin +PS Tinzaparin + PS UFH + PS 10 min 1.00 1.00 1.00 0 1.00 1.00 1.00 20 min 0.85 0.86 0.70 0 0.80 0.85 0.89 Protamine 25 min 0.71 0.75 0.69 0 0.45 0.29 0.01 35 min 0.68 0.64 0.48 0 0.40 0.28 0 50 min 0.48 0.32

Novel concatameric heparin-binding peptides reverse heparin and low-molecular-weight heparin anticoagulant activities in patient plasma in vitro and in rats in vivo

Blood ◽  
2004 ◽  
Vol 103 (4) ◽  
pp. 1356-1363 ◽  
Author(s):  
Barbara P. Schick ◽  
David Maslow ◽  
Adrianna Moshinski ◽  
James D. San Antonio
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Tandem Repeats ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
High Affinity ◽  
Binding Peptides

Abstract Patients given unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) for prophylaxis or treatment of thrombosis sometimes suffer serious bleeding. We showed previously that peptides containing 3 or more tandem repeats of heparin-binding consensus sequences have high affinity for LMWH and neutralize LMWH (enoxaparin) in vivo in rats and in vitro in citrate. We have now modified the (ARKKAAKA)n tandem repeat peptides by cyclization or by inclusion of hydrophobic tails or cysteines to promote multimerization. These peptides exhibit high-affinity binding to LMWH (dissociation constant [Kd], ≈ 50 nM), similar potencies in neutralizing anti–Factor Xa activity of UFH and enoxaparin added to normal plasma in vitro, and efficacy equivalent to or greater than protamine. Peptide (ARKKAAKA)3VLVLVLVL was most effective in all plasmas from enoxaparin-treated patients, and was 4- to 20-fold more effective than protamine. Several other peptide structures were effective in some patients' plasmas. All high-affinity peptides reversed inhibition of thrombin-induced clot formation by UFH. These peptides (1 mg/300 g rat) neutralized 1 U/mL anti–Factor Xa activity of enoxaparin in rats within 1 to 2 minutes. Direct blood pressure and heart rate measurements showed little or no hemodynamic effect. These heparin-binding peptides, singly or in combination, are potential candidates for clinical reversal of UFH and LMWH in humans.

Novel Design of Peptides to Reverse the Anticoagulant Activities of Heparin and other Glycosaminoglycans

2001 ◽  
Vol 85 (03) ◽  
pp. 482-487 ◽  
Author(s):  
Joel Gradowski ◽  
James San Antonio ◽  
Jose Martinez ◽  
Barbara Schick
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Heparin Binding ◽  
Antifactor Xa ◽  
Novel Design

SummaryPatients undergoing anticoagulation with unfractionated heparin, low molecular weight heparin, or danaparoid may experience excess bleeding which requires reversal of the anticoagulant agent. Protamine is at present the only agent available for reversal of unfractionated heparin. Protamine is not effective in patients who have received low molecular weight heparin or danaparoid. We have developed a series of peptides based on consensus heparin binding sequences (Verrecchio et al., J Biol Chem 2000; 275: 7701-7707) that are capable of neutralizing the anti-thrombin activity of unfractionated heparin in vitro, the antifactor Xa activity of unfractionated heparin, Enoxaparin (Lovenox) and danaparoid (Orgaran) in vitro and the anti-Factor Xa activity of Enoxaparin in vivo in rats. These peptides may serve as alternatives for Protamine reversal of UFH and may be useful for neutralization of enoxaparin and danaparoid in humans.

Studies in Man and Experimental Animals of a Low Molecular Weight Heparin Fraction

1981 ◽  
Vol 45 (03) ◽  
pp. 214-218 ◽  
Author(s):  
D P Thomas ◽  
R E Merton ◽  
W E Lewis ◽  
T W Barrowcliffe
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Specific Activity ◽  
Ex Vivo ◽  
High Specific Activity ◽  
Factor Xa ◽  
In Vivo Studies ◽  
Low Molecular Weight

SummaryIn vitro and in vivo studies were carried out on a commercially prepared low molecular weight heparin fraction. By APTT assay the fraction had a specific activity of half that of unfractionated mucosal heparin, yet retained full potency by anti-Xa assay (both clotting and chromogenic substrate). When administered intravenously to human volunteers, the anti-Xa/APTT ratio remained the same as it was in vitro. However, after subcutaneous injection, the ratio increased and anti-Xa activity could not be fully neutralized ex vivo by PF4. The fraction was as effective as unfractionated heparin in preventing experimental serum-induced thrombosis, suggesting that a heparin fraction with high specific activity by anti-Factor Xa assay compared to APTT activity may be an effective drug for the prophylaxis of venous thrombosis.

