Interference of DOACs in different DRVVT assays for diagnosis of lupus anticoagulants.

OBJECTIVE: Determination of lupus anticoagulants (LA) is an important, but still challenging test in the diagnosis of antiphospholipid syndrome (APS). This is especially the case in patients using one of the direct oral anticoagulants (DOACs). The aim of our study was to examine the influence of these drugs on DRVVT assays from two companies (in each case: screening test, confirming test and calculated ratio) and on aPTT and lupus-sensitive aPTT. METHODS: We used plasma samples from healthy volunteers spiked with the DOACs dabigatran, rivaroxaban and apixaban (0, 10, 30, 50, 100 ng/mL) for testing. Furthermore, samples from patients receiving a DOAC were investigated. The plasma concentrations of the DOACs were determined using ultra-performance liquid chromatography/electrospray ionization-tandem mass spectrometry (UPLC-MS/MS). RESULTS: Depending on type and concentration, all the DOACs resulted in pathological values in the DRVVT screening assays. In samples spiked with apixaban, no influence on the DRVVT normalized ratio of the two assays was observed, but 7 to 15% of samples from patients receiving apixaban displayed pathological values. In contrast, up to 71% of dabigatran-spiked samples showed normalized ratio values above the cut-off, whereas there was no influence in the patients' samples. In both spiked and patient samples containing rivaroxaban, the DRVVT assays were influenced. CONCLUSION: LA diagnostics should, under DOAC therapy, be limited to situations in which time-critical evaluation is warranted. It is crucial to take into account the finding that even samples containing DOAC concentrations below the limit of detection of the drugs may lead to false-positive DRVVT measurements.

A Highly Effective Ruthenium System for the Catalyzed Dehydrogenative Cyclization of Amine-Boranes to Cyclic Boranes under Mild Conditions.

We recently disclosed a new ruthenium-catalyzed dehydrogenative cyclization process (CDC) of diamine-monoboranes leading to cyclic diaminoboranes. In the present study, the CDC reaction has been successfully extended to a larger number of diamine-monoboranes (4-7) and to one amine-borane alcohol precursor (8). The corresponding NB(H)N- and NB(H)O-containing cyclic diaminoboranes (12-15) and oxazaborolidine (16) were obtained in good to high yields. Multiple substitution patterns on the starting amine-borane substrates were evaluated and the reaction was also performed with chiral substrates. Efforts have been spent to understand the mechanism of the ruthenium CDC process. In addition to a computational approach, a strategy enabling the kinetic discrimination on successive events of the catalytic process leading to the formation of the NB(H)N linkage was performed on the six-carbon chain diamine-monoborane 21 and completed with a (15) N NMR study. The long-life bis-σ-borane ruthenium intermediate 23 possessing a reactive NHMe ending was characterized in situ and proved to catalyze the dehydrogenative cyclization of 1, ascertaining that bis σ-borane ruthenium complexes are key intermediates in the CDC process.

Electronic structure and formation of simple ferryloxo complexes: mechanism of the Fenton reaction.

The Fenton reaction is a famous reaction in inorganic chemistry, with relevance to topics such as bioinorganic oxidation and fundamental redox chemistry of water and oxygen. It is also a reaction concerning which there has been very extensive mechanistic debate, with experimental and computational work leading to extensive evidence concerning its mechanism-not all of which is consistent. Here, we use this reaction as a challenge to modern electronic structure theory methods and show that density functional theory, when validated by accurate ab initio methods, can yield a picture of this reaction that is consistent with experiment. The article also highlights some of the challenges in accurate studies of reaction mechanisms of ionic species in water solution.

Association of antibodies against Neospora caninum in mares with reproductive problems and presence of seropositive dogs as a risk factor.

