Low Molecular Weight Heparin Preparations Research Articles

A retrospective review on importance and various preparation of low molecular weight heparin for cardio vascular diseases

Low-molecular-weight heparin (LMWH) are most importantly used in the clinical practice for cardio vascular diseases from 1990. The LMWH was a sulfated polysaccharide obtained from animal sources and some in natural especially from marine sources. The actual anticoagulant has the molecular weight about 25000 Daltons which have bleeding as side effect in the cardiovascular diseases. The LMWH are prepared by enzymatic or chemical hydrolysis the length of the heparin chain is reduced and also in same manner the molecular weight also reduced below 10000 Daltons. So, it has main advantage to reduce the bleeding in the cardiovascular diseases. The LMWH eliminated through the renal and it was not given to the patients with renal dysfunction. The LMWH of some product are still in clinical trials in order to reduce its side effects. The commercial LMWH preparation has concentrated in animal sources by killing them and lungs, intestine etc are used to prepare heparin. Here the alternate sources are discussed in order from killing the animal. This review summarizes the importance, difference between commercial heparin and LMWH, mechanism, preparation, LMWH products, clinical trials and LMWH from marine sources, Enzymatic degradation in shrimp species, Preparation of LMW heparin by chemically modified Fractions, Chromatography separation of LMW Heparin are discussed in this review.

Comparative Analysis of Two Enoxaparin Sodium Drugs by Heptest.

The optimal conditions for analysis of low-molecular-weight heparin (LMWH) drugs by the Heptest and the metrological characteristics of the Heptest method were determined. The effects of two LMWH preparations of enoxaparin sodium, the domestic drug Enoxaparin sodium (Technologiya Lekarstv, Russia) and the original drug Clexane (Sanofi, France), on the clotting time of human plasma in the Heptest were comparatively analyzed. The mean blood clotting times were calculated for each dilution of each series of Enoxaparin sodium and Clexane based on the results of the Heptest. Graphs of the dependence of the plasma clotting time on the degree of dilution of these drugs were plotted. The shapes of the curves of the dependence of the blood plasma clotting time on the activity of enoxaparin sodium were shown to be similar for both Enoxaparin sodium and Clexane. The Heptest method showed that Enoxaparin sodium (Technologiya Lekarstv, Russia) was comparable to the original drug Clexane (Sanofi, France).

Practice of pharmaceutical thrombosis prophylaxis and anticoagulation in patients with sepsis and pre-existing anticoagulation or heparin-induced type II thrombocytopenia-Results of a nationwide survey in German intensive care units