Effects of Unfractionated and Low Molecular Weight Heparins on Platelet Thromboxane Biosynthesis “In Vivo”

1994 ◽  
Vol 72 (06) ◽  
pp. 942-946 ◽  
Author(s):  
Raffaele Landolfi ◽  
Erica De Candia ◽  
Bianca Rocca ◽  
Giovanni Ciabattoni ◽  
Armando Antinori ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Low Molecular Weight Heparin ◽  
Primary Source ◽  
In Vivo Studies ◽  
Low Molecular Weight ◽  
Heparin Administration ◽  
To Receive

SummarySeveral “in vitro” and “in vivo” studies indicate that heparin administration may affect platelet function. In this study we investigated the effects of prophylactic heparin on thromboxane (Tx)A2 biosynthesis “in vivo”, as assessed by the urinary excretion of major enzymatic metabolites 11-dehydro-TxB2 and 2,3-dinor-TxB2. Twenty-four patients who were candidates for cholecystectomy because of uncomplicated lithiasis were randomly assigned to receive placebo, unfractionated heparin, low molecular weight heparin or unfractionaed heparin plus 100 mg aspirin. Measurements of daily excretion of Tx metabolites were performed before and during the treatment. In the groups assigned to placebo and to low molecular weight heparin there was no statistically significant modification of Tx metabolite excretion while patients receiving unfractionated heparin had a significant increase of both metabolites (11-dehydro-TxB2: 3844 ± 1388 vs 2092 ±777, p <0.05; 2,3-dinor-TxB2: 2737 ± 808 vs 1535 ± 771 pg/mg creatinine, p <0.05). In patients randomized to receive low-dose aspirin plus unfractionated heparin the excretion of the two metabolites was largely suppressed thus suggesting that platelets are the primary source of enhanced thromboxane biosynthesis associated with heparin administration. These data indicate that unfractionated heparin causes platelet activation “in vivo” and suggest that the use of low molecular weight heparin may avoid this complication.

Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

1986 ◽  
Vol 56 (03) ◽  
pp. 318-322 ◽  
Author(s):  
V Diness ◽  
P B Østergaard
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Thrombus Formation ◽  
Experimental Models ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

scholarly journals Inhibition of Plasmin Generation in Plasma By Heparin, Low Molecular Weight Heparin and a Covalent Antithrombin-Heparin Complex (ATH)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4217-4217
Author(s):  
Gabriela Chang ◽  
Helen M. Atkinson ◽  
Leslie R. Berry ◽  
Anthony K.C. Chan
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Low Molecular Weight Heparin ◽  
Conflicts Of Interest ◽  
Area Under The Curve ◽  
Low Molecular Weight ◽  
Plasminogen Activator Inhibitor 1 ◽  
Significant Difference

Abstract Introduction: Unfractionated heparin (UFH) and low molecular weight heparin (LMWH) are widely used anticoagulants for thrombosis treatment. However, these anticoagulants have limitations such as increased bleeding, variable dose response, required frequent monitoring, and, in the case of LMWH, inability to inhibit thrombin. This has led to the development of a covalent complex of antithrombin and heparin (ATH), which has been shown to overcome many of these shortcomings. ATH has faster rates of inhibition of many coagulation factors, is able to inhibit clot-bound thrombin, and is a more effective inhibitor of both venous and arterial thrombosis in animal models. Moreover, in a rabbit thrombosis model, ATH has been shown to decrease clot mass and fibrin accretion, while the contrary was observed for UFH. From these observations, it was suggested that ATH may enhance fibrin breakdown and thus led to investigations into the effects of UFH and ATH on fibrinolysis. In vitro studies have shown that UFH enhances antithrombin inhibition of plasmin. In addition, ATH displays a slightly greater inhibition of plasmin generation and activity. Such studies were conducted in purified systems, in the absence of other plasmin inhibitors naturally present in plasma. Therefore, the aim of the present study was to compare the effects of UFH, LMWH, and ATH on plasmin generation in plasma. Methods: At 37°C tissue plasminogen activator (tPA) and soluble fibrin fragments (fib) were added to normal adult pooled platelet poor plasma supplemented with 0.35, 0.7, 1.4, or 2.1 U anti-Xa/ml UFH, LMWH, or ATH, to initiate plasmin generation (8.93nM tPA and 300µg/ml fib). At various time points, subsamples were mixed with excess plasminogen activator inhibitor 1 (PAI-1) (55.12nM) to stop further plasmin generation. The plasmin concentration at each time point was determined using a plasmin-specific chromogenic substrate and a standard curve produced from purified plasmin. Results: Comparisons of mean area under the curve (AUC) for plasmin generation displayed a significant decrease in plasmin generation in the presence of all three anticoagulants at all doses tested (p<0.05). Comparing the anticoagulants at similar doses, plasmin generation was significantly decreased in the presence of ATH (15384.66±1930.23nM/min) compared to LMWH (23892.28±3090.54nM/min) at 0.7 U/ml (p<0.05). At a dose of 1.4 U/ml, there was significantly less plasmin generated, over time, in the presence of UFH (20089.49±3022.1623nM/min) and ATH (19273.86±1805.7323nM/min) when compared to LMWH (24743.18±1265.1023nM/min) (p<0.05). There was no significant difference in plasmin inhibition between UFH and ATH at any of the doses tested. Conclusion: The present study supports previous findings that UFH and ATH can facilitate antithrombin inhibition of plasmin. It is also observed that LMWH catalyzes the inhibition of plasmin by antithrombin but possibly to a lesser extent. These findings suggest that ATH has a similar inhibitory effect on plasmin generation and activity in plasma compared to UFH, despite its overall superior anticoagulant properties. Therefore, previous in vivo observations displaying decrease in clot mass with administration of ATH was due to its enhanced anticoagulant abilities and not fibrinolysis enhancement. These findings add to our understanding of ATH mechanisms of action and aid in its development for clinical use. Disclosures No relevant conflicts of interest to declare.