Sera from 112 mares from 5 horse-breeding farms was examined for the presence of antibodies to Neospora caninum and Toxoplasma gondii by an indirect fluorescent antibody test (IFAT), as well as from dogs and cattle present on these properties for the presence of antibodies to N. caninum. Among the 112 mares, 35 had a history of reproductive problems in the last breeding season and 77 had no reproductive problems. The rates of seroprevalence of N. caninum in mares with and without a history of reproductive problems were 25.71% and 6.49% and from T. gondii 2.85% and 1.29%, respectively. In dogs and cattle, the rates of seroprevalence of N. caninum were 10.52% and 15.55%, respectively. A positive correlation was found between the presence of antibodies against N. caninum (p=0.010) in mares and the occurrence of reproductive problems using the Fisher's exact test. Significantly higher seroprevalence for N. caninum in mares was observed on the farm that had seropositive dogs (p=0.018). Cattle on this farm were also seropositive. No significant differences in seropositivity were found on farms where dogs were seronegative, or absent. This result suggests, for the first time, the presence of seropositive dogs as a risk factor for N. caninum in mares and the necessity for further investigation of the epidemiology of this parasite in horse-breeding farms with reproductive problems and the presence of cattle and dogs. This is the first report on the occurrence of antibodies against N. caninum in horses from the state of Santa Catarina, Brazil.

Adverse effects of heparin.

All the adverse effects of heparins are related to their wide variety of biological activities, with bleeding being the most important safety issue, resulting directly from the potency of heparin as an anticoagulant. However, it is hard to define the bleeding risk, since it depends on numerous parameters including the indication, dosage, method, and duration of heparin application, the clinical study design and definition of bleeding as well as patient characteristics and determinants of bleeding such as type of surgery and co-medication. Nonbleeding complications of heparins are caused by binding of heparin molecules to proteins other than antithrombin and to cells, which is generally more pronounced with unfractionated heparin than with low-molecular-weight heparins. Accordingly, heparin-induced thrombocytopenia, the most severe nonbleeding adverse reaction, occurs about 10 times less with low-molecular-weight heparins than with unfractionated heparin. Frequent and therefore important adverse reactions of heparins are skin lesions resulting from delayed-type hypersensitivity reactions. All the other undesirable effects are discussed as well, but they are mostly clinically irrelevant.

Atmospheric hydrocarbon activation by the hydroxyl radical: a simple yet accurate computational protocol for calculating rate coefficients.

The overall rate coefficient at standard temperature and pressure for the hydrogen abstraction reaction by the hydroxyl radical (HO˙) from common saturated volatile organic compounds (VOCs) is derived theoretically using electronic structure calculations and transition state theory (TST). The computational approach used is based on relatively efficient methods, and hence is applicable to a large number of compounds with only a modest use of computer resources. The key methods used are density functional theory (for the calculation of barrier heights) and simple transition state theory (TST), including a simple correction for tunnelling. All thermally relevant conformers of the reactant and the abstraction TS are included in the study. For all compounds in a test set of thirty-four, the calculated rate coefficient agrees with the experimental value to within better than an order of magnitude, and to within better than a factor of three for all but six cases, so that the accuracy is of predictive utility.

Ruthenium agostic (phosphinoaryl)borane complexes: multinuclear solid-state and solution NMR, X-ray, and DFT studies.

The reactivity of the (o-phosphinophenyl)(amino)borane compound HB(N(i)Pr(2))C(6)H(4)(o-PPh(2)) prepared from Li(C(6)H(4))PPh(2) and HBCl(N(i)Pr(2)) toward the bis(dihydrogen) complex RuH(2)(H(2))(2)(PCy(3))(2) (1) was studied by a combination of DFT, X-ray, and multinuclear NMR techniques including solid-state NMR, a technique rarely employed in organometallic chemistry. The study showed that the complex RuH(2){HB(N(i)Pr(2))C(6)H(4)(o-PPh(2))}(PCy(3))(2) (3), isolated in excellent yield as yellow crystals and characterized by X-ray diffraction, led in solution to PCy(3) dissociation and formation of an unsaturated 16-electron complex RuH(2){HB(N(i)Pr(2))C(6)H(4)(o-PPh(2))}(PCy(3)) (4), with a hydride trans to a vacant site. In both cases, the (phosphinoaryl)(amino)borane acts as a bifunctional ligand through the phosphine moiety and a Ru-H-B interaction, thus featuring an agostic interaction.