HintergrundSowohl eine vorbestehende Antikoagulation als auch prädisponierende Vorerkrankungen für Thromboembolien stellen ein häufiges Problem bei Patienten mit Sepsis dar, wenngleich der Umgang mit diesen Rahmenbedingungen von den aktuellen nationalen und internationalen Leitlinien für Sepsis und septischen Schock nicht adressiert wird. Ein Ziel der hier vorliegenden deutschlandweiten Umfrage war es, den Umgang von Intensivmedizinern mit derartigen Problemen zu eruieren.MethodenVon Oktober 2019 bis Mai 2020 führten wir eine deutschlandweite Umfrage unter ärztlichen Leitern von Intensivstationen zum Thema Antikoagulation und medikamentöser Thromboseprophylaxe bei Sepsis und Sepsis-induzierter Koagulopathie durch. Ein Fokus war hierbei, das Vorgehen bei Patienten mit vorbestehender Indikation zur therapeutischen Antikoagulation sowie bei vorbekannter Heparin-induzierter Thrombopenie Typ II (HIT-II) (akut-symptomatisch vs. Jahre zurückliegend) zu eruieren.ErgebnisseDie Umfrageergebnisse zeigen, dass auf den meisten der teilnehmenden Intensivstationen eine vorbestehende Antikoagulation größtenteils mit niedermolekularen Heparinpräparaten oder unfraktioniertem Heparin fortgeführt wird. Bei bekannter HIT-II wird Argatroban bevorzugt, unabhängig davon, ob es sich um eine Jahre zurückliegende oder eine akut-symptomatische HIT-II handelt. Eine hohe Variabilität besteht bei der Festlegung der Zielwerte für die Antikoagulation, wobei diese größtenteils deutlich über dem Bereich einer reinen venösen Thromboembolie(VTE)-Prophylaxe zu liegen kommen.SchlussfolgerungDie Datenlage zur Fortführung einer über die reine VTE-Prophylaxe hinausgehenden Antikoagulation mit konsekutiv erhöhtem Blutungsrisiko bei Patienten mit Sepsis und septischem Schock ist eingeschränkt, und Therapieentscheidungen unterliegen in vielen Fällen einer individuellen Abwägung des Behandlungsteams. Die Ergebnisse unserer Umfrage implizieren die Notwendigkeit einer systematischen Aufarbeitung dieses Themenfeldes, um die auf vielen Intensivstationen gelebte Praxis mit der gebotenen Evidenz zu unterlegen.Zusatzmaterial onlineDie Online-Version dieses Beitrags (10.1007/s00101-021-01011-9) enthält weitere Tabellen, Abbildungen und Auswertungen der Fragebögen.Beitrag und Zusatzmaterial stehen Ihnen auf www.springermedizin.de zur Verfügung. Bitte geben Sie dort den Beitragstitel in die Suche ein oder nutzen Sie den QR-Code auf der ersten Seite des Artikels. Das Zusatzmaterial finden Sie beim Beitrag unter „Ergänzende Inhalte“.

Cloning, expression, and characterization of a novel heparinase I from Bacteroides eggerthii

Heparinase I (Hep I) specifically degrades heparin to oligosaccharide or unsaturated disaccharide and has been widely used in preparation of low molecular weight heparin (LMWH). In this work, a novel Hep I from Bacteroides eggerthii VPI T5-42B-1 was cloned and overexpressed in Escherichia coli BL21 (DE3). The enzyme has specific activity of 480 IU·mg−1 at the optimal temperature and pH of 30 °C and pH 7.5, and the Km and Vmax were 3.6 mg·mL−1 and 647.93 U·mg−1, respectively. The Hep I has good stability with t1/2 values of 350 and 60 min at 30 and 37 °C, respectively. And it showed a residual relative activity of 70.8% after 21 days incubation at 4 °C. Substrate docking study revealed that Lys99, Arg101, Gln241, Lys270, Asn275, and Lys292 were mainly involved in the substrate binding of Hep I. The shorter hydrogen bonds formed between heparin and these residues suggested the higher specific activity of BeHep I. And the minimum conformational entropy value of 756 J·K−1 provides an evidence for the improved stability of this enzyme. This Hep I could be of interest in the industrial preparation of LMWH for its high specific activity and good stability.

Allergic heparin hypersensitivity – recommendations for diagnostic work up and patient management

Heparins are unproblematic drugs from an allergist’s perspective, since despite frequent application heparin hypersensitivity is quite rarely in clinical routine. This article provides an overview of selected scientific articles and is based on a literature search in PubMed and specialist databases. Most commonly, heparin allergic patients develop lymphocyte-mediated, eczematous local reactions directly at the injection sites (usually on the lower abdomen) hours to days after initiating regular subcutaneous heparin injections. The high sensitivity of skin-prick and intradermal testing procedures means that subcutaneous provocation tests are seldom necessary for diagnosis. The “compartment allergy” phenomenon is a particular feature of heparin-induced local allergic reactions. Here, the route of application determines whether an allergic inflammatory reaction develops. In case of heparin-allergic individuals, injections into the subcutaneous tissue cause eczematous plaques, whereas intravenous injections or infusions are well tolerated. Since the introduction of low molecular weight heparin preparations, the incidence of potentially life-threatening heparin-induced thrombocytopenia caused by activating IgG (Ig: immunoglobulin) antibodies against heparin-platelet factor 4 (PF4) complexes on platelet membranes has declined. IgE-mediated heparin allergy with immediate-type anaphylactic reactions seems to be extremely rare.

