Heparin Preparations Research Articles

Immobilized high-performance heparin lyase III for efficient preparation of low molecular weight heparin

Heparin lyase III has garnered widespread attention due to its high specificity and minimal loss of anticoagulant activity during the preparation of low molecular weight heparin (LMWH), a crucial anticoagulant drug in clinical practice. However, low expression levels and complex preparation processes limit its practical application. To address these challenges, high-performance Bacteroides thetaiotaomicron heparin lyase III (Bhep III) variants were engineered and immobilized for LMWH preparation. First, we enhanced enzyme expression by adding a solubility-enhancing tag and optimizing the N-terminal coding sequence, which resulted in a Bhep III activity level of 2.9 × 103 U/L with 8-fold increase. After evolution guided the design of rational mutations, the variant Bhep III K85A/Q95F/S471T generated higher activity (5.4 × 104 U/L in 5-L fermenter), which is, to our knowledge, the highest reported to date in the literature, being 1.7-fold that of the wild type and demonstrating 2-fold increase of the thermal stability. By screening and optimizing the C-terminal self-assembling tag, we successfully immobilized Bhep III, further increasing its thermal stability by 12-fold, and allowing for the multi-batch preparation of LMWH with simple centrifugation. The immobilized heparin lyase III demonstrated sufficient reusability in enzymatic reactions, facilitating efficient industrial-scale production of LMWH.

Study of the possibility of controlling the production of low-molecular-weight heparin preparations by domestic test systems (review)

Introduction. The data on the wide clinical use of low-molecular-weight heparin (LMWH) preparations are presented, necessitating the need for efficient and high-quality production of appropriate pharmaceuticals. The revealed variability of anticoagulant activity of LMWH preparations implies the provision of production with validated methods of control and certification. The aim of the work is to study the possibility of control of anticoagulant activity and assessment of functional compatibility of LMWH preparations when using domestic test systems.Text. The data on analytical characteristics of chromogenic and coagulometric methods performed with the help of domestic test systems are presented. Their parameters correspond with the existing international requirements. The possibility of the mentioned methods to certify LMWH preparations correctly and reproducibly is shown. The given research results testify to the possibility of clotting methods to determine the bioequivalence of the studied preparations of LMWH.Conclusion. The presented data prove the expediency of production control and certification of LMWH preparations by domestic test systems.

Development and attestation of a standard sample of calcium nadroparin

Introduction. The widespread use of heparin preparations for the treatment and prevention of venous thromboembolic complications is known. One of the low molecular weight heparin drugs successfully used in clinical practice is calcium nadroparin. The modern approach to assessing the quality of medicines involves the use of standards, in comparison with which the quality control of the drug is carried out – standard samples (SS). The most important specific parameter in assessing the authenticity of low molecular weight heparins is the characteristic of the fractional composition.Aim. The aim of the work was to develop, attestation and determine the stability during the shelf life of a secondary standard sample to determine the authenticity of calcium nadroparin as part of improving the quality control system of the enterprise with the prospect of creating a domestic pharmacopoeia standard sample.Materials and methods. The object of development and attestation was the standard sample of the enterprise – low molecular weight heparin, obtained on the basis of the Technology implementation center of Siberian state medical university. The certified standard sample was obtained by purification of calcium nadroparin by solid-phase extraction. The completeness of the purification was confirmed by comparing the chromatograms of the purified samples with the chromatograms of the international standard sample. The characteristics of the molecular weight distribution were determined chromatographically. The stability of the certified SSE (the standard sample of the enterprise) was studied under two storage conditions – at a temperature below 8 °C in a dry state for 16 months and in solution at –40 °C for 6 months by evaluating trends in molecular weight distribution.Results and discussion. For each batch of the certified standard sample, the molecular weight and molecular weight characteristics are calculated in 2 parallel definitions, and a representative chromatogram is presented. The values of the relative standard deviation of the molecular weight of the samples and the content of the controlled fractions did not exceed 2.5 %. A detailed analysis of the molecular weight distribution showed the "classical" dependence of RSD (relative standard deviation) from the average value with a significant increase up to 30 % for small fraction values (less than 0.5 %). It was found that during 16 months at a storage temperature below 8 ºC, the molecular weight and molecular weight characteristics of standard samples of low molecular weight heparin did not significantly change. However, in the SSE solution at a storage temperature of minus 40 °C for 6 months, they degraded by more than 10 %.Conclusion. According to the results of the study, a protocol has been developed for obtaining a standard sample of low-molecular-weight heparin for the standardization of the calcium nadroparin substance according to the indicator "molecular weight distribution". The established shelf life of the sample is at least 16 months at temperatures below 8 °C. The stability of solutions of the standard sample at a storage temperature of –40 °C for 3 months is shown.

