Concentrations Of Low Molecular Weight Heparin Research Articles

E0169 In vitro effects of low molecular weight heparin on clot rate

ObjectiveThis study was designed to determine the in vitro effects of different doses and different kinds of Low Molecular Weight Heparin (LMWH) on clot rate (CR), and to determine whether...

E0170 The sensitivity of different reagents for laboratory monitoring of low molecular weight heparin: an in vitro study

ObjectiveBecause of the lack of Point-of-care testing, the use of low-molecular-weight heparin (LMWH)therapy in some special patients is restricted. This study was designed to determine the in vitro sensitivity to...

Heptest-STAT, a new assay for monitoring of low-molecular-weight heparins, is not influenced by pregnancy-related changes of blood plasma

Monitoring of anti-factor Xa activity is often performed during treatment with low-molecular-weight heparins (LMWHs) in pregnancy because the anticoagulant effect may decrease as pregnancy progresses, but assays for anti-factor Xa activity are unavailable in many clinical institutions caring for pregnant women. Heptest-STAT is a new clotting assay for monitoring of LMWHs, which has been optimised for use in near-patient laboratory instrumentation. It has been suggested that monitoring of LMWHs requires the use of individual calibration curves for each LMWH. We compared the dose response of four conventional LMWHs and fondaparinux in normal plasma, and plasma from women in first, second and third trimester of pregnancy. Three concentrations of LMWHs, fondaparinux, or unfractionated heparin were added to pooled plasma samples from non-pregnant women (n=10), and pregnant women in first (n=10), second (n=10) and third (n=10) trimester of pregnancy. Heptest results are not influenced by the stage of pregnancy. In contrast, dose-related aPTT prolongation declines during pregnancy. All LMWHs tested, as well as fondaparinux, display a similar dose-response in Heptest compared to the chromogenic anti-factor Xa assay. Heptest-STAT can be used with the same standard calibration for non-pregnant and pregnant patients and for all LMWHs under investigation, including fondparinux. No individual calibrations are necessary.

Heparin-induced thrombocytopenia: A stoichiometry-based model to explain the differing immunogenicities of unfractionated heparin, low-molecular-weight heparin, and fondaparinux in different clinical settings

IntroductionHeparin-induced thrombocytopenia (HIT) is caused by platelet-activating antibodies that recognize platelet factor 4 (PF4)/heparin complexes. The frequency of HIT is highly variable in different clinical settings, and is more frequent with unfractionated heparin (UFH) than with low-molecular-weight heparin (LMWH), despite the in vitro observation that HIT antibodies activate platelets similarly well with LMWH as with UFH. An important difference between UFH, LMWH, and fondaparinux is their widely differing plasma concentrations. We aimed to provide a model that included anticoagulant concentrations and PF4 availability as risk factors influencing the anti-PF4/heparin immune response. Materials and methodsBy photon correlation spectroscopy we determined the concentrations at which UFH, LMWH, and fondaparinux form complexes optimally with PF4. Plasma concentrations of UFH and LMWH were calculated based on ex vivo pharmacokinetic data, with information on fondaparinux and PF4 concentrations taken from the literature. Results and conclusionsThe main features of our model are: optimal complex formation occurs at prophylactic-dose UFH and high PF4 levels, whereas therapeutic-dose LMWH concentrations are too high for optimal complex formation; in contrast, concentrations of fondaparinux are usually below the optimal stoichiometric range. Thus, immunization should occur more often in situations with major rather than minor platelet activation, and—for a given degree of platelet activation (PF4 availability)—as: prophylactic-dose UFH>therapeutic-dose UFH>prophylactic-dose LMWH, fondaparinux>therapeutic-dose LMWH. Our model provides a framework for explaining empirical observations that LMWH induces less anti-PF4/heparin antibodies than does UFH, and that anti-PF4/heparin antibodies are more often found in patients undergoing major surgery than in medical patients.