Получение низкомолекулярных гепаринов методом радиационной деструкции

Низкомолекулярные гепарины (НМГ) широко применяются в медицинский практике. Для получения НМГ существуют различные способы контролируемой деполимеризации нефракционированного гепарина (НФГ). В данной работе представлен метод радиационной деструкции водных растворов гепарина с помощью источника гамма-излучения на основе изотопа 60Co. Установлены оптимальные условия деполимеризации исходного гепарина. Изучена антикоагулянтная активность НМГ в зависимости от дозы облучения. Результаты исследования позволяют рекомендовать данный метод получения НМГ для разработки новых антикоагулянтов.

Empirically Reduced Dosages of Tinzaparin in Patients with Moderate-to-Severe Renal Insufficiency Lead to Inadequate Anti-Xa Levels

Background: Due to the higher molecular weight of tinzaparin, the low molecular weight heparin (LMWH) is less dependent on renal excretion than other LMWH preparations. However, several international guidelines recommend the same preemptive dosage reduction for all therapeutic dose LMWHs prescribed in renal insufficient patients, to ensure that there is no accumulation of anticoagulant activity and increased risk of bleeding. This study is aimed at assessing whether a preemptive dosage reduction of tinzaparin in all renal insufficient patients (comprising 25% reduction in patients with Modification of Diet in Renal Disease - estimated glomerular filtration rate (MDRD-eGFR) 30-60 mL/min/1.73 m² and 50% reduction in patients with MDRD-eGFR <30 mL/min/1.73 m²) leads to adequate anti-Xa levels. Methods: We selected the anti-Xa levels of in-hospital patients (≥18 years) with moderate-to-severe renal insufficiency (MDRD-eGFR <60 mL/min/1.73 m²), on therapeutic dosages of tinzaparin. Anti-Xa levels were measured using a chromogenic assay. Results: Preemptive dosage reduction resulted in a median anti-Xa activity of 0.50 IU/mL (interquartile range [IQR] 0.38-0.60). In 92.3% of patients the anti-Xa level was below the target anti-Xa level of >0.85 IU/mL for therapeutic indications. Unadjusted dosages led to a median anti-Xa activity of 0.74 IU/mL (IQR 0.56-0.92). The preemptive dosage reduction was significantly associated with anti-Xa activity below therapeutic range (p = 0.007). No difference in anti-Xa activity was observed between patients with moderate (0.71 IU/mL, IQR 0.61-0.95) versus severe (0.65 IU/mL, IQR 0.41-1.06) renal insufficiency in whom an unadjusted dose had been administered (p = 0.77). None of the anti-Xa levels were above the upper margin of the presumed therapeutic range of 2.0 IU/mL. Conclusion: In renal insufficient patients, the preemptive dosage reduction of tinzaparin leads to inadequate anti-Xa levels.

Complete Molecular Weight Profiling of Low-Molecular Weight Heparins Using Size Exclusion Chromatography-Ion Suppressor-High-Resolution Mass Spectrometry.

Low-molecular weight heparins (LMWH) prepared by partial depolymerization of unfractionated heparin are used globally to treat coagulation disorders on an outpatient basis. Patent protection for several LMWH has expired and abbreviated new drug applications have been approved by the Food and Drug Administration. As a result, reverse engineering of LMWH for biosimilar LMWH has become an active global endeavor. Traditionally, the molecular weight distributions of LMWH preparations have been determined using size exclusion chromatography (SEC) with optical detection. Recent advances in liquid chromatography-mass spectrometry methods have enabled exact mass measurements of heparin saccharides roughly up to degree-of-polymerization 20, leaving the high molecular weight half of the LMWH preparation unassigned. We demonstrate a new LC-MS system capable of determining the exact masses of complete LMWH preparations, up to dp30. This system employed an ion suppressor cell to desalt the chromatographic effluent online prior to the electrospray mass spectrometry source. We expect this new capability will impact the ability to define LMWH mixtures favorably.

