Pentasaccharide Fondaparinux Research Articles

Synthesis of Anticoagulant Pentasaccharide Fondaparinux via 3,5-Dimethyl-4-(2'-phenylethynylphenyl)phenyl Glycosides.

Here, we disclosed a convenient procedure for the preparation of EPP [3,5-dimethyl-4-(2'-phenylethynylphenyl)phenyl] glycosides and their application to an effective synthesis of fondaparinux, the clinically approved anticoagulant heparin pentasaccharide. The use of EPP glycosides in the one-pot orthogonal glycosylation for the synthesis of heparin-like tetrasaccharides has also been achieved.

OKL-1111, A modified cyclodextrin as a potential universal reversal agent for anticoagulants

BackgroundAntithrombotic therapy is inevitably associated with a risk for bleeding and these bleeding complications can be life-threatening. Recently, specific reversal agents were developed for the direct factor Xa and thrombin inhibitors (DOACs). However, next to the fact that these agents are relatively expensive, the use of selective reversal agents complicates treatment of bleeding patients in practice. In a series of screening experiments, we discovered a class of cyclodextrins with procoagulant properties. In this study we characterize a lead compound, OKL-1111, and demonstrate its potential use as a universal reversal agent. ObjectivesTo assess the anticoagulant reversal properties of OKL-1111, in vitro and in vivo. MethodsThe effect of OKL-1111 on coagulation in the absence and presence of DOACs was investigated in a thrombin generation assay. Its reversal effect on a variety of anticoagulants in vivo was investigated in a rat tail cut bleeding model. A possible prothrombotic action of OKL-1111 was assessed in a Wessler model in rabbits. ResultsOKL-1111 concentration-dependently reversed the in vitro anticoagulant effects of dabigatran, rivaroxaban, apixaban and edoxaban in the thrombin generation assay. Also in the absence of a DOAC, OKL-1111 concentration-dependently accelerated coagulation in this assay, but did not initiate coagulation. The reversal effect was also seen for all DOACs in the rat tail cut bleeding model. In addition, when tested with other anticoagulants, OKL-1111 also reversed the anticoagulant effect of the vitamin K antagonist warfarin, the low molecular weight heparin enoxaparin, the pentasaccharide fondaparinux and the platelet inhibitor clopidogrel in vivo. OKL-1111 did not have prothrombotic effects in the Wessler model. ConclusionOKL-1111 is a procoagulant cyclodextrin with a currently unknown working mechanism that has potential to become a universal reversal agent for anticoagulants and platelet inhibitors.

When Will Fondaparinux Induce Thrombocytopenia?

The pentasaccharide Fondaparinux, a synthetic selective factor Xa inhibitor, is one of the safest anticoagulants in the heparin family that is recommended as an alternative drug for patients with hypersensitivity to other drugs such as heparin-induced thrombocytopenia (HIT). However, some observations of Fondaparinux-induced thrombocytopenia (FIT) have been reported while others claimed that FIT does not occur in patients with fondaparinux therapy, indicating that the mechanism of FIT remains controversial. Here, we utilized different methodologies including dynamic light scattering, immunosorbent and platelet aggregation assays, confocal laser scanning microscopy, and flow cytometry to gain insights into FIT. We found that at a certain concentration, Fondaparinux formed sufficient large and stable complexes with PF4 that facilitated binding of the HIT-like monoclonal KKO antibody and enhanced platelet aggregation and activation. We proposed a model to describe the role of Fondaparinux concentration in the formation of complexes with platelet factor 4 and how it promotes the binding of KKO. Our results clarify controversial observations of FIT in patients as each contains a dissimilar PF4:Fondaparinux concentration ratio.