A MOUSE MODEL OF MATERNAL THROMBOPHILIAASSOCIATED FETAL LOSS: EFFICACY OF ANTICOAGULATION THERAPY

Introduction: Maternal thrombophilia, such as that caused by the Leiden polymorphism in blood coagulation factor V, contributes to the pathogenesis of fetal loss and other pregnancy disorders. Clinical trials suggest that heparin anticoagulation mitigates pregnancy failure in prothrombotic mothers. Due to multifactorial nature of the disorder, its uncertain etiology and the lack of established criteria for risk stratification, prophylactic anticoagulation during pregnancy is a subject of intense debate. We describe a mouse model of pregnancy disorder in factor V Leiden mothers, in which fetal loss is triggered when maternal thrombophilia coincides with fetal gene defects that reduce activation of the protein C anticoagulant pathway within the placenta. Fetal loss is caused by a disruption of placental morphogenesis at the stage of labyrinth layer formation, and occurs in the absence of overt thrombosis. Platelet depletion or genetic elimination of thrombin receptor Par4 from the mother allows normal placentation and prevents fetal loss demon- strating a critical role of maternal platelets in this phenomenon. We exploit this model to test the efficacy of anti-thrombotic treatments in the prevention of fetal loss. Methods and Results: We tested three different anticoagulants; hirudin, low molecular weight heparin (LMWH) and the synthetic pentasaccharide, Fondaparinux. Pregnant females were continuously infused with the anticoagulant through subcutaneously implanted pumps from day 5.5 of pregnancy. Each of these treatments effectively anticoagulated pregnant females, as measured by significant prolongations in partial thromboplastin times (PTT). However, only LMWH treatment afforded rescue from fetal loss. We evaluated the possibility that LMWH rescues pregnancies via inhibition of the complement pathway rather than anticoagulation. Daily bolus subcutaneous injections of 30 or 100 microgram LMWH, previously shown to block complement activation, were ineffective in maintaining an anticoagulant state in the animals and in preventing fetal loss. Conclusions: Our data suggests that although genetic absence of Par4 from maternal platelets rescues pregnancies, anticoagulation therapies that target thrombin or free Xa activity are ineffective in preventing fetal loss. The absence of rescue by these treatments, despite high sustained anticoagulant levels in maternal blood, may reflect their inability to inhibit the prothrombinase complex, resulting in localized availability of active thrombin on platelet surface in the milieu of the placenta. LMWH, on the other hand, affords protection from pregnancy loss in the mouse model, consistent with its reported efficacy in improving pregnancy outcome in prothrombotic women. Widely different pharmokinetics of this drug in humans and mice prevents direct comparison of doses used in human clinical trials and mouse studies. Anticoagulation independent activities of LMWH, such as inhibition of pselectin mediated platelet tethering to trophoblast cells operational at high LMWH concentrations, may be involved in rescue in the mouse model. The newly developed model of inherited thrombophilia-associated pregnancy disorder provides an opportunity to test these hypotheses and identify novel targets of therapeutic intervention. (platform)

Capillary zone electrophoresis characterization of low molecular weight heparin binding to interleukin 2

A method based on capillary zone electrophoresis (CZE) was used to study the interaction between low molecular weight heparin (LMWH) and interleukin 2 (IL-2). The results showed that the increase of the concentration of LMWH led to the decrease of the peak height and the increase of the peak width of IL-2, but the peak areas were kept constant. The binding constant of IL-2 with LMWH was calculated as 1.2 × 10 6 M −1 by Scatchard analysis, which is in good agreement with the results found in the references using enzyme-linked immunosorbent assay (ELISA). The results demonstrated that the interaction between IL-2 and LMWH is of fast on-and-off kinetic binding reaction. CZE might be used to study not only slow on-and-off rates interactions, but also fast on-and-off rates ones. The binding constant can be calculated easily, and the method can be applied to study a wide range of heparin–protein interactions.

Effect of 1,6-Anhydro Bicyclic Ring Structure on the Pharmacokinetic and Pharmacodynamic Behavior of Low Molecular Weight Heparin.