[New oral anticoagulants: better than vitamin K antagonists?]. / Neue orale Antikoagulanzien: Werden sie die Vitamin-K-Antagonisten verdrängen?

Many years of practical use and intensive scientific research have allowed vitamin K antagonists to become a cornerstone of treatment of internal diseases. Nevertheless, limitations in pharmacokinetics and -dynamics of vitamin K antagonists and the availability of new drugs in regard to a targeted anticoagulation therapy ask for a new review of the situation. Proof of effectiveness for the perioperative prophylaxis of venous thrombosis after hip and knee replacement has already been achieved for the direct thrombin inhibitor dabigatran etexilate as well as for the factor Xa inhibitors rivaroxaban und apixaban compared to low molecular weight heparins. These new drugs are now also investigated in patients with internal diseases. For the long-term application (6 or 12 months) concerning the treatment of venous thrombosis and/or stroke prophylaxis in patients with atrial fibrillation data is already available for the direct thrombin inhibitor dabigatran etexilate. Depending on its dosage its effectiveness in comparison with vitamin K antagonists is equal or even better without disadvantages in safety. However, vitamin K antagonists will remain the standard oral anticoagulation until open questions regarding e.g. insufficient therapy adherence (with termination rates up to 20%) or problems with drug interactions of the new competitive products have been completely answered.

Pharmacological strategies for inhibition of thrombin activity.

For decades, the options for therapeutic anticoagulation were limited to unfractionated heparin (UFH) and vitamin K antagonists (VKA), and their well-known limitations had to be accepted. With the introduction of the various LMWHs, the short-term anticoagulation could be much improved. The heparins delivered the proof of concept that FXa and thrombin represent suitable targets for therapeutic anticoagulation. Consequently, the search for new anticoagulants focus on inhibitors of thrombin or FXa. Apart from the VKA, the anticoagulants presently available or in an advanced stage of development can thus be divided in two classes: One are the glyco-anticoagulants with the natural sulfated glycosaminoglycans (GAGs) (UFH, LMWHs, and danaparoid) and the synthetic oligosaccharides (OS) (fondaparinux, idraparinux, and SR123781A). The other class are the xenobiotic anticoagulants, i.e. proteins and synthetic chemical entities. Die glyco-anticoagulants act partially (GAGs) or exclusively (oligosaccharides) by catalysing antithrombin, whereas the xenobiotic anticoagulants are direct inhibitors of either thrombin or FXa. At present, three direct thrombin inhibitors (DTI) (lepirudin, argatroban, and bivalirudin) are clinically used for limited indications, whereas there is still no direct FXa inhibitor available. The DTI ximelagatran represented the first oral anticoagulant since the introduction of VKA, but was withdrawn due to safety concerns. Among numerous drug candidates in the clinical development, two orally active anticoagulants dabigatran etexilate, a DTI, and rivaroxaban, the direct FXa inhibitor, are in the most advanced stage of development and may allow a paradigm change in anticoagulation in the foreseeable future. This review describes the pharmacological profile of all these anticoagulants.

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.

INTRODUCTION: Heparin-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 METHODS: By 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 CONCLUSIONS: The 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.

[Natural and synthetic glycosaminoglycans. Molecular characteristics as the basis of distinct drug profiles]. / Natürliche und synthetische Glykosaminoglykane. Molekulare Charakteristika als Grundlage unterschiedlicher Arzneistoffprofile.