LMWH in the prevention of preeclampsia and fetal growth restriction in women without thrombophilia. A systematic review and meta-analysis.

Placental mediated pregnancy complications such as preeclampsia and fetal growth restriction (FGR) are common, serious, and associated with increased morbidity and mortality. We conducted a systematic review and meta-analysis to determine the effect of treatment with low-molecular-weight heparins (LMWHs) for secondary prevention of these complications in non thrombophilic women. We searched the electronic databases PubMed, Scopus, and Cochrane Library for randomised controlled trials addressing this question. Five studies including 403 patients met the inclusion criteria, 68 developed preeclampsia and 118 FGR. The studies were very heterogeneous in terms of inclusion criteria, LMWH preparation, and dosage. Meta-analyses were performed using random-effect models. The overall use of LMWHs was associated with a risk reduction for preeclampsia (Relative risk (RR) 0.366; 95 % confidence interval (CI), 0.219-0.614) and FGR (RR 0.409; 95 % CI, 0.195-0.932) vs. no treatment. From the data available for analysis it appears that the use of Dalteparin is associated with a risk reduction for preeclampsia (p=0.002) and FGR (p<0.001); while Enoxaparin is associated with risk reduction for preeclampsia (p=0.013) but not for FGR (p=0.3). In spite of the small number of studies addressing the research question, and the high variability among them, our meta-analysis found a modest beneficial effect of LMWH for secondary prevention of preeclampsia and FGR. Further studies are needed to address these questions before a definite conclusion can be reached.

Structure and Activity of a New Low-Molecular-Weight Heparin Produced by Enzymatic Ultrafiltration

The standard process for preparing the low-molecular-weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield because of the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin's core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity.

低分子ヘパリン中の夾雑ムコ多糖類の工業的除去方法

低分子ヘパリン（LMWH）であるダルテパリンナトリウム（DN）の純度試験法と，その中の他のムコ多糖類（MS）夾雑物の工業的除去方法について検討した．高純度の DN (DN-W）に高分子デルマタン硫酸（DSH）および高分子過硫酸コンドロイチン硫酸（OSH）を添加して粗DN（DN-C）を調製し，2段階のエタノール（EtOH）分画処理を行い，各不純物の挙動と DN-C の精製度を確認した．加えて，商業用 DN についても同様の EtOH 分画処理を行い，EtOH 分画処理の有効性を確認した．各分画物を HPLC法と1H-NMR（NMR）法により分析した結果，全ての検体で純度が向上した．また，本試験で用いた商業用 DN 中の高分子 MS 不純物はおもに高分子 DS であることが確認された．以上の結果から，LMWH の調製工程において，亜硝酸分解抵抗性不純物を高感度で検出できる亜硝酸分解/HPLC 法による純度検査を行い，さらに，2段階の EtOH 分画処理を実施することで，高品質な LMWH 原料を安定的に調製できると考えられた．

Reversed-phase ion-pair ultra-high-performance-liquid chromatography–mass spectrometry for fingerprinting low-molecular-weight heparins

Heparin is a complex mixture of sulfated linear carbohydrate polymers. It is widely used as an antithrombotic drug, though it has been shown to have a myriad of additional biological activities. Heparin is often partially depolymerized in order to decrease the average molecular weight, as it has been shown that low molecular weight heparins (LMWH) possess more desirable pharmacokinetic and pharmacodynamic properties than unfractionated heparin (UFH). Due to the prevalence of LMWHs in the market and the emerging availability of generic LMWH products, it is important that analytical methods be developed to ensure the drug quality. This work explores the use of tributylamine (TrBA), dibutylamine (DBA), and pentylamine (PTA) as ion-pairing reagents in conjunction with acetonitrile and methanol modified mobile phases for reversed-phase ion-pairing ultraperformance liquid chromatography coupled to mass spectrometry (RPIP-UPLC–MS) for fingerprint analysis of LMWH preparations. RPIP-UPLC–MS fingerprints are presented and compared for tinzaparinand enoxaparin.

