Potent Antithrombotic Activity Research Articles

Mutant IDH1 and thrombosis in gliomas.

Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30% of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0%). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90% in wild-type versus 2-6% in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

Antithrombotic and Antioxidant Activity of Amaranth Hydrolysate Obtained by Activation of an Endogenous Protease.

Ingestion of diets with antithrombotic and antioxidant components offer a convenient and effective way to prevent and reduce the incidence of cardiovascular diseases. The aim of the present work was to obtain an amaranth hydrolysate by the activation of an endogenous aspartic protease, to establish adequate experimental conditions, and to evaluate its antithrombotic and antioxidant activity in order to assess its potential application as an ingredient in functional foods. The results obtained not only confirmed the presence of an endogenous protease in the amaranth isolate, but also allowed us to select an adequate incubation conditions (pH2, 40°C, 16h). The hydrolysate obtained (degree of hydrolysis 5.3±0.4%) showed potential antithrombotic activity (IC50=5.9±0.1mg soluble protein/mL) and had more antioxidant activity than the isolate, indicating that the activation of the protease released bioactive peptides from amaranth proteins. Decreasing the pH is a simple and cheap process and is another way to obtain potential functional ingredients with bioactive compounds.

Amaranth peptides with antithrombotic activity released by simulated gastrointestinal digestion

Amaranth protein isolate was obtained and subjected to simulated gastrointestinal digestion to evaluate its potential antithrombotic activity. The protein isolate did not present fibrin clotting inhibition at the concentrations studied, whereas the hydrolysate (DH% = 51.1 ± 3.8%) exhibited inhibition of fibrin coagulation, showing a dose–response behaviour (IC50 = 0.23 ± 0.02 mg/mL), confirming that the enzymatic treatment was able to release bioactive peptides from amaranth proteins. A fraction with high antithrombotic activity was obtained from this hydrolysate, and resulted to be three times more potent than its original sample (IC50 = 0.07 ± 0.01 mg/mL). The absorption of this active fraction was studied with an in vitro peptides transport assay through intestinal epithelium and it was observed that some peptides are able to cross the Caco2-TC7 cell monolayer. Potentially bioactive peptides were found after sequencing them, and informatics tools allowed us to select and locate in their native molecules those peptides prone to inhibit thrombin activity.

TMIC-13MUTANTIDH1/2SUPPRESSES LOCAL AND SYSTEMIC THROMBOSIS IN GLIOMA PATIENTS

Mutations in isocitrate dehydrogenase 1 and 2 (mutIDH1/2) are common in gliomas, resulting in enzymes that produce D-2-hydroxyglutarate (D-2-HG). The effects of mutIDH1/2 on the microenvironment, including thrombosis, are not known. In discovery and validation cohorts of World Health Organization (WHO) grade II-IV gliomas from two institutions (total N = 317), microthrombi were present in 85-90% of wild-type gliomas but in only 2-6% of mutant gliomas (P < 0.0001), and their absence was an independent predictor of mutIDH1/2. Systemic hypercoagulation occurred in 25-30% of patients with wild-type IDH1/2 gliomas, but in 0% of patients with mutIDH1/2 gliomas (P < 0.0001). The prognostic strength of microthrombi was comparable to necrosis and microvascular proliferation, the current WHO criteria for a diagnosis of glioblastoma. In silico analysis of 428 gliomas from The Cancer Genome Atlas (TCGA) revealed that, in the coagulome, the mRNA with the strongest inverse relationship to mutIDH1/2 was F3, encoding tissue factor procoagulant (P = 1.06E-52). MutIDH1/2 gliomas in the TCGA also had significantly elevated F3 promoter methylation (P < 0.0001). In a separate tissue microarray containing 95 gliomas, mutIDH1/2 tumors showed dramatically reduced tissue factor protein expression (P < 0.0001). Additionally, D-2-HG inhibited human platelet aggregation by blocking intracellular calcium accumulation. In human in vitro and mouse in vivo models, mutIDH1 and D-2-HG lowered free serum calcium by up to 40% and markedly impaired coagulation (P < 0.0001). In sum, mutIDH1/2 enzymes have potent antithrombotic activity within gliomas and systemically. These findings have profound implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment and calcium homeostasis, and the pre-and postoperative management of these patients.

Synthesis and biological evaluation of methylated scutellarein analogs based on metabolic mechanism of scutellarin in vivo

Scutellarin (1) could be hydrolyzed into scutellarein (2) in vivo and then converted into methylated, sulfated and glucuronidated forms. In order to investigate the biological activities of these methylated metabolites, eight methylated analogs of scutellarein (2) were synthesized via semi-synthetic methods. The antithrombotic activities of these compounds were evaluated through the analyzation of prothrombin time (PT), activated partial thromboplastin time (APTT), thrombin time (TT) and fibrinogen (FIB). Their antioxidant activities were assessed by measuring their scavenging capacities toward 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and the ability to protect PC12 cells against H2O2-induced cytotoxicity. Furthermore, the physicochemical properties of these compounds including aqueous solubility and lipophilicity were also investigated. The results showed that 6-O-methylscutellarein (5) demonstrated potent antithrombotic activity, stronger antioxidant activity and balanced solubility and permeability compared with scutellarin (1), which warrants further development of 5 as a promising lead for the treatment of ischemic cerebrovascular disease.