For several decades, anticoagulants based on glycosaminoglycans (GAG) are drugs of choice in the therapy and prophylaxis of thromboembolic diseases. In principle, it has to be differentiated between the natural GAG-anticoagulants, which are molecular mixtures with complex composition, and the synthetic GAG-anticoagulants, which are chemically defined oligosaccharides. The former include unfractionated heparin, the various low molecular weight heparins and danaparoid. Representatives of the second group are fondaparinux, idraparinux and the hexadecasaccharide SR123781A. They share a common mechanism of action together with the endogenous antithrombotic heparan sulfate, i.e. the catalysis of the antithrombin-mediated inhibition of factor Xa. Besides, considerable structural differences between the various GAG-anticoagulants result in rather distinct product characteristics. This concerns their pharmacodynamics, pharmacokinetics as well as practice-related aspects such as dosage, monitoring, accumulation tendency, antagonisation and HIT-Typ II.

[Pharmacology of heparins and direct anticoagulants]. / Pharmakologie der Heparine und der direkten Antikoagulanzien.

For decades, the options for therapeutic anticoagulation were limited to unfractionated heparin (UFH) and vitamin K antagonists (VKA), and their well-known limitations had to be accepted. With the introduction of the various LMWHs, the short- and medium-term anticoagulation could be much improved, but an alternative to VKA is still missing The heparins provided the proof of concept that FXa and thrombin represent suitable targets for therapeutic anticoagulation. Consequently, the search for new anticoagulants focuses on inhibitors of thrombin (DTI) or FXa (DXI). Apart from the VKA, the anticoagulants presently available or in an advanced stage of development can thus be divided in two classes: One are the glyco-anticoagulants with the natural sulfated glycosaminoglycans (GAGs) (UFH, LMWHs, and danaparoid) and the synthetic oligosaccharides (OS) (fondaparinux, idraparinux, and SR123781A). The other class are the xenobiotic anticoagulants, i.e. proteins and synthetic chemical entities. Die glyco-anticoagulants act partially (GAGs) or exclusively (oligosaccharides) by catalysing antithrombin, whereas the xenobiotic anticoagulants are direct inhibitors of either thrombin or FXa. At present, three parenteral DTI (lepirudin, argatroban, and bivalirudin) and since March 2008 one oral DTI (dabigatran etexilate) are clinically used for limited indications. In September 2008 rivaroxaban has been approved as the first oral DXI. This review describes the development of the anticoagualants as well as the pharmacological profile of the clinically used anticoagualants.

Fixed dosage of low-molecular-weight heparins causes large individual variation in coagulability, only partly correlated to body weight.

BACKGROUND: Low-molecular-weight heparins (LMWHs) are routinely given without the control of their effect on coagulation. The endogenous thrombin potential (ETP) is a sensitive detector of the heparin effect. QUESTION: What is the interindividual variation in TG after a fixed dose of LMWH in normal volunteers, is it explained by variation in weight? METHODS: Subcutaneous (s.c.) injection, in 12 healthy volunteers, of 9000 aXa-units of unfractionated heparin (UFH) and of three heparins with narrow MW distribution around 10.5, 6.0 and 4.5 kD. Measurement of anti-thrombin (aIIa) and antifactor Xa (aXa)-activities and ETP at 11 time points over 24 h. RESULTS: The coefficient of variation (CV) of the AUCs of aXa- and aIIa-activities is 50% for UFH and 22-37% for LMWHs. Because of the hyperbolic form of the dose-response curve, the CV of the inhibition of the ETP is lower: 32% for UFH and 13-21% for the LMWHs. Fixed dosage of LMWH caused under-dosage in 10-13% of the samples and over-dosage in 5-11%. High or low response is an individual property independent of the type of heparin injected and only partially explained by variation in body weight. CONCLUSION: Optimized individual dosage of LMWH is possible through recognition of high and low responders, which requires one measurement of the heparin concentration or, preferably, the heparin effect on the ETP, 2-5 h after a first injection.

[Chromogenic assay for functional determination of TFP]. / Chromogene Methode zur funktionellen. TFPI-Bestimmung.