A Unique Enoxaparin Derived from Bovine Intestinal Heparin: A Single Purification Step of the Starting Material Assures a Bovine Enoxaparin Like the Standard from Porcine Origin.

Low-molecular-weight heparin represent a significant advancement in anticoagulant therapy with enoxaparin being a prominent example obtained exclusively through the fragmentation of porcine intestinal heparin. However, escalating demand and limited resources have raised concerns about enoxaparin supplementation. The current challenge involves exploring alternative heparin sources for large-scale enoxaparin production with bovine intestinal heparin emerging as a promising option. Our study demonstrates that enoxaparin derived from the available bovine heparin preparation differs significantly from the reference compound. Yet, the implementation of a straightforward purification step yields a preparation termed "high-anticoagulant bovine heparin". Fragmentation of this purified product through β-elimination produces enoxaparin akin to the standard from a porcine origin. To ensure physicochemical similarity, we employed various spectroscopic, enzymatic, and chromatographic tests to compare the new bovine-derived enoxaparin with the original porcine compound. Biological activity was confirmed through in vitro coagulation assays and assessments using an animal model of venous thrombosis. Our study affirms that the β-elimination reaction cleaves the bovine heparin chain without preferential breaks in regions with different sulfation patterns. Additionally, we scrutinized decasaccharides purified from enoxaparin preparations, providing a comprehensive demonstration of the similarity between products obtained from porcine and bovine heparin. In summary, our findings indicate that an enoxaparin equivalent to the original porcine-derived product can be derived from bovine heparin, given that the starting material undergoes a simple purification step.

PB0579 Approaches to Assure Similarity of Pharmaceutical Preparations of Unfractionated Heparin

PB0579 Approaches to Assure Similarity of Pharmaceutical Preparations of Unfractionated Heparin

Potential targets of heparin during progression and metastasis of malignant neoplasms

In the modern world, oncological diseases occupy the leading positions in the structure of mortality. An integrated approach to oncotherapy is not only aimed at immediate affection of malignant tumors, but also directed at reducing the risk of tumor recurrence and metastasis, as well as alleviating side effects of chemotherapy and radiotherapy of the disease. In oncologic disorders, blood viscosity increases, thus being associated with hypercoagulation syndrome. To prevent its consequences, the direct and indirect anticoagulants, especially heparin and its derivatives, are actively used. Biological functions and structural features of heparin make it a potential universal platform of a drug development for broad application, including oncology. With the advent of heparin fractionation technology and preparation of low-molecular weight forms and their derivatives, it has become possible to focus not only on anticoagulant activity but also to obtain fractions with targeted pharmacological activity. Usage of the anticoagulants has shown their antitumor activity in some cases, thus providing a basis for a more detailed study of pharmacotherapeutic effects of this group of drugs. Currently, some data suggest various pathways of interaction between heparin and tumor cells. There are multiple common features in development of a primary tumor and formation of secondary distant metastases, which may be attributed to similar molecular cellular mechanisms. The molecules mediating intercellular interactions, both between the tumor cells and between malignant cells and tumor-associated immune cells (e.g., lymphocytes and macrophages) may serve as targets for heparin thus helping the tumor to evade immune surveillance. The cytokines that stimulate tumor angiogenesis represent another important therapeutic target. Heparin derivatives are able to suppress tumor activity and prevent metastatic processes at various stages by inhibiting heparanase, P-/L-selectin, and angiogenesis activity, modulating the CXCL12-CXCR4 chemokine axis, and regulating OAM activity.This brief review addresses the current understanding and application of the potentially antimetastatic properties of heparin and its derivatives in malignant bone tumors since the heparin-based drugs are used as anticoagulants in arthroplasty of large joints and bone defects in patients with osteosarcoma.