Characterizing the microstructure of heparin and heparan sulfate using N-sulfoglucosamine 1H and 15N NMR chemical shift analysis.

Heparin and heparan sulfate (HS) are members of a biologically important group of highly anionic linear polysaccharides called glycosaminoglycans (GAGs). Because of their structural complexity, the molecular-level characterization of heparin and HS continues to be a challenge. The work presented herein describes an emerging approach for the analysis of unfractionated and low molecular weight heparins, as well as porcine and human-derived HS. This approach utilizes the untapped potential of (15)N NMR to characterize these preparations through detection of the NH resonances of N-sulfo-glucosamine residues. The sulfamate group (1)H and (15)N chemical shifts of six GAG microenvironments were assigned based on the critical comparison of selectively modified heparin derivatives, NMR measurements for a library of heparin-derived oligosaccharide standards, and an in-depth NMR analysis of the low molecular weight heparin enoxaparin through systematic investigation of the chemical exchange properties of NH resonances and residue-specific assignments using the [(1)H,(15)N] HSQC-TOCSY experiment. The sulfamate microenvironments characterized in this study include GlcNS(6S)-UA(2S), ΔUA(2S)-GlcNS(6S), GlcNS(3S)(6S)-UA(2S), GlcNS-UA, GlcNS(6S)-red(α), and 1,6-anhydro GlcNS demonstrating the utility of [(1)H,(15)N] HSQC NMR spectra to provide a spectroscopic fingerprint reflecting the composition of intact GAGs and low molecular weight heparin preparations.

Parenteral Anticoagulants: Antithrombotic Therapy and Prevention of Thrombosis, 9th ed: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines

This article describes the pharmacology of approved parenteral anticoagulants. These include the indirect anticoagulants, unfractionated heparin (UFH), low-molecular-weight heparins (LMWHs), fondaparinux, and danaparoid, as well as the direct thrombin inhibitors hirudin, bivalirudin, and argatroban. UFH is a heterogeneous mixture of glycosaminoglycans that bind to antithrombin via a unique pentasaccharide sequence and catalyze the inactivation of thrombin, factor Xa, and other clotting enzymes. Heparin also binds to cells and plasma proteins other than antithrombin causing unpredictable pharmacokinetic and pharmacodynamic properties and triggering nonhemorrhagic side effects, such as heparin-induced thrombocytopenia (HIT) and osteoporosis. LMWHs have greater inhibitory activity against factor Xa than thrombin and exhibit less binding to cells and plasma proteins than heparin. Consequently, LMWH preparations have more predictable pharmacokinetic and pharmacodynamic properties, have a longer half-life than heparin, and are associated with a lower risk of nonhemorrhagic side effects. LMWHs can be administered once daily or bid by subcutaneous injection, without coagulation monitoring. Based on their greater convenience, LMWHs have replaced UFH for many clinical indications. Fondaparinux, a synthetic pentasaccharide, catalyzes the inhibition of factor Xa, but not thrombin, in an antithrombin-dependent fashion. Fondaparinux binds only to antithrombin. Therefore, fondaparinux-associated HIT or osteoporosis is unlikely to occur. Fondaparinux exhibits complete bioavailability when administered subcutaneously, has a longer half-life than LMWHs, and is given once daily by subcutaneous injection in fixed doses, without coagulation monitoring. Three additional parenteral direct thrombin inhibitors and danaparoid are approved as alternatives to heparin in patients with HIT.

US Food and Drug Administration approval of generic versions of complex biologics: implications for the practicing physician using low molecular weight heparins

Low-molecular-weight heparins (LMWHs) have shown equivalent or superior efficacy and safety to unfractionated heparin as antithrombotic therapy for patients with acute coronary syndromes. Each approved LMWH is a pleotropic biological agent with a unique chemical, biochemical, biophysical and biological profile and displays different pharmacodynamic and pharmacokinetic profiles. As a result, LMWHs are neither equipotent in preclinical assays nor equivalent in terms of their clinical efficacy and safety. Previously, the US Food and Drug Administration (FDA) cautioned against using various LMWHs interchangeably, however recently, the FDA approved generic versions of LMWH that have not been tested in large clinical trials. This paper highlights the bio-chemical and pharmacological differences between the LMWH preparations that may result in different clinical outcomes, and also reviews the implications and challenges physicians face when generic versions of the original/innovator agents are approved for clinical use.