Chemical synthesis and pharmacological properties of heparin pentasaccharide analogues

The pentasaccharide fondaparinux is a synthetic anticoagulant based on heparin antithrombin-binding sequence. Fondaparinux improves safety and predictable pharmacodynamics compared with heparins; however, it requires a complicate synthesis process which contain more than 50 steps of synthesis. Herein, we designed and synthesized four fondaparinux analogues (compounds 1, 2, 3, 4) using a [2+3] convergent synthetic method, which greatly simplified the synthetic process, improved the product yield, and curtailed the expenditures. These synthesized compounds showed stronger anticoagulant activities by factor Xa inhibition (IC50 725–1126 nM vs. 1909 nM for fondaparinux) in the AT-dependent manner. After subcutaneous (s.c.) administration to rats, the compounds displayed long-lasting anti-factor Xa activities and inhibition of thrombin generation ex vivo. Compared with fondaparinux, these compounds were slowly eliminated after s.c. administration to rats, the half-lies (t1/2) were more than 2-fold of that of fondaparinux. These results suggested the pentasaccharide analogues may exhibit better pharmacokinetic and predictable pharmacodynamic characteristics.

Treatment of ChAdOx1 nCoV-19 Vaccine-Induced Immune Thrombotic Thrombocytopenia Related Acute Ischemic Stroke

Recently cases of vaccine-induced immune thrombotic thrombocytopenia (VITT) and thrombosis following the adenoviral vector vaccine against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were reported. A mechanism similar to heparin-induced thrombocytopenia was proposed with antibodies to platelet factor 4 (PF4). Vaccine related arterial thrombosis in the brain is rare but life-threatening and optimal treatment is not established. We report clinical, laboratory, imaging findings and treatment in a 51-year-old female presenting with acute left middle cerebral artery (MCA) occlusion 7 days after the first dose of ChAdOx1 nCoV-19 vaccine. Due to low platelet count and suspicion of VITT she was not eligible for intravenous thrombolysis (IVT) and proceeded to mechanical thrombectomy (MER) with successful recanalization four hours after onset of symptoms. Treatment with intravenous immunoglobulin (IVIG) and heparin pentasaccharide fondaparinux was initiated. Presence of anti-PF4 antibodies was confirmed. The patient improved clinically with normalization of platelet count. Clinicians should be alert of VITT in patients with acute ischemic stroke after ChAdOx1 nCov-19 vaccination and low platelet counts. MER showed to be feasible and effective. We propose considering MER in patients with VITT and large vessel occlusion despite thrombocytopenia. High-dose IVIG should be started immediately. Alternative anticoagulation to heparin should be started 24 hours after stroke onset unless significant hemorrhagic transformation occurred. Platelet transfusion is contraindicated and should be considered only in severe hemorrhagic complications. Restenosis or reocclusion of the revascularized artery is possible due to the hypercoagulable state in VITT and angiographic surveillance after the procedure is reasonable.

The challenge of structural heterogeneity in the native mass spectrometry studies of the SARS-CoV-2 spike protein interactions with its host cell-surface receptor.

Native mass spectrometry (MS) enjoyed tremendous success in the past two decades in a wide range of studies aiming at understanding the molecular mechanisms of physiological processes underlying a variety of pathologies and accelerating the drug discovery process. However, the success record of native MS has been surprisingly modest with respect to the most recent challenge facing the biomedical community—the novel coronavirus infection (COVID-19). The major reason for the paucity of successful studies that use native MS to target various aspects of SARS-CoV-2 interaction with its host is the extreme degree of heterogeneity of the viral protein playing a key role in the host cell invasion. Indeed, the SARS-CoV-2 spike protein (S-protein) is extensively glycosylated, presenting a formidable challenge for native MS as a means of characterizing its interactions with both the host cell–surface receptor ACE2 and the drug candidates capable of disrupting this interaction. In this work, we evaluate the utility of native MS complemented with the experimental methods using gas-phase chemistry (limited charge reduction) to obtain meaningful information on the association of the S1 domain of the S-protein with the ACE2 ectodomain, and the influence of a small synthetic heparinoid on this interaction. Native MS reveals the presence of several different S1 oligomers in solution and allows the stoichiometry of the most prominent S1/ACE2 complexes to be determined. This enables meaningful interpretation of the changes in native MS that are observed upon addition of a small synthetic heparinoid (the pentasaccharide fondaparinux) to the S1/ACE2 solution, confirming that the small polyanion destabilizes the protein/receptor binding.Graphical abstract