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.

Leriche syndrome diagnosed due to polytrauma: a case report

BackgroundLeriche syndrome is caused by atherosclerosis and is often characterized by symptoms such as intermittent claudication and numbness and coldness of the lower limbs. Its exact prevalence and incidence are unknown because it is a rare disease. We report a case of Leriche syndrome diagnosed incidentally on trauma pan-scan computed tomography (CT).Case presentationA 61-year-old Asian male was driving a passenger car and had a head-on collision with a dump truck that required an emergency call. The patient was transported to our hospital in a doctor’s helicopter. Physical examination revealed the following vital signs: respiratory rate, 23 breaths per min; SpO2, 98% under a 10-L administration mask; pulse rate, 133 beats per min; blood pressure, 142/128 mmHg; Focused Assessment with Sonography for Trauma, positive; Glasgow Coma Scale assessment, E3V5M6; and body temperature, 35.9 °C.Trauma pan-scan CT showed bilateral mandibular fractures, bilateral multiple rib fractures, bilateral pneumothorax, sternal fractures, hematoma around thoracic spine, small bowel perforation, mesenteric injury, right clavicle fracture, right ankle debridement injury, and thrombotic occlusion from just above the abdominal aortic bifurcation to the bilateral common iliac arteries. Although thrombotic occlusion needed to be differentiated from traumatic aortic injury, the presence of collateral blood vessels led to the diagnosis of Leriche syndrome, and conservative treatment was performed.Damage control surgery was required for the small bowel injuries. From the second day of admission, the patient was treated with continuous intravenous heparin and prostaglandin preparations. However, impaired blood flow and reperfusion injury in the right lower extremity progressed. On the fifth day of admission, right thigh amputation was performed. The patient required renal replacement therapy for 2 weeks starting from the third day of admission.ConclusionsIn this case, conservative therapy was initially chosen for Leriche syndrome. However, the complex factors in the acute phase of trauma led to development of hemorrhagic necrosis, requiring amputation of the lower extremity. Our findings indicate the need to carefully consider the unique factors affecting Leriche syndrome patients when considering treatment indications and choices for trauma.

Current Trends in the Duration of Anticoagulant Therapy for Venous Thromboembolism: A Systematic Review.

Anticoagulation therapy is the first line and drug of choice for both the treatment and prophylaxis of venous thromboembolism (deep vein thrombosis and/or pulmonary embolism). Anticoagulation drugs, ranging from different preparations of heparin, warfarin, and newer direct oral drugs such as rivaroxaban and dabigatran, work mainly by inhibiting important factors and enzymes in the coagulation cascade by preventing fibrin formation, platelet aggregation, and clot assembly. With recurrent thrombosis and embolisms being a feared complication for many physicians treating such cases, anticoagulation is often extended beyond the initial three- to six-month acute phase after an incident of venous thromboembolism. For some groups of patients, anticoagulation needs to be offered indefinitely to decrease the risk of a recurrent thrombosis. However, this concomitantly increases obvious and dangerous adverse effects such as increased risk of hemorrhage, as the ability to clot is hindered. This tradeoff between recurrent venous thromboembolism and bleeding is what underscores the controversy of the clinical question: for how long should anticoagulation be administered for venous thromboembolism? This review analyzes the use of anticoagulants in different types of venous thromboembolism and remarks on current consensus and trends on the length of anticoagulation treatment. We are doing so while acknowledging that venous thromboembolism management is an active area of research that is rapidly evolving. A literature search was performed looking at recent studies on anticoagulant administration for the treatment of venous thromboembolism with a focus on varying durations and patient populations. Factors that affect clinical decisions of duration are also elucidated. The most clinically relevant anticoagulants were discussed and their effects on the risk of recurrent thrombosis and embolism, and the risk of bleeding in relation to other drugs were analyzed. Ultimately, this article discussed patterns of anticoagulant treatments duration and which patient groups are likely to benefit the most from certain durations, shedding light on the ambiguity in how physicians should approach administering anticoagulation therapy over time for a broad range of presentations of venous thromboembolism.

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“.

Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

Unfractionated heparin inhibits live wild type SARS-CoV-2 cell infectivity at therapeutically relevant concentrations.

Background and PurposeCurrently, there are no licensed vaccines and limited antivirals for the treatment of COVID‐19. Heparin (delivered systemically) is currently used to treat anticoagulant anomalies in COVID‐19 patients. Additionally, in the United Kingdom, Brazil and Australia, nebulised unfractionated heparin (UFH) is being trialled in COVID‐19 patients as a potential treatment. A systematic comparison of the potential antiviral effect of various heparin preparations on live wild type SARS‐CoV‐2, in vitro, is needed.Experimental ApproachSeven different heparin preparations including UFH and low MW heparins (LMWH) of porcine or bovine origin were screened for antiviral activity against live SARS‐CoV‐2 (Australia/VIC01/2020) using a plaque inhibition assay with Vero E6 cells. Interaction of heparin with spike protein RBD was studied using differential scanning fluorimetry and the inhibition of RBD binding to human ACE2 protein using elisa assays was examined.Key ResultsAll the UFH preparations had potent antiviral effects, with IC50 values ranging between 25 and 41 μg·ml−1, whereas LMWHs were less inhibitory by ~150‐fold (IC50 range 3.4–7.8 mg·ml−1). Mechanistically, we observed that heparin binds and destabilizes the RBD protein and furthermore, we show heparin directly inhibits the binding of RBD to the human ACE2 protein receptor.Conclusion and ImplicationsThis comparison of clinically relevant heparins shows that UFH has significantly stronger SARS‐CoV‐2 antiviral activity compared to LMWHs. UFH acts to directly inhibit binding of spike protein to the human ACE2 protein receptor. Overall, the data strongly support further clinical investigation of UFH as a potential treatment for patients with COVID‐19.

Preparation and Application of Nanosensor in Safeguarding Heparin Supply Chain

Heparin has been in clinical use as an anticoagulant for the last eight decades and used worldwide in more than 100 million medical procedures every year. This lifesaving drug is predominantly obtained from ~700 million pig intestines or bovine organs through millions of small and medium-sized slaughterhouses. However, the preparations from animal sources have raised many safety concerns, including the contamination of heparin with potential pathogens, proteins, and other impurities. In fact, contaminated heparin preparations caused 149 deaths in several countries, including the United States, Germany, and Japan in 2008, highlighting the need for implementing sensitive and simple analytical techniques to monitor and safeguard the heparin supply chain. The contaminant responsible for the adverse effects in 2008 was identified as oversulfated chondroitin sulfate (OSCS). We have developed a very sensitive, facile method of detecting OSCS in heparin lots using a nanosensor, a gold nanoparticle-heparin dye conjugate. The sensor is an excellent substrate for heparitinase enzyme, which cleaves the heparin polymer into smaller disaccharide fragments, and therefore facilitates recovery of fluorescence from the dye upon heparitinase treatment. However, the presence of OSCS results in diminished fluorescence recovery from the nanosensor upon heparitinase treatment, because OSCS inhibits the enzyme. The newly designed nanosensor can detect as low as 1 × 10-9% (w/w) OSCS, making it the most sensitive tool available to date for the detection of trace amounts of OSCS in pharmaceutical heparins. In this report, we describe a simple methodology for the preparation of nanosensor and its application in the detection of OSCS contaminants.

Construction and evaluation of an antibody phage display library targeting heparan sulfate

Heparan sulfate (HS) is a linear polysaccharide with high structural diversity. Different HS epitopes have been detected and localized using single chain variable fragment (scFv) antibodies from a ‘single pot’ phage display library containing a randomized complementarity determining region of the heavy chain (CDR3). In this study, we created a new library containing anti-HS scFvs that all harbor a dp-38 heavy chain segment where the CDR3 region was engineered to contain the XBBXBX heparin binding consensus site (X = any amino acid, B = R, K or H). The library contained ~1.73 × 106 unique antibodies and was biopanned against HS from several sources. The selected antibodies were sequenced and chemically/immunohistologically characterized. A number of 67 anti-HS scFv antibodies were selected, of which 31 contained a XBBXBX CDR3 sequence. There was a clear preference for glycine at the first and proline at the fourth position of the CDR3. The sequence GZZP(R/K)X (Z = R, K or H, but may also contain N, S, or Q) was unusually overrepresented. Selected antibodies reacted with HS/heparin, but not with other glycosaminoglycans. Antibodies reacted differentially with respect to N-, 2-O, or 6-O-desulfated heparin preparations, and showed distinct topologies of HS epitopes in rat kidney sections. The library may be instrumental in the selection of a large pool of HS epitope-specific antibodies, and - since all antibodies differ only in their 6 amino acid CDR region - may be a tool for a rational design of antibodies recognizing specific HS sulfation patterns.