PHARMACOKINETICS OF A LOW MOLECULAR WEIGHT HEPARIN (KABI 2165, FRAGMIN) ATFER INTRAVENOUS AND SUBCUTANEOUS ADMINISTRATION IN HUMAN VOLUNTEERS AND ITS IN VIVO NEUTRALIZATION BY PROTAMINE SULFATE

1987 ◽  
Author(s):  
J Albada ◽  
K K Nieuwenhuis ◽  
J J Sixma
Keyword(s):  
Molecular Weight ◽  
Healthy Volunteers ◽  
Half Life ◽  
Low Molecular Weight Heparin ◽  
Subcutaneous Administration ◽  
Low Molecular Weight ◽  
Protamine Sulphate ◽  
Original Curve ◽  
Rebound Phenomenon

Pharmacokinetics of a low molecular weight heparin (LMWH) were studied in healthy volunteers. After an intravenous bolus injection of 5000 anti-Xa U in 5 healthy volunteers anti Xa activity disappeared according to the combination of saturable and a linear mechanism, preceded by a rapid initial disappearance. The apparent half-life of the anti Xa activity is about twice as long as that of standard heparin. In another set of experiments 5000 anti Xa U of LMWH were immediately followed by 50 mgr of Protamine Sulphate (PS). The curve of the anti Xa-activity parallelled the original curve at a level of about 30-40%. No rebound phenomenon was observed. The same dose of the LMWH followed by 100 mg of PS resulted in an anti Xa disappearance curve at an obvious higher level of about 50%. Also at this dose no rebound phenomenon was noticed.A continuous infusion of 10.000 anti Xa U/24 h during 10 hours was followed by 15.000 anti Xa U/24 h for another 10 hours after which the dose was raised to 20.000 anti Xa U/24 h for another 10 hours. Only the first infusion period resulted in a plateau fase. At the end of these experiments anti Xa activity was neutralized by 50 mg P.S. i.v. resulting in the disappearance of less than 50% of anti Xa activity. After subcutaneous administration of 15.000 anti Xa U (corresponding to the dose for i.v. treatment per day with this LMWH) peak levels of 1,1-1,8 anti Xa were reached after 3-4 hours. Supra-optimal anti Xa levels (higher than 0.9) were observed in all volunteers during a period of 5 hours. After 24 hours in none of the volunteers any anti Xa-activity could be detected.Conclusions:In contrast to previous reports pharmacokinetics of this LMWH do not essentially differ from those of standard heparin apart from its longer half-life and its high bioavialability after subcutaneous injection.

Protamine neutralization of a very low molecular weight heparin fragment CY 222 in vitro and in vivo study

1986 ◽  
Vol 41 ◽  
pp. 88 ◽  
Author(s):  
C. Doutremepuich ◽  
F. Bonini ◽  
O. de Seze ◽  
A. Masse ◽  
F. Toulemonde ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
In Vivo Study ◽  
Low Molecular Weight

PHARMACOLOGIC INEQUIVALENCE OF LOW MOLECULAR WEIGHT HEPARINS

1987 ◽  
Author(s):  
J Fareed ◽  
J M Walenga ◽  
D Hoppensteadt ◽  
R N Emanuele ◽  
A Racanell
Keyword(s):  
Molecular Weight ◽  
Animal Models ◽  
Comparative Study ◽  
Factor Xa ◽  
Low Molecular Weight ◽  
Low Molecular Weight Heparins ◽  
In Vivo Test ◽  
In Vitro Effects

Compared to unfractionated heparin, low molecular weight heparins (LMWHs) have been found to exhibit marked variations in in vitro effects due to variations in molecular weight and structure. Moreover, when the in vitro potency of these agents is equally adjusted bypharmacopeial assay (current and proposed) wide variations in the in vivo responses have been noted. These variations were strongly dependent on the route of administration. Utilizing defined animal models, a systematic comparative study of the in vivo responses of seven commercial LMWHs was undertaken. Choay Fraxiparine (CY 216} Choay CY 222, NovoLHN, Kabi Fragmin, Opocrin 2123 (OP), Hepar RD 11885 (RD), Pharmuka Enoxaparin (PK) and Choay porcine mucosal heparin (PMH) were tested in identical settings at equigravimetric dosages. The graded results are given in the following.Wide variations in the in vivo pharmacologic and toxicity responseswere noted suggesting that different LMWHs are not bioequivalent at equigravimetric levels. When these responses were expressed in anti-factor Xa or pharmacopeial potency, these differences were further magnified. The clinically reported dosimetric and safety problems may be minimized by profiling LMWHs in defined in vivo test systems to optimize their safety/efficacy ratio.

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