Synthetic Pentasaccharide Fondaparinux (Arixtra®) Reduces Sepsis Induced Coagulopathy and Promotes Survival in the Baboon Model of E. coli Sepsis

Bacterial sepsis triggers profound dysfunction of the coagulation and fibrinolytic systems leading to disseminated intravascular coagulation (DIC), characterized by consumptive coagulopathy and bleeding. DIC may contribute to multiple organ failures due to occlusion of microvasculature and tissue hypoperfusion. Bacteria and/or bacteria-derived molecules initiate the activation of both the intrinsic/contact phase and extrinsic pathways of coagulation that activate factor X (FX). Active FX (FXa) is the enzyme of the prothrombinase complex that leads to thrombin generation and subsequent formation of fibrin clots. Arixtra® (Fondaparinux Sodium), a synthetic pentasaccharide, binds to antithrombin and thereby selectively inhibits FXa. Here, a baboon model of E. coli sepsis was used to investigate the effect of FXa inhibition on sepsis-induced activation of coagulation and fibrinolytic pathways.Two experimental baboon groups were studied:(i) E. coli challenge (n=4) , (ii) E. coli + Arixtra®(n=4). E. coli (1-2 x 1010 CFU/kg, LD100 dose) was infused intravenously over a 2 hour period. Arixtra® (0.08mg/kg) was administered subcutaneously to achive therapeutic concentration at the time of E. coli challenge (T0). The activation of coagulation was studied by measuring plasma levels of inhibitory complexes of antithrombin (AT) with activated factors: FXa-AT, FXIa-AT, thrombin-AT (TAT) and FVIIa-AT.E. coli challenge induced the activation of coagulation pathway as early as 2 hours post-challenge as evident by increase in plasma level of FXa-AT and TAT complexes. Contact pathway activation (FXIa-AT), as well as FVII activation (FVIIa-AT), were significantly reduced in Arixtra® treated animals. FXa-AT and TAT complexes were maximally present at 4-8 hours post-challenge and were reduced by more than 50% in Arixtra® treated animals (Fig. 1, A and B). Similarly, Arixtra® reduced activated partial thromboplastin time and prothrombin time as well as fibrinogen consumption following E. coli challenge. In line with the above observation, the plasma levels of fibrinolysis markers, fibrinogen/fibrin degradation products, D-Dimer and plasmin-antiplasmin complex (PAP) were significantly reduced in the Arixtra® treated group (Fig. 1, C and D). Platelets consumption/thrombocytopenia was similar in both the Arixtra® treated and the untreated groups. Moreover, neutrophil activation as measured by myeloperoxidase activity in plasma was reduced in the Arixtra® treated group. Two out of four animals survived in Arixtra® treated groups compared to 100% mortality in untreated group [Log-rank (Mantel-Cox) test P=0.0037]. Notably, Arixtra® treated non-survivor animals demonstrated prolonged survival times.Overall, the data suggests that Arixtra® reduced both coagulation and fibrinolytic pathway activation, thereby preventing DIC and providing survival benefits in E. coli sepsis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Towards better understanding of the heparin role in NETosis: Feasibility of using native mass spectrometry to monitor interactions of neutrophil elastase with heparin oligomers