UNLABELLED: Due to the manifold functions of tissue factor (TF), also tissue factor pathway inhibitor (TFPI), its endogenous antagonist, is of interest. For the determination of TFPI, either its antigen concentration or its activity can be measured. METHOD: A chromogenic assay for the functional determination of TFPI is presented and modified for its application in the routine laboratory. Its suitability for daily use was evaluated by testing more than 7000 plasma samples from a heparin study in healthy volunteers. In addition, our data confirm that the method records the activity of both free- and lipoprotein-associated TFPI. CONCLUSION: The chromogenic assay proved to be rapid and easy to perform and features a high reproducibility. Due to excellent correlation of the results with the total TFPI antigen concentrations, the method represents a more rapid and economic alternative to the determination of total TFPI antigen by ELISA in plasma samples after heparin application.

From heparins to factor Xa inhibitors and beyond.

Despite some disadvantages, unfractionated heparin (UFH) and oral anticoagulants have been the only anticoagulants for prophylaxis and therapy of thromboembolic disorders for several decades. Based on the increasing knowledge of the structure and pharmacology of heparin, low molecular weight heparins (LMWH) have been developed in the 1980s. Compared to UFH, their advantages are mainly based on their reduced nonspecific binding to proteins and cells resulting in improved pharmacokinetics. In 1991, LMWH were declared as the most efficient prophylaxis in high-risk patients. Although the use of LMWH is increasing and they are today also applied for therapy and in other indications like acute coronary syndrome, they are considered not optimal concerning efficacy and safety. With the approval of fondaparinux for the prevention of venous thromboembolic disease in high-risk orthopedic patients, there might be a paradigm shift in the field of anticoagulants. Fondaparinux, a synthetic, chemically defined pentasaccharide, is the first selective inhibitor of factor Xa. By its highly specific binding to antithrombin, it selectively inhibits factor Xa and consequently prevents thrombin generation. In contrast to UFH and LMWH, it does not bind to any other cells and other proteins than antithrombin. This leads to a favourable linear pharmacokinetic profile, allowing once-daily subcutaneous application of a fixed dose without monitoring in thromboembolism prophylaxis. In addition to the evaluation of fondaparinux for further indications, chemical modifications of this pentasaccharide such as the long-acting idraparinux are currently under investigation.

The 'precautionary principle' as a guide for future drug development.

During the last decade, the 'precautionary principle' health has gained importance. It is an approach to manage uncertain risks and to prevent any damage to the environment or human. A key element is to take action, even if some cause and effect relationships are not fully established scientifically. Although there are also critics of this principle, it is meanwhile, also increasingly implemented in medicine. An important subject is medicinal products of human or animal origin. Manifold official precaution-guided regulations have been stated to improve their safety, particularly to avoid any infection by viruses and pathogens causing transmissible spongiform encephalopathies. In addition to numerous regulations and decisions, it is generally recommended to substitute animal and human-derived products with adequate alternatives wherever possible. This is a great challenge for research and drug development. One option is recombinant proteins, which however, are not generally free of any risk of contamination. Therefore, the best strategy might be the development of synthetic, specifically acting drugs. The most widely used medicinal product of animal origin at present is heparin. Although there has been no indication of any viral contamination, many other reasons suggest its substitution by alternative antithrombotics. These actually promoted the research on new anticoagulants. With the approval of fondaparinux, the first synthetic, selective factor Xa, a first alternative to the porcine-derived heparin has become available. In addition, other synthetic antithrombotics are currently in clinical development. In principle, it is thus possible that the prophylaxis and therapy of thromboembolic diseases will become completely independent of animal-derived drugs, which would be in line with the precautionary principle.

Thrombin generation for the control of heparin treatment, comparison with the activated partial thromboplastin time.