Introduction: Heparin cleavage under alkaline conditions results in low molecular weight heparin (LMWH) chains, a significant proportion of which contain 1,6-anhydromannosamine and/or 1,6-anhydroglucosamine at the reducing end. Despite the widespread use of the LMWHs for the prophylaxis and treatment of thrombosis, it remains unclear whether such structural modifications impact the pharmacologic activity of the drug. This study examined the in vitro anticoagulant and in vivo pharmacokinetic/pharmacodynamic (PK/PD) behavior of LMWHs containing varying levels of 1,6-anhydrosugar content.Materials and Methods: By altering the temperature and pH of the depolymerization reaction, LMWHs containing 0, 5, 10, 20 and 40% 1,6-anhydrosugar were produced. These compounds were supplemented to normal human plasma and normal primate plasma and assayed for anticoagulant (APTT and Heptest) and antiprotease (anti-IIa and anti-Xa) activity. The effect of 1,6-anhydrosugar on the PK/PD profile of LMWHs was assessed by administering the 40% 1,6-anhydro LMWH or enoxaparin (~20% 1,6-anhydrosugar) intravenously to groups of non-human primates (n=4–6) at a dose of 1 mg/kg. Blood samples were collected at baseline and at various time points up to 24 hours post-administration for determination of Heptest clotting times, anti-IIa and anti-Xa activity. The biologic activities were converted to equivalent LMWH concentrations using calibration curves prepared in normal primate plasma.Results: The molecular weight profiles of these LMWHs were comparable. No effect on anticoagulant or antiprotease activity was observed when the 1,6-anhydro content varied between 0 and 10%. When the 1,6-anhydro content was increased to 20 and 40%, a content-dependent reduction in anticoagulant activity was observed such that the prolongation of the APTT and Heptest by the 40% 1,6-anhydro LMWH was 58 and 23% less, respectively, than that produced by the LMWH lacking the 1,6-anhydro group when tested in the linear range of the concentration-response curve. This effect appears to be related primarily to an interference with antithrombin activity. Inhibition of thrombin activity in an amidolytic assay was 35% lower with the 40%-anhydro LMWH compared to the 0% anhydro compound (10 mg/ml), whereas anti-Xa activity was only 7% lower. Assay dependent variations were observed in the PK/PD profiles of the 40% anhydro LMWH and enoxaparin. As expected, the half-life of antithrombin activity was considerably shorter than that of the anti-Xa activity. The pharmacokinetic behavior of the 40% 1,6-anhydro LMWH and enoxaparin in terms of half-life, area under the curve, systemic clearance and volume of distribution was not significantly different when calculated using plasma concentrations determined by anti-IIa or anti-Xa assay. When concentrations determined by Heptest were used, the AUC determined for enoxaparin was approximately 2-fold higher than that determined with the 40% anhydro LMWH.Conclusions: Microchemical changes in the structure of low molecular weight heparin oligosaccharides can induce measurable changes in the biologic activity of LMWHs. While the pharmacokinetic profile does not appear to be altered by an enhanced 1,6-anhydro content, the effect of 1,6-anhydro content on the clinical efficacy and safety of LMWHs is unknown. Such findings may have particular impact on the development of generic LMWHs.

Heparin penetration into and permeation through human skin from aqueous and liposomal formulations in vitro

The transport of unfractionated (UH) and low molecular weight Heparin (LMWH) in human skin was investigated in vitro using heat separated epidermal membrane and dermis and the effect of liposomal formulations with Phospholipon® 80 (PL80) and Sphingomyelin (SM) was assessed. The distribution of Heparin within skin tissue was studied by the tape stripping method. Heparin concentrations were measured with a biological assay. Transepidermal water loss was determined to characterize barrier properties of skin. No consistent permeation of Heparin through epidermal membrane was detected. Penetration into the epidermal membrane was for LMWH significantly greater than for UH. Accumulation of UH was largely restricted to the outermost layers of the stratum corneum while LMWH penetrated into deeper epidermal layers. UH penetration into epidermis was detected for the PL80 liposomal formulation only. The extent of LMWH penetration was independent of the formulation, LMWH, however, showed a trend to accumulate in deeper epidermal layers for the PL80 compared to the aqueous formulation. Thus, molecular weight and liposomal formulations influenced the penetration pattern of Heparin in the epidermis. It can not be concluded whether the concentration of LMWH achieved at the blood capillaries is sufficient to exert a pharmacological effect. UH permeated readily through dermis irrespectively of formulation and its accumulation in the dermis was significantly enhanced and its lag time of permeation increased in the presence of SM liposomes.