Neutrophil elastase is a serine protease released by neutrophils, and its dysregulation has been associated with a variety of debilitating pathologies, most notably cystic fibrosis. This protein is also a prominent component of the so-called neutrophil extracellular traps (NETs), whose formation is a part of the innate immunity response to invading pathogens, but also contributes to a variety of pathologies ranging from autoimmune disorders and inflammation to cancer to thrombotic complications in COVID-19. Retention of neutrophil elastase within NETs is provided by ejected DNA chains, although this protein is also capable of interacting with a range of other endogenous polyanions, such as heparin and heparan sulfate. In this work, we evaluate the feasibility of using native mass spectrometry (MS) as a means of studying interactions of neutrophil elastase with heparin oligomers ranging from structurally homogeneous synthetic pentasaccharide fondaparinux to relatively long (up to twenty saccharide units) and structurally heterogeneous chains produced by partial depolymerization of heparin. The presence of heterogeneous N-glycan chains within neutrophil elastase and the structural heterogeneity of heparin oligomers render the use of standard MS to study their complexes impractical. However, supplementing MS with limited charge reduction in the gas phase allows meaningful data to be extracted from MS measurements. In contrast to earlier molecular modeling studies where a single heparin-binding site was identified, our work reveals the existence of multiple binding sites, with a single protein molecule being able to accommodate up to three decasaccharides. The measurements also reveal the ability of even relatively short heparin oligomers to bridge two protein molecules, suggesting that characterization of these complexes using native MS can shed light on the structural properties of NETs. Lastly, the use of MS allows the binding preferences of heparin oligomers to neutrophil elastase to be studied with respect to specific structural properties of heparin, such as the level of sulfation (i.e., charge density). All experimental measurements are carried out in parallel with molecular dynamics simulations of the protein/heparin oligomer systems, which are in remarkable agreement with the experimental data and highlight the role of electrostatic interactions as dominant forces governing the formation of these complexes.

Structural characterization of fondaparinux interaction with per-6-amino-beta-cyclodextrin: An NMR and MS study

The highly anionic synthetic pentasaccharide fondaparinux (FDPX) – representing the antithrombin binding sequence of heparin – is in clinical use as a potent anticoagulant. Contrary to the unfractionated heparin, FDPX lacks potent antidote completely reversing its anticoagulant activity, therefore it is of great importance to identify new structures exhibiting strong intermolecular interactions towards FDPX. The polycationic heptakis(6-amino-6-deoxy)-beta-cyclodextrin (NH2-β-CD) can serve as an excellent model compound to mimic these interactions between the oppositely charged oligosaccharides. Herein, extensive NMR spectroscopic and nano-electrospray ionization mass spectrometric (nESI-MS) studies were conducted to understand the molecular-level interactions in the FDPX - NH2-β-CD systems. NMR experiments were performed at pD 7.4 and 2.0. Job’s method of continuous variation and 1H NMR titration experiments suggested the formation of FDPX∙NH2-β-CD complex at pD 7.4, while the presence of multiple complexes was assumed at pD 2.0. Stability constants were determined by separate 1H NMR titrations, yielding log β11=3.65 ± 0.02 at pD 7.4, while log β11 ≥ 4.9 value suggested a high-affinity system at pD 2.0. 2D NOESY NMR studies indicated spatial proximities between the anomeric resonance α-l-iduronic acid residue and the cyclodextrin’s methylene unit in the proximity of the cationic amino function. Acidic degradation of FDPX was investigated by NMR and MS for the first time in detail confirming that desulfation occurs involving one to two sulfate moieties. The desulfation of FDPX was inhibited by the cationic cyclodextrin in the case of equimolar ratio at pD 2.0. This is the first report on the stabilizing effect of cyclodextrin complexation on heparin degradation.

Characterization of defined sulfated heparin-like oligosaccharides by electrospray ionization ion trap mass spectrometry.