(Plenary) Electrochemical/Optical Sensors in Medicine: Meeting Needs for the 21st Century

Over the past 40 years, miniaturized potentiometric and amperometric sensors for ions (K+, Ca++, Na+, Mg++, Cl-, H+), gases (O2 and CO2), and nutrients/metabolites (glucose, lactate, creatinine, urea) have revolutionized the practice of critical care medicine by providing tools to measure an array of these physiologically important species, simultaneously, in small volumes of undiluted whole blood. Indeed, all modern point-of-care whole blood analyzers used in hospitals worldwide now employ electrochemical sensor arrays as either single-use or multi-use devices for near-patient testing, especially in operating rooms, emergency rooms, intensive care units, etc. Some of the same or similar chemistries have also been adapted to create either single-use or continuous monitoring optical sensors (fluorescence, etc.). Further, nearly all blood glucometers as well as newer implantable subcutaneous glucose sensors now use electrochemical measurement principles to provide accurate glucose concentrations for millions of diabetic patients each and every day. A brief overview of these existing electrochemical/optical sensor technologies that have already had such a great impact in medicine will be provided during the introductory portion of this lecture.At the same time, there remain a number of unmet needs in medicine where electrochemical/optical chemical sensing devices could still play important analytical roles. Therefore, in the major portion of this presentation, the following research projects ongoing in our laboratories at the University of Michigan will be highlighted: 1) recent efforts to utilize electrochemical sensors for measurement of polyionic drugs and associated contaminants (including the anticoagulant heparin, low-molecular weight heparin, and inflammatory over-sulfated chondroitin sulfate (OSCS) contaminants in biomedical heparin preparations);1-2 2) research aimed at utilizing the same chemistry employed to make ion and polyion selective electrochemical sensors to create optical sensing films as well as optical sensing microfluidic devices that are able to quantitate species in volumes of << 1 µL;3 3) research related to the development of implantable electrochemical sensors for ions, gases, glucose, lactate, etc. that emit low levels of nitric oxide (NO) (a potent anti-platelet and antimicrobial agent) and that can potentially be used to continuously monitor critical care species intravenously in ICU patients with improved accuracy (see Fig. 1);4 and 4) efforts to use improved electrochemical/amperometric gas phase sensors for detecting nitric oxide (NO) in exhaled nasal/oral breath and also the use of NO and nitrogen dioxide (NO2) sensors to monitor and feedback control the levels of therapeutic gas phase NO being generated by novel photochemical and electrochemical gas phase generators.5-6 These gas phase NO generators can potentially replace use of very high cost compressed gas cylinders containing NO to provide controlled levels of NO gas on-demand for inhalation therapy of patients with pulmonary hypertension (including infants) and for use in the sweep gas of oxygenators to prevent clotting and Systemic Inflammatory Response Syndrome (SIRS) in patients undergoing open heart surgery.Literature Cited: 1. L. Wang, S. Buchanan and M. E. Meyerhoff, “Rapid Detection of High Charge Density Polyanion Contaminants in Biomedical Heparin Preparations Using Potentiometric Polyanion Sensors,” Anal. Chem., 80, 9845-9847 (2008).2. K. Gemene and M. E. Meyerhoff, “Reversible Detection of Heparin and Other Polyanions by Pulsed Chronopotentiometric Polymer Membrane Electrode,” Anal. Chem., 82, 1612-1615 (2010).3. X. Wang, M. Sun, S. A. Ferguson, J. D. Hoff, Y. Qin, R. C. Bailey, and M. E. Meyerhoff, “Ionophore-Based Biphasic Chemical Sensing in Droplet Microfluidics,” Angew. Chem. Int. Ed., 58, 1-6 (2019).4. M. Frost and M. E. Meyerhoff, “Real-Time Monitoring of Critical Care Analytes in the Bloodstream with Chemical Sensors: Progress and Challenges,” Ann. Rev. Anal. Chem., 8, 171-192 (2015).5. J. Zajda, N. J. Schmidt, Z. Zheng, X. Wang and M. E. Meyerhoff, “Evaluation of Amperometric Platinized-Nafion-Based Gas Phase Sensor for Determining Nitric Oxide (NO) Levels in Exhaled Human Nasal Breath,” Electroanalysis 30, 1602-1607 (2018).6. Y. Qin, J. Zajda, H. Ren, J. Toomasian, T. Major, A. Rojas-Pena, B. Carr, T. Johnson , J. Haft, R. H. Bartlett, A. Hunt, N. Lehnert, and M. E. Meyerhoff, “Low Cost Portable Gas Phase Nitric Oxide (NO) Generator Based on Electrochemical Reduction of Nitrite for Potential Applications in Inhaled NO Therapy and Within the Sweep Gas/Cardiotomy Suction Air During Cardiopulmonary Bypass Surgery,” Mol. Pharmaceutics, 14, 3762-3771 (2017). Figure 1