Monitoring of low-molecular-weight heparins in cardiovascular disease

Thrombin generation is a key event in the pathophysiology of coronary syndromes and provides the rationale for treatment with anticoagulants. Unlike standard heparin, low-molecular-weight heparin (LMWH) has little effect on activated partial thromboplastin time. LMWH treatment has been monitored by measurement of anti-Factor Xa activity, but this may not accurately reflect the anticoagulant action because LMWHs also inhibit Factor II. The Heptest is a clotting assay that is sensitive to both anti-Xa and anti-IIa activity, as well as inhibition of the extrinsic pathway by LMWH-stimulated release of tissue factor pathway inhibitor. The plasma thrombin neutralization assay has also been used to measure LMWH and to detect low concentrations to which chromogenic assays are insensitive. In the clinical setting, monitoring the anti-Xa activity in patients treated with LMWH after acute deep vein thrombosis offered no advantages over a standard weight-adjusted dose. Moreover, in acute coronary syndromes there is no increase in major hemorrhage rates with weight-adjusted LMWH. Monitoring of LMWH concentrations may be advisable in the presence of comorbid conditions carrying an increased risk of hemorrhage, such as renal disease, advanced age, severe over- or underweight, or a history of previous bleeding episodes.

The Effects of Standard and Low Molecular Weight Heparin on Bone Nodule Formation In Vitro

Previously, we demonstrated in a rat model of heparin-induced osteoporosis that low molecular weight heparin (LMWH) produces less bone loss than unfractionated heparin, and that only heparin increases osteoclast number and activity. In contrast, both heparin and LMWH were found to decrease osteoblast function to a similar extent, possibly because at the doses tested both agents produced maximal inhibition. To examine the relative effects of heparin and LMWH on osteoblast function more closely we used an in vitro bone nodule assay, together with measurements of alkaline phosphatase (ALP) activity. Both agents inhibited bone nodule formation and ALP activity in a concentration-dependent manner, but 6 to 8-fold higher concentrations of LMWH were required to achieve equivalent effects. The effect of heparin on osteoblast function was both chain-length and negative charge-dependent because the ability of defined heparin fragments to inhibit nodule formation correlated with their molecular weight (r = 0.98), and N-desulfated heparin was less inhibitory than heparin. In contrast, the effect of heparin on osteoblast function was pentasaccharide-independent because heparin with low affinity for antithrombin had similar activity to heparin with high antithrombin activity. These findings help to explain mounting clinical evidence that the risk of osteoporosis is lower with LMWH than with heparin.

In vitro and ex vivo Activities of CY216: Comparison with Other Low Molecular Weight Heparins

Low molecular weight (LMW) heparins achieve their anticoagulant effects by inhibiting prothrombin activation, catalyzing endogenous thrombin inhibition, and binding thrombin. The extent of which each of the three actions of LMW heparins contributes to the antithrombotic effectiveness of LMW in man has not been defined. Several studies have reported that ex vivo anti-factor Xa activities of LMW heparins correlate with efficacy and potential haemorrhagic complications. However, critical review of the data suggests that anti-factor Xa assays are basically surrogate tests for the mass of LMW heparins in patients' plasmas rather than reliable estimates of total catalytic concentrations of LMW heparins. Antithrombotic effectiveness of LMW heparins probably reflects their ability to inhibit coagulation in vivo. If the last concept proves correct, then results of tests which directly assess the extents to which LMW heparins have suppressed in vivo coagulation may provide more reliable markers for their antithrombotic effectiveness or lack thereof.