Glycosaminoglycans (GAG) as long, unbranched polysaccharides are major components of the extracellular matrix. Many studies provided additional evidence of a specific binding between mediators and sulfated GAG, at which the sulfation code-which means the number and positions of sulfate groups along the polysaccharide chain-plays an important role. GAG from natural sources are very inhomogeneous regarding their sulfation patterns and molecular weight. Additionally, there is a high risk of contamination. This results in a growing interest in the careful characterization of native GAG and the synthesis of artificial GAG. Additionally, chemically oversulfated GAG analogues show many favorable properties. However, the structural characterization of these carbohydrates by mass spectrometry remains challenging. One significant problem is the sulfate loss during the ionization, which increases with the number of sulfate residues. We used the sulfated pentasaccharide fondaparinux as model substance to optimize sample preparation and measurement conditions, compared different established desalination methods and already existing protocols for sulfated oligosaccharides, and investigated their impact on the quality of the mass spectra. After optimization of the measurement conditions, we could establish a gentle and fast protocol for the mass spectrometry characterization of (fully) sulfated, artificial GAG-like oligosaccharides with minimized sulfate loss in the positive and negative ion mode. Here, the negative ion mode was more sensitive in comparison with the positive one, and fondaparinux species with sulfate loss were not detectable under the optimized conditions in the positive ion mode.

Conformational Modulation of Iduronic Acid-Containing Sulfated Glycosaminoglycans by a Polynuclear Platinum Compound and Implications for Development of Antimetastatic Platinum Drugs.

1 H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution-inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1 C4 :2 S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA-MB-231 triple-negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti-metastatic potential.

Conformational Modulation of Iduronic Acid‐Containing Sulfated Glycosaminoglycans by a Polynuclear Platinum Compound and Implications for Development of Antimetastatic Platinum Drugs

Abstract1H NMR spectroscopic studies on the 1:1 adduct of the pentasaccharide Fondaparinux (FPX) and the substitution‐inert polynuclear platinum complex TriplatinNC show significant modulation of geometry around the glycosidic linkages of the FPX constituent monosaccharides. FPX is a valid model for the highly sulfated cell signalling molecule heparan sulfate (HS). The conformational ratio of the 1C4:2S0 forms of the FPX residue IdoA(2S) is altered from ca. 35:65 (free FPX) to ca. 75:25 in the adduct; the first demonstration of a small molecule affecting conformational changes on a HS oligosaccharide. Functional consequences of such binding are suggested to be inhibition of HS cleavage in MDA‐MB‐231 triple‐negative breast cancer (TNBC) cells. We further describe inhibition of metastasis by TriplatinNC in the TNBC 4T1 syngeneic tumour model. Our work provides insight into a novel approach for design of platinum drugs (and coordination compounds in general) with intrinsic anti‐metastatic potential.

Peracylation Coupled with Tandem Mass Spectrometry for Structural Sequencing of Sulfated Glycosaminoglycan Mixtures without Depolymerization.

The structures of glycosaminoglycans (GAGs), especially the patterns of modification, are crucial to modulate interactions with various protein targets. It is very challenging to determine the fine structures using liquid chromatography-mass spectrometry (LC-MS) due in large part to the gas-phase sulfate losses upon collisional activation. Previously, our group reported a method for fine structure analysis that required permethylation of the GAG oligosaccharide. However, uncontrolled depolymerization during the permethylation process due to esterification of uronic acid lowers the reliability of the method to resolve structures of GAGs, especially for larger oligosaccharides. Here, we describe a simplified derivatization method using propionylation and desulfation. The oligosaccharides have all hydroxyl and amine groups protected with propionyl groups and then have sulfate groups removed to generate unprotected hydroxyl and amine groups at all sites that were previously sulfated. This derivatized oligosaccharide generates informative fragments during collision-induced dissociation that resolve the original sulfation patterns. This method is demonstrated to enable accurate determination of sulfation patterns of even the highly sulfated pentasaccharide fondaparinux by MS2 and MS3. Using a mixture of dp6 from porcine heparin, we demonstrate that this method allows for structural characterization of complex mixtures, including clear chromatographic separation and sequencing of structural isomers, all at high yields without evidence of depolymerization. This represents a marked improvement in the reliability to structurally characterize GAG oligosaccharides over permethylation-based derivatization schemes.

Glycosaminoglycans as Tools to Decipher the Platelet Tumor Cell Interaction: A Focus on P-Selectin.