Anticoagulant activity and pleiotropic effects of heparin.

The recognized therapeutic effect of heparins is an anticoagulant activity (anti-Xa and anti-IIa) acting in an indirect manner (cofactor of antithrombin) but which is carried by only 20% at best of the glycan chains composing any commercial preparation of heparin, whether unfractionated or low molecular weight. However, the effects of glycan chains that participate in the therapeutic but also potentially adverse effects of heparin preparations must also be considered. These specific effects of glycans are potentially different for each commercial preparation of heparins and, in particular, low molecular weight heparins (LMWH) compared with unfractionated heparin (UFH) and LMWH between them. The glycanic nature of heparin is responsible for its very particular pharmacology: exchange with the glycocalyx of cells in particular endothelial. Exchanges which depend on the length and structure of the glycan chains therefore different between UFH and LMWH between the different heparin preparations between them but also according to the state of glycocalyx differently altered according to the underlying diseases and their degree of evolution. If the anticoagulant effects of heparins can potentially be replaced with those of new oral anticoagulants, the glycan effects of heparins cannot be replaced by synthetic non-glycan molecules. This replacement will undoubtedly limit certain risks such as heparin-induced thrombocytopenia (HIT) but other beneficial effects participating to the overall efficacy of heparin (whose relative importance remains to be ascertained), will also disappear: effects on surfaces, anti-inflammatory effects, antineoplastic and anti-metastatic effects, ancillary anticoagulant effects (not dependent on antithrombin), effect on endothelial dysfunction. This review will be focused on all of these related/pleiotropic effects of heparins that are in fact the effects of the glycan nature of heparin. Among the antithrombotic effects not dependent on antithrombin one has been more recently highlighted: the passivation/neutralization of the positively charged fibrils of Netosis, by the negatively charged glycan chains of heparin. This also has clinical implications: in the era of generics and biosimilars where biosimilar heparins begin to appear, it is important to know that accordingly to FDA and EMEA rules: their biosimilarity is judged only on the "classical" anticoagulation effect cofactor of antithrombin (anti-IIa/anti-Xa) but that all glycan effects that are potentially beneficial or potentially deleterious are not taken into consideration in their assessment.