Comparison of the Pharmacokinetic and Pharmacodynamic Behavior of Branded and Generic Enoxaparin in Non-Human Primates

Abstract 1254 Introduction:Low molecular weight heparins (LMWHs) have been shown to be safe and effective in the prevention and treatment of venous thrombosis and in the treatment of patients with pulmonary embolism or acute coronary syndrome. Commercially available LMWHs are distinct agents in terms of their molecular weight profile and in vitro potency. Molecular weight influences the pharmacokinetics (PK) and pharmacodynamics (PD) of heparin oligosaccharides. Although there continues to be interest in developing biosimilar versions of LMWHs, defining biosimilarity for LMWHs is challenging because chemical means of measuring plasma heparin levels do not exist, potency measures typically only measure AT-dependent activities, bioequivalence in terms of one activity does not ensure identical pharmacokinetic behavior in terms of other biological activities and identification and quantification of the various oligosaccharide components in a LMWH preparation is difficult. In vitro studies have shown that biosimilar LMWHs can differ considerably from the branded product as well as from each other. In the current study, the PD behavior of branded and biosimilar enoxaparin was compared in a cross-over study following a single subcutaneous dose to non-human primates. Methods:Lovenox (lot #514956, sanofi-aventis, Paris, France) and Fibrinox (lot#0002, Sandoz S.A., Buenos Aires, Argentina) were obtained in pre-filled syringes at a concentration of 100 mg/ml. Upon anesthesia, the non-human primates (Macaca mulatta) were weighed and a baseline blood sample was collected. A site on the abdomen was shaved and cleansed prior to the subcutaneous administration of LMWH. Blood samples were collected at 1, 4, 6 and 28 hours post-administration and were centrifuged to make platelet poor plasma which was stored in aliquots at −70°C until analysis. Primates were randomly dosed with one of the two test agents. After a minimum of one week wash-out, the same primates were dosed with the other test agent. Plasma samples were evaluated using clot-based and amidolytic assays. LMWH concentrations in the blood samples were determined by extrapolation from in vitro concentration-response curves and used to calculate values for PK parameters. p-values < 0.05 determined by two-way repeated measures analysis of variance were considered to be statistically significant. Results:LMWH levels determined by circulating anti-Xa activity following Fibrinox and Lovenox administration were not observed to be significantly different. Circulating anti-IIa levels, however, were significantly higher in Lovenox-treated animals at 1, 4 and 6 hours post-administration. Using the Heptest assay, higher circulating drug levels were measured in Fibrinox-treated animals at 4 and 6 hours. A high degree of variability was observed in the absorption and elimination of the LMWHs. Statistically significant differences in pharmacokinetic behavior was not observed when circulating drug concentrations were determined using anti-Xa activity or Heptest prolongation. Using drug concentrations determined from anti-IIa activity, the AUC following Lovenox treatment was significantly larger than following Fibrinox treatment. Plotting mean drug levels based on anti-IIa activity versus mean drug levels based on anti-Xa activity at the various sample times indicated a hysteretic relationship which was distinct for Fibrinox and Lovenox-treated primates. Clear differentiation of the two LMWHs was also observed when drug levels measured by anti-IIa activity were plotted against those determined using the prolongation of the Heptest. Conclusions:Potency of LMWHs can be expressed in terms of a number of AT-dependent and AT-independent activities, which may not be proportional. The PK/PD behavior of LMWHs is different when drug levels are determined using distinct biologic activities. The measured concentration of a drug and its corresponding effect can be asynchronous, that is, peak drug levels may not occur at the same time as peak effect levels. In this study, plotting drug concentrations determined by one functional parameter against those derived from a different parameter provided clear visual evidence that the two LMWHs tested exhibit distinct pharmacodynamic behavior. In vivo behavior is an important consideration for defining pharmacoequivalence of complex biologic drugs. Disclosures:No relevant conflicts of interest to declare.