Tumor cell–platelet interactions are regarded as an initial crucial step in hematogenous metastasis. Platelets protect tumor cells from immune surveillance in the blood, mediate vascular arrest, facilitate tumor extravasation, growth, and finally angiogenesis in the metastatic foci. Tumor cells aggregate platelets in the bloodstream by activation of the plasmatic coagulation cascade and by direct contact formation. Antimetastatic activities of unfractionated or low molecular weight heparin (UFH/LMWH) can undoubtedly be related to attenuated platelet activation, but molecular mechanisms and contribution of contact formation vs. coagulation remain to be elucidated. Using a set of non-anticoagulant heparin derivatives varying in size or degree of sulfation as compared with UFH, we provide insight into the relevance of contact formation for platelet activation. Light transmission aggregometry and ATP release assays confirmed that only those heparin derivatives with P-selectin blocking capacities were able to attenuate breast cancer cell-induced platelet activation, while pentasaccharide fondaparinux was without effects. Furthermore, a role of P-selectin in platelet activation and signaling could be confirmed by proteome profiler arrays detecting platelet kinases. In this study, we demonstrate that heparin blocks tumor cell-induced coagulation. Moreover, we identify platelet P-selectin, which obviously acts as molecular switch and controls aggregation and secretion of procoagulant platelets.

Anticoagulation strategies in patients with deep vein thrombosis and pulmonary artery embolisms

Deep vein thrombosis and pulmonary artery embolisms share pathophysiological features and are therefore collectively referred to as venous thromboembolisms (VTE). While the incidence of VTE has been increasing for years as aresult of demographic changes and improved diagnostics, the morbidity and mortality are decreasing. This is particularly due to more sensitive diagnostics, improvements in risk stratification and more effective anticoagulation strategies. The aim of effective anticoagulation therapy is the avoidance of early events up to death and prevention of recurrent events. Anticoagulation treatment should be started with either heparins (unfractionated or low molecular weight), the pentasaccharide fondaparinux or direct oral anticoagulants. Patients with recurrent events qualify for indefinite anticoagulation treatment. For afirst episode of VTE anticoagulation treatment for at least 3months is recommended (maintenance therapy). Subsequently, prolonged maintenance therapy for secondary prevention can be meaningful, depending on the individual patient risk (provoked event, risk for recurrence or bleeding). The non-vitamin K antagonist oral anticoagulants (NOACs) have now also been approved for this indication. As aresult of a probably permanently high risk for recurrent events of up to 10% per year after cessation of anticoagulation, insufficient scores for estimation of the risk of bleeding and recent data documenting the safety and efficacy of NOACs for secondary prevention, ashift towards prolonged anticoagulation of 3-6months or even indefinite (>1 year) treatment can be anticipated for patients after thromboembolic diseases.

Use of Fondaparinux Off-Label or Approved Anticoagulants for Management of Heparin-Induced Thrombocytopenia

BackgroundLife-threatening heparin-induced thrombocytopenia (HIT) is treated with the alternative nonheparin anticoagulants argatroban, lepirudin, or danaparoid. Frequently, the pentasaccharide fondaparinux is used off-label. ObjectivesThe authors sought to investigate the safety and efficacy of the different anticoagulants for treating HIT. MethodsIn a national, multicenter registry study, hospitalized patients who were diagnosed with HIT, an at least intermediate clinical HIT-risk (4Ts score ≥4 points), and received treatment with ≥1 dose of the aforementioned anticoagulants were included. Main outcome measures were the incidences of HIT-specific complications (thromboembolic venous/arterial events, amputations, recurrent/persistent thrombocytopenia, skin lesions) and bleedings. ResultsOf 195 patients, 46 (23.6%), 4 (2.1%), 61 (31.3%), and 84 (43.1%) had been treated first-line with argatroban, lepirudin, danaparoid, and fondaparinux, respectively. The composite endpoint of HIT-specific complications (thromboembolic events, amputation, skin necrosis) occurred in 11.7% of patients treated with approved alternative anticoagulation and in 0.0% of fondaparinux-treated patients. The all-cause in-hospital mortality rates were 14.4% during approved alternative anticoagulation and 0.0% during fondaparinux treatment. Bleeding complications occurred in alternatively anticoagulated patients and in fondaparinux-treated patients in 6.3% and 4.8%, respectively. Post hoc analysis of clinical and laboratory features confirmed “true“ HIT in at least 74 of 195 (38.0%) patients; 35 of 74 (47.3%) were treated with fondaparinux. ConclusionsFondaparinux is effective and safe in suspected acute HIT; no HIT-specific complications occurred in the fondaparinux-treated patients, even among those with a high clinical HIT probability. Further data from randomized controlled trials are urgently needed because lepirudin was recalled from the market; danaparoid access has been limited and is not approved in the United States; and argatroban is contraindicated in patients with impaired liver function, and activated partial thromboplastin time confounding may interfere with monitoring. (Retrospective Registry of Patients With Acute Heparin-induced Thrombocytopenia Type II; NCT01304238)

