Susanne Alban

Anticoagulant sulfated polysaccharides: Part I. Synthesis and structure–activity relationships of new pullulan sulfates

In order to develop new anticoagulants as potential heparin alternatives, two pullulans with different molecular weight (MW) were used as starting polymers for the partial synthesis of a structurally new class of sulfated polysaccharides. Sulfation of these linear α-1,4-/1,6-glucans was carried out by a method with a SO3–pyridine complex in DMF, which had been optimized for the modification of β-1,3-glucans. Modifications of this methods resulted in pullulan sulfates with degrees of sulfation (DS) ranging from 0.17 to 1.99 and MW between 15 and 250 kDa. More than 50% of the sulfate groups were bound to the secondary C atoms in positions 2, 3 and 4 of the glucose monomers. The anticoagulant activity of the obtained pullulan sulfates was determined in the coagulation assays prothrombin time (PT), activated partial thromboplastin time (APTT), Heptest® and thrombin time (TT). They represent potent anticoagulants reaching the efficacy of heparin. Their activity not only improves with increasing DS and MW, but also with increasing part of sulfate groups in positions 2, 3 and 4. In addition, their action profile changes in dependence on their individual structure as reflected by the ratio of the TT- to the APTT-activity. The pullulan sulfates specifically interfere with different stages of the coagulation cascade, and these interactions have different requirements on the chemical structure.

β-1,3 Glucan Sulfate, but Not β-1,3 Glucan, Induces the Salicylic Acid Signaling Pathway in Tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a β-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated β-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.

The 37-kDa/67-kDa Laminin Receptor Acts as a Receptor for Infectious Prions and Is Inhibited by Polysulfated Glycanes

BACKGROUND Recently, we showed that the 37-kDa/67-kDa laminin receptor (LRP/LR) acts as the receptor of the cellular prion protein. METHODS For the present study, we investigated the binding of the murine scrapie prion protein (moPrP27-30) to baby hamster kidney (BHK) cells, using the Semliki Forest virus system. RESULTS The enhanced binding of moPrP27-30 to BHK cells expressing moLRP::FLAG was inhibited by the LRP/LR-specific antibody W3, which suggests that LRP/LR acts as a receptor for the scrapie form of the prion protein, PrP(Sc). This finding was confirmed by a parallel study that showed that bovine prions are internalized by human enterocytes via LRP/LR. The heparan sulfate mimetics HM5004 and HM2602 reduced PrP27-30 binding to moLRP-expressing cells to approximately 30% and approximately 20%, respectively, at a concentration of 10 microg/mL, whereas pentosan polysulfate (SP54) and phycarin sulfate (PS3) both reduced the binding to approximately 40% at a concentration of 100 microg/mL. CONCLUSIONS We suggest that the inhibition reported elsewhere of PrP(Sc) synthesis and the incubation times prolonged in rodent models by these sulfated glycans are due to the inhibition of the LRP/LR-dependent binding of prions to the target cells.

The ability of different forms of heparins to suppress P-selectin function in vitro correlates to their inhibitory capacity on bloodborne metastasis in vivo

Ample evidence suggests that many of the in vivo anti-metastatic effects by heparins reflect their actions on P-selectin-mediated binding. We hypothesized that the ability of widely used heparins and derivatives to interfere with P-selectin-dependent tumour cell interactions under flow in vitro could be used to identify anticoagulants with advanced inhibitory functions on experimental blood-borne metastasis in vivo. To test this assumption, the impact of unfractionated heparin, the low-molecular-weight heparins (LMWH) nadroparin and enoxaparin, and the synthetic pentasaccharide fondaparinux on P-selectin-dependent tumour interactions in vitro and metastasis formation in vivo were evaluated. Our data revealed that these commonly used anticoagulants widely differ in their potential to interfere with P-selectinmediated cell binding. Importantly, the superior inhibitory capacity on P-selectin function of unfractionated heparin and LMWH nadroparin as opposed to LMWH enoxaparin and synthetic heparin pentasaccharide fondaparinux strongly correlated to the inhibitory potency of each in inhibiting experimental lung metastasis in vivo. Hence, P-selectin inhibition may constitute a valuable feature to identify anticoagulants that are suitable for anticancer therapy.

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.

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

Summary.  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.

Dabigatran, rivaroxaban, apixaban, argatroban and fondaparinux and their effects on coagulation POC and platelet function tests.

Abstract Background: In recent years, several selectively acting anticoagulants, including the direct thrombin inhibitors (DTI; argatroban, dabigatran) and the factor Xa inhibitors (rivaroxaban, apixaban, fondaparinux), have been developed. With their clinical application increasing, it is of interest to evaluate their interference with classical haemostaseological point-of-care tests. Additionally, the effect of the investigated anticoagulants on platelet function tests will come increasingly more into focus for monitoring not only hereditary platelet dysfunction, but also antiplatelet therapy. Methods: Blood samples from healthy volunteers were spiked with therapeutic and supratherapeutic concentrations of the drugs listed above and investigated with regard to their effects on the following POCTs: activated clotting time (ACT), thromboelastometry with ROTEM®, PFA® and Multiplate®. Light-transmission aggregometry (LTA) was used for a platelet function assay. Results: At supratherapeutic concentrations, ACT and ROTEM® analysis were always influenced after administration of the drugs listed above (except fondaparinux in EXTEM-CT). Therapeutic concentrations showed differential effects on these assays. LTA measurements revealed a distinct decrease in α-thrombin-induced platelet aggregation for both DTIs (therapeutic and supratherapeutic concentrations), while argatroban reduced platelet function in supratherapeutic concentrations. None of the drugs seemed to have any influence on PFA® or Multiplate®. Conclusions: Selective thrombin and factor Xa inhibitors exhibit distinct effects on POCTs and platelet function tests. This must be considered in assessing assay results when taking medical decisions.

Structure-activity relationship of antithrombotic polysaccharide derivatives.

Heparin has been the drug of choice in clinical pre-surgical and post-surgical prophylaxis of thrombotic events. However, because of its side-effects, such as bleeding and other disadvantages (i.e. chemical inhomogeneity and variability of its physiological activities), alternatives to heparin are an important field of research. A necessary procedure in the development of new drugs is the evaluation of structure-activity relationships. Genuine neutral polysaccharides were chemically modified and examined for their anticoagulant activities. The linear beta-1,3-glucan curdlan, an easily available bacterial polysaccharide, served as the basic polymer. It could be established that the anticoagulant activity was dependent on the degree of sulfation and the molecular weight. For heparin, the sulfation pattern, i.e. the actual location of the sulfate groups along the heparin chain, was of importance in addition to the degree of sulfation. Therefore, we investigated whether there was also a relationship between the substitution pattern of the curdlan sulfates and their anticoagulant activity. For determination of the substitution pattern of the sulfated polysaccharides, a method was developed that is based on synthesis of the partially alkylated alditol acetates of the polymer and examination of these derivatives using combined gas chromatography-mass spectrometry. In addition to the analytical data, the structure-activity relationship of anticoagulative curdlan sulfates is presented.

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

Summary.  Backgrounds: 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.

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.