Heparin can be quantified with antifactor Xa and IIa tests (aXa, aIIa) but the anticoagulant power of heparin depends upon plasma properties as well as upon heparin concentrations and thus differs between subjects. Measuring the effect, as with the activated partial thromboplastin time (APTT) therefore is clinically more relevant. Here we investigate the use of the endogenous thrombin potential (ETP) for this purpose. In 12 volunteers 9000 IU of four heparins of different mol. wt distributions were injected. Samples were taken at 11 time points between 0 and 24 h. With the exception of the 0 and 24-h time points, heparin could be demonstrated by its aIIa and aXa activity in virtually all samples. The APTT showed the effect of this heparin in 34% of the samples; the ETP in 80%. This is partly due to the wide margins of the normal values, caused by large interindividual variation [coefficient of variation (CV) approximately 12% for the APTT, approximately 17% for the ETP]. The intraindividual variation is much smaller (CV approximately 4% for the APTT, approximately 5% for the ETP). Relative to the baseline value of the individual, the heparin effect was recognized by the APTT in 55% of the cases and by the ETP in 98%. There were no large differences between the different types of heparin.

Selectin-blocking semisynthetic sulfated polysaccharides as promising anti-inflammatory agents.

Selectin-induced leucocytes rolling along the endothelial surface of blood vessels initiate a complex adhesion cascade, which is an essential step in the cellular immune response. Consequently, blocking the binding between the selectins and their ligands represents a promising strategy for suppressing pathological inflammatory reactions. This study describes the effects of an unfractionated heparin and a low-molecular-weight heparin and a series of structurally well-defined semisynthetic glucan sulfates on selectin-mediated cell-rolling with respect to inhibition. To simulate the blood flow characteristics of postcapillary venules, the rolling experiments were performed in a dynamic-flow-chamber system with immobilized selectins and selectin ligand-carrying U937 cells. The influence of the test compounds on cell rolling was measured by the percentage of adherent cells after a certain flow time and the velocity of the rolling cells. Whereas the test compounds displayed no inhibitory effect on E-selectin-mediated cell rolling, they efficiently blocked the rolling induced by P-selectin. The glucan sulfates were much more active than either unfractionated heparin or low-molecular-weight heparin, or the standard inhibitor Sialyl Lewis(X). Their inhibitory potency turned out to be strongly dependent on various structural parameters, such as sulfation pattern and molecular weight. In conclusion, the semisysnthetic glucan sulfates represent promising candidates in the development of selectin blocking agents.

Partial synthetic glucan sulfates as potential new antithrombotics: a review.

Structurally defined sulfated polysaccharides were produced by partial synthesis to develop new antithrombotics as potential heparin alternatives. Glucans of different natural origins were used as starting polymers. The resulting glucan sulfates display pronounced anticoagulant effects; some of them are as active as heparin. According to studies on the structure-activity relationships, besides the molecular weight (MW) and the degree of sulfation (DS), the sulfation pattern and the polysaccharide basic structure are crucial parameters for their anticoagulant potency. Their mode of action differs from that of heparin. Depending on their individual structure, they specifically interfere with various stages of the coagulation process. In vivo, they partly exhibit antithrombotic activity similar to that of heparin. But the in vivo efficacy is not just based on their anticoagulant activity. Their profibrinolytic actions and their strong TFPI-releasing effect may considerably contribute to this overall effect. Due to their manifold interactions with the system of hemostasis, each glucan sulfate shows a structure-dependent, individual action profile. From the investigated glucan sulfates, mainly C2- and C4-sulfated, linear beta-1,3-glucan sulfates with DS > 1.0 and MW between 18 and 50 kDa proved to be most suitable for a potential use as heparin alternatives. The results of this study demonstrate the impact of the various structural parameters on the antithrombotic activity of sulfated polysaccharides. However, the biological actions of sulfated polysaccharides are not limited to hemostasis, but they also show manifold modulating effects on other biological systems. Therefore, the approach of using highly sophisticated carbohydrate drug design might be a possibility to obtain new drugs with specific action profiles.