Extended Physicochemical Characterization of the Synthetic Anticoagulant Pentasaccharide Fondaparinux Sodium by Quantitative NMR and Single Crystal X-ray Analysis.

Fondaparinux sodium is a synthetic pentasaccharide representing the high affinity antithrombin III binding site in heparin. It is the active pharmaceutical ingredient of the anticoagulant drug Arixtra®. The single crystal X-ray structure of Fondaparinux sodium is reported, unequivocally confirming both structure and absolute configuration. The iduronic acid adopts a somewhat distorted chair conformation. Due to the presence of many sulfur atoms in the highly sulfated pentasaccharide, anomalous dispersion could be applied to determine the absolute configuration. A comparison with the conformation of Fondaparinux in solution, as well as complexed with proteins is presented. The content of the solution reference standard was determined by quantitative NMR using an internal standard both in 1999 and in 2016. A comparison of the results allows the conclusion that this method shows remarkable precision over time, instrumentation and analysts.

Polyguluronate sulfate, polymannuronate sulfate, and their oligosaccharides have antithrombin III- and heparin cofactor II-independent anticoagulant activity

Cardiovascular disease is the leading causes of death. However, the complications can be treated with heparin and heparinoids, such as heparin pentasaccharide Fondaparinux, dermatan sulfate, and PSS made from alginate extracted from brown seaweeds by chemical sulfation. Alginate is composed of a linear backbone of polymannuronate (PM), polyguluronate (PG), and alternate residues of mannuronic acid and guluronic acid. It is unknown if heparin and sulfated PG (PGS)/PM (PMS) have the same or different anticoagulant molecular targets. In the current study, the anticoagulant activities of PGS, PMS, and their oligosaccharides were directly compared to that of heparin, Fondaparinux, and dermatan sulfate by the activated partial thrombinplastin time (aPTT) assay using normal, antithrombin III (ATIII)-deficient, heparin co-factor II (HCII)-deficient, and ATIII- and HCII-double deficient human plasmas. Our results showed that PGS, PMS, and their oligosaccharides had better anticoagulant activity than that of Fondaparinux in all four human plasmas tested. As expected, heparin was the best anticoagulant in normal plasma. Moreover, PGS, PGS6, PGS12, PGS25, PMS6, PMS12, and PMS25 were better anticoagulants than dermatan sulfate in HCII-deficient plasma. Most strikingly, PGS, PGS12, PGS25, PMS6, PMS12, and PMS25 were better anticoagulants than that of heparin in ATIII- and HCII-double deficient human plasma. The results revealed for the first time that sulfated alginate had ATIII- and HCII-independent anticoagulant activities. Therefore, developing PGS and PMS-based anticoagulants might require to discover their major molecular targets and to develop target-specific anticoagulant assays.

Antiangiogenic platinum through glycan targeting.