International abstracts

The recognized therapeutic effect of heparins is an anticoagulant activity (anti-Xa and anti-IIa) acting in an indirect manner (cofactor of antithrombin) but which is carried by only 20% at best of the glycan chains composing any commercial preparation of heparin, whether unfractionated or low molecular weight. However, the effects of glycan chains that participate in the therapeutic but also potentially adverse effects of heparin preparations must also be considered. These specific effects of glycans are potentially different for each commercial preparation of heparins and, in particular, low molecular weight heparins (LMWH) compared with unfractionated heparin (UFH) and LMWH between them. The glycanic nature of heparin is responsible for its very particular pharmacology: exchange with the glycocalyx of cells in particular endothelial. Exchanges which depend on the length and structure of the glycan chains therefore different between UFH and LMWH between the different heparin preparations between them but also according to the state of glycocalyx differently altered according to the underlying diseases and their degree of evolution. If the anticoagulant effects of heparins can potentially be replaced with those of new oral anticoagulants, the glycan effects of heparins cannot be replaced by synthetic non-glycan molecules. This replacement will undoubtedly limit certain risks such as heparin-induced thrombocytopenia (HIT) but other beneficial effects participating to the overall efficacy of heparin (whose relative importance remains to be ascertained), will also disappear: effects on surfaces, anti-inflammatory effects, antineoplastic and anti-metastatic effects, ancillary anticoagulant effects (not dependent on antithrombin), effect on endothelial dysfunction. This review will be focused on all of these related/pleiotropic effects of heparins that are in fact the effects of the glycan nature of heparin. Among the antithrombotic effects not dependent on antithrombin one has been more recently highlighted: the passivation/neutralization of the positively charged fibrils of Netosis, by the negatively charged glycan chains of heparin. This also has clinical implications: in the era of generics and biosimilars where biosimilar heparins begin to appear, it is important to know that accordingly to FDA and EMEA rules: their biosimilarity is judged only on the “classical” anticoagulation effect cofactor of antithrombin (anti-IIa/anti-Xa) but that all glycan effects that are potentially beneficial or potentially deleterious are not taken into consideration in their assessment.

Identification of DNA variation in callus derived from Zingiber officinale and anticoagulation activities of ginger rhizome and callus

BackgroundThe medicinal plants have been used as alternative treatments for many diseases in many countries. Thus, the possibility of the alteration of some naturally relatively cheap sources into highly valuable products for pharmaceutical and biological importance via tissue culture is investigated in this study. Special attention is needed to estimate the molecular genetic variation between the studied plant and the callus.ResultsGinger (Zingiber officinale Roscoe) rhizome was affected by various concentrations of cytokinin and auxin for the induction of callus. The highest percentage of callus induction and maximal callus fresh weight was achieved when Murashige and Skoog (MS) medium was supplemented with 2 mg/l 2, 4-D + 1mg/l BA. The genetic variations accompanied with in vitro conditions of callus induction was evaluated by four primers of inter-simple sequence repeat (ISSR) that amplified 36 bands. The highest readings for clotting times were found by using the sulfated neutral extract of ginger rhizome at a concentration of 400 μg/ml, and the extract of sulfated alkaline plant callus had an anticoagulation activity at (200 μg/ml) comparable to that of a standard preparation of heparin sodium.ConclusionThese results showed that ginger (Zingiber officinale Roscoe) rhizome was affected by various concentrations of cytokinin and auxin for induction of callus especially when MS was supplemented with 2 mg/l 2, 4-D + 1 mg/l BA. Also, the results of ISSR markers confirmed the occurrence of genetic variations during callus induction process. The results indicated that the sulfated alkaline of ginger rhizome and sulfated aqueous extracts of ginger rhizome and callus exhibited anticoagulant activity. So, it was clear that the addition of sulfate group into the investigated extracts enhances the anticoagulation activities.

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.

A procedure for calibration transfer of DOSY NMR measurements: An example of molecular weight of heparin preparations

AbstractCalibration transfer is commonly used for spectra obtained on different spectrometers or other conditions. This paper reports a multivariate transfer approach for 2D diffusion‐ordered spectroscopy (DOSY) measurements among high‐resolution nuclear magnetic resonance (NMR) spectrometers on the basis of partial least squares (PLS) regression. As the test system previously published quantitative model to predict molecular weight of heparin, low molecular weight heparin (LMWH) was used.For multivariate modeling, piecewise direct standardization (PDS) and direct standardization (DS) were employed. PDS showed the best performance for estimating heparin molecular weight resulting in a significant decrease in root mean square error of prediction (RMSEP) from 647 and 393 Da without standardization to 513 and 135 Da when PDS was applied for heparin and LMWH, respectively. DS is not recommended for high‐resolution NMR data.The study showed the success of standardization procedure in cases when multivariate models have to be applied to the 2D NMR spectra recorded on different NMR spectrometers. The proposed approach appears to be a valuable achievement towards application of transfer models also to 2D NMR data.