Low Molecular Weight Heparin Inhibits Plasma Thrombin Generation Via Direct Targeting of Factor IXa: Contribution of the Serpin-Independent Mechanism

Abstract 2242The relevance of factor IXa as a therapeutic target for heparin therapy remains incompletely defined. To evaluate the contribution of factor IXa inhibition, particularly serpin-independent inhibition of the intrinsic tenase complex (factor IXa-factor VIIIa), the ability of heparin preparations to inhibit tissue factor (TF)-triggered thrombin generation was examined in human plasma. Thrombin generation was determined by a fluorogenic substrate assay employing Technothrombin TGA evaluation software to determine lag time, time to thrombin peak, peak thrombin concentration, and velocity index (slope). The inhibitory potency (EC50) of each heparin was determined by plotting concentration versus relative velocity index (ratio of the velocity index in presence and absence of heparin). Inhibition of thrombin generation was initially compared under factor IX-dependent (limiting TF) and independent (excess TF) conditions, respectively, by addition of either 0.2 or 4 pM TF to pooled normal human plasma containing increasing concentrations of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), Fondaparinux, or unfractionated heparin (UFH). UFH and Fondaparinux demonstrated complete or near complete inhibition with identical EC50 values at both tissue factor concentrations, suggesting that inhibition of intrinsic tenase activity does not significantly contribute to their mechanism of action in plasma. In contrast, LMWH and ssLMWH demonstrated 2.9- and 5.1-fold lower EC50 values, respectively, in the presence of the limiting TF concentration. These results suggest that inhibition of intrinsic tenase activity contributes to the mechanism of action for LMWH in plasma. The effect of heparins on the time course of plasma thrombin generation by Western blotting under similar conditions correlated well with results of the fluorogenic substrate assay. At their EC50 values, LMWH, ssLMWH and Fondaparinux clearly reduced prothrombin/meizothrombin consumption in plasma, while UFH primarily accelerated formation of the thrombin-antithrombin complex. The contribution of antithrombin to inhibition of thrombin generation was determined by comparison of antithrombin- and mock-depleted human plasmas. As expected, antithrombin depletion markedly increased the EC50 values for UFH (62–fold) and Fondaparinux (42-fold) to levels that are ∼3–8 fold higher than their expected therapeutic ranges, confirming an antithrombin-dependent mechanism of action. In contrast, antithrombin depletion increased the EC50 values more modestly for LMWH (9.4-fold) and ssLMWH (2-fold), with the EC50 for LMWH (∼ 0.7 U/ml) remaining within the therapeutic range. In the absence of antithrombin, LMWH demonstrated partial inhibition of plasma thrombin generation with a plateau representing ∼8–10% of the starting activity, remarkably similar to the inhibition of intrinsic tenase activity with purified components. The molecular target for LMWH and ssLMWH was evaluated in dual factor IX/antithrombin-depleted plasma supplemented with 90 nM recombinant factor IX possessing mutations in the heparin-binding exosite. Based on both relative EC50 values for reduction in the velocity index and the maximal degree of inhibition, plasma supplemented with mutant factor IX demonstrated relative resistance to inhibition of thrombin generation by LMWH as follows: R233A > (K126A/R165A/K132A) > R170A > WT. Factor IX R233A demonstrated an 11.4-fold increase in the EC50 for LMWH relative to the wild type protease. The magnitude of differences between mutant factor IX proteins was reduced for ssLMWH relative to LMWH, but the rank order was similar. The effect of these mutations in human plasma recapitulates their effects on in vitro inhibition of intrinsic tenase activity, confirming the factor IXa heparin-binding exosite as the molecular target for antithrombin-independent inhibition of thrombin generation by LMWH and ssLMWH. The ability of therapeutic concentrations of LMWH to directly target factor IXa in antithrombin-depleted plasma suggests that this mechanism may contribute to antithrombotic effects. Furthermore, chemo-enzymatic synthesis of “non-anticoagulant” LMWH preparations acting via this exosite-mediated antithrombotic mechanism may offer advantages in high-risk populations, particularly patients with underlying malignancy. Disclosures:Buyue:Biogen Idec Hemophilia: Employment.