Heparan sulfate is identified as a ligand receptor for polynuclear platinum anti-cancer agents through sulfate cluster binding. We present a new biological role for platinum and coordination compounds and a new target for metal-based drugs while presenting a new chemotype for heparanase and growth factor inhibition through modulation (metalloshielding) of their interactions. Masking of extracellular (ECM)-resident heparan sulfate (HS) through metalloshielding results in very effective inhibition of physiologically critical HS functions including enzyme (heparanase, HPSE) and protein growth factor recognition. The interaction of the highly cationic polynuclear platinum complexes (PPCs) with the highly sulfated pentasaccharide Fondaparinux (FPX, in this case as a model HS-like substrate) results in inhibition of its cleavage by the HS-related enzyme heparanase. Binding of the fibroblast growth factor FGF-2 to HS is also inhibited with consequences for downstream signalling events as measured by a reduction in accumulation of phospho-S6 ribosomal protein in human colon tumor HCT-116 cells. The end-point of inhibition of HPSE activity and growth factor growth factor signaling is the prevention of cell invasion and angiogenesis. Finally these events culminate in inhibition of HCT-116 cell invasion at sub-cytotoxic concentrations and the process of angiogenesis. A competition assay shows that Fondaparinux can sequester the 8+ TriplatinNC from bound DNA, emphasising the strength of PPC-HS interactions. Altering the profile of platinum agents from cytotoxic to anti-metastatic has profound implications for future directions in the development of platinum-based chemotherapeutics.

Coagulation disorders

For clinicians, who are not experienced in hemostasis and thrombosis, coagulation and coagulation disorders remain a ‘book of seven seals’ (1). This may be due, at least in part, to the complexity of the hemostatic apparatus and its interaction with inflammatory processes and immune mechanisms, all of which require detailed theoretical knowledge, practical experience, and continuous medical education (CME). More recently, the use of direct oral anticoagulants, which have specific pharmacological targets and pharmacodynamics distinct from those of vitamin K antagonists, heparins, or the pentasaccharide fondaparinux, has created insecurities and concerns among clinicians. Some of these concerns result from the fact that these novel agents with their superior safety profile impact on standard coagulation screening tests (such as prothrombin time and activated partial thromboplastin time) in a way that is different from conventional anticoagulants. This theme issue of Hamostaseologie should facilitate the readers’ access to several acquired coagulation disorders and assist to overcome fundamental problems of understanding hemostasis or, metaphorically spoken, to ‘break some of the seals’ of hemostasis and thrombosis textbooks. As third part in a series of reviews, the current edition also reflects several highlights of the GTH Congress 2015 (2, 3, 4). In his article, Medcalf provides an upto-date view on fibrinolysis, demonstrating that tissue-type plasminogen activator (t-PA)-mediated conversion of plasminogen into plasmin is not solely designed to remove fibrin deposits and blood clots in a self-regulated proteolytic process (5). Importantly, it is shown that t-PA and plasmin have pleotrophic effects and other substrates than fibrin such as prion proteins, amyloid-β, and misfolded or aggregated proteins. Thus, t-PA can act in a nonfibrinolytic (i.e., plasmin-independent) manner and have multiple effects on cerebral functions (e.g., synaptic plasticity, neurotransmission, visual processing, learning and memory). The author therefore concludes that the “original concept for the fibrinolytic system, being focused on fibrin, is a massive understatement” (5). Tiede et al. report on recent advances in the management of acquired hemophilia A (AHA) (6). At an incidence of about 1.3 cases per million people (7), AHA is a rare but challenging bleeding disorder caused by inhibitory autoantibodies against coagulation factor (F) VIII. Apart from hemostatic management to control active bleeding, immunosuppressive treatment is mandatory in AHA to achieve remission. However, the outcome of AHA remains highly variable. Therefore, clinically valid prognostic factors for remission are urgently required. Recently, the GTH-AH (01/2010) study, a multicenter trial conducted under the leadership of Andreas Tiede, has demonstrated for the first time that residual FVIII activity and inhibitor