Low-molecular-weight heparins do not modify obliterative airway disease in rat tracheal allografts

ABSTRACTImmunosuppressive and antiproliferative effects of heparin may be beneficial in the field of solid organ transplantation. The aim of this study was to examine the effect of low-molecular-weight heparin (LMWH) compounds on the development of obliterative airway disease (OAD) in the rat tracheal transplant model. Allogenic heterotopic tracheal transplantations were performed from Brown-Norway into Lewis rats. Recipients were treated either with nadroparin, enoxaparin, parnaparin, or vehicle from day 0 until harvesting at day 7 or 21. Graft rejection was morphometrically assessed to determine the extent of luminal obliteration end epithelial necrosis. All tracheal grafts harvested at day 7 demonstrated nearly equivalent degree of luminal obstruction regardless of treatment regimen. Likewise, at day 21 the extent of airway narrowing and the degree of inflammatory cell infiltration were similar among the groups. Moreover, loss of airway epithelium was not prevented by LMWH treatments. Finally, intragraft mRNA expression for transforming growth factor-β1 and platelet-derived growth factor-A, interleukin-2, interferon-γ, and monocyte chemoattractant protein-1 did not differ between the groups. In contrast with findings in other animal models, treatment with LMWH preparations did not modify the development of OAD in rat tracheal allografts.

Polyion-sensitive membrane-based electrodes for heparin-binding foldamer analysis

Polymer membrane-based electrodes sensitive to low molecular weight heparin (LMWH) have been used to examine the binding between several preparations of LMWH and heparin-binding foldamers, which have recently been developed as potential inhibitors of the anticoagulant activity of LMWHs. It was found that the structure of the heparin-binding foldamer affects the equilibrium binding constant, K eq, determined by analysis of the titration curves of the foldamers with LMWHs monitored with these electrodes, and further, the strength of binding depends on the specific LMWH preparation. Additionally, polymer membrane-based electrodes utilizing dinonylnaphthalene sulfonate as the ion-exchanger were developed to measure the heparin-binding foldamers directly in whole blood, and the response was found to depend on the lipophilicity and charge density of the foldamer.

Differences Among Low-Molecular-Weight Heparins: Evidence in Patients With Acute Coronary Syndromes

Low-molecular-weight heparins (LMWHs) have shown equivalent or superior efficacy and safety to unfractionated heparin as antithrombotic agents for the treatment of patients with acute coronary syndromes (ACS). In patients with unstable angina and non-ST segment elevation myocardial infarction (MI), many data support the use of the LMWH enoxaparin to reduce cardiovascular events and death. LMWHs also appear more effective than unfractionated heparin in reducing the composite end point of acute MI, recurrent ischemia, or death in patients with ST segment elevation MI, and can also be used effectively in patients undergoing thrombolysis reperfusion and percutaneous coronary intervention. However, the various LMWH preparations should not be used interchangeably. Each LMWH is a pleiotropic biological agent with a unique chemical, biochemical, biophysical, and biological profile, and it displays a unique pharmacodynamic and pharmacokinetic profile. As a result, LMWHs are not equipotent in preclinical assays or equivalent in terms of their clinical efficacy and safety. Therefore, it is essential that new, emerging, generic versions of LMWHs demonstrate clinical equivalence, in specific indications, with the existing approved LMWHs. This article highlights the chemical, biological, and pharmacological differences between the LMWH preparations that may result in different clinical outcomes during the treatment of patients with acute coronary syndromes.