A tryptophan derivative TD-26 attenuates thrombus formation by inhibiting both PI3K/Akt signaling and binding of fibrinogen to integrin αIIbβ3

The incidence and mortality of thrombotic disorders are rapidly increasing worldwide. The existing antithrombotic drugs, however, are associated with side effects, especially bleeding complications. Therefore, there remains a need for the development of more effective and safer antithrombotic agents. In this study, we discovered a new synthetic tryptophan derivative TD-26, producing potent inhibitory effect on platelet aggregation while without causing obvious bleeding risk. It has been shown that TD-26 inhibited platelet aggregation induced by ADP, thrombin, U46619 and collagen in vitro and suppressed the platelet aggregation induced by ADP ex vivo. Mechanism studies indicated that TD-26 inhibited platelet adhesion to fibrinogen-coated surfaces, blocked the binding of fibrinogen to integrin αIIbβ3 and reduced AktSer473 phosphorylation in platelet phosphatidylinositol 3-kinase (PI3K) signaling. Furthermore, TD-26 exhibited potent antithrombotic activity in vivo. In animal models, it decreased death of mice with acute pulmonary thrombosis by 90% and attenuated thrombosis weight by 60.3%, both at a dose of 3 mg/kg. Additionally, TD-26 did not obviously prolong bleeding time in mice. Taken together, our results reveal that TD-26 is a novel antithrombotic compound exhibiting both integrin αIIbβ3 inhibition and PI3K signaling blockage, with a low bleeding risk.

A Novel Direct Factor Xa Inhibitory Peptide with Anti-Platelet Aggregation Activity from Agkistrodon acutus Venom Hydrolysates.

Snake venom is a natural substance that contains numerous bioactive proteins and peptides, nearly all of which have been identified over the last several decades. In this study, we subjected snake venom to enzymatic hydrolysis to identify previously unreported bioactive peptides. The novel peptide ACH-11 with the sequence LTFPRIVFVLG was identified with both FXa inhibition and anti-platelet aggregation activities. ACH-11 inhibited the catalytic function of FXa towards its substrate S-2222 via a mixed model with a Ki value of 9.02 μM and inhibited platelet aggregation induced by ADP and U46619 in a dose-dependent manner. Furthermore, ACH-11 exhibited potent antithrombotic activity in vivo. It reduced paralysis and death in an acute pulmonary thrombosis model by 90% and attenuated thrombosis weight in an arterio-venous shunt thrombosis model by 57.91%, both at a dose of 3 mg/kg. Additionally, a tail cutting bleeding time assay revealed that ACH-11 did not prolong bleeding time in mice at a dose of 3 mg/kg. Together, our results reveal that ACH-11 is a novel antithrombotic peptide exhibiting both FXa inhibition and anti-platelet aggregation activities, with a low bleeding risk. We believe that it could be a candidate or lead compound for new antithrombotic drug development.

Monoterpenes and flavones from the leaves of Crataegus pinnatifida with anticoagulant activities

Hawthorn is becoming increasingly popular as a functional food that has numerous medicinal and nutritional properties. Antithrombotic activity-guided fractionation of hawthorn leaves resulted in the isolation of four new monoterpene glycosides (1–4) and a new phenolic glycoside (10) in addition to 10 known ones (2–9, 11–15). Based on physicochemical properties and spectral data, such as NMR and HRESIMS, the isolated compounds identified as pinnatifidanoside A–E (1–4, 10), byzantionoside B (5), (3S,5R,6R,7E,9R)-3,6-epoxy-7-megastigmen-5, 9-diol-9-O-β-D-glucopyranoside (6), (6S,7Z,9R)-roseoside (7), icariside B6 (8), linalool oxide β-D-glucoside (9), shanyenoside A (11), dihydrocharcone-2′-β-D-glucopyranoside (12), eriodectyol (13), vitexin (14), and 2″-O-rhamnosyl vitexin (15). In vitro and in vivo antithrombotic assays of compounds 1–15 were carried out and found that compounds 7, 13 and 15 exerted potent antithrombotic activity in vitro, which was in agreement with the in vivo results of their antithrombotic activity in a transgenic zebrafish system.

Structures and Anticoagulant Activities of the Partially Mild Acidic Hydrolysis Products of the Fucosylated Chondroitin Sulfate from Sea Cucumber Pearsonothuria graeffei

Fucosylated chondroitin sulfate from Pearsonothuria graeffei (fCS-Pg) is a unique glycosaminoglycan (GAG), which was reported to have potent antithrombotic and anticoagulant activities. In the present study, the native fCS-Pg was hydrolyzed by mild acid to improve its bioavailability. The structures of the acid-released and acid-resistant sulfated fucose branches of fCS-Pg before and after acidic hydrolysis were characterized by nuclear magnetic resonance spectroscopy (NMR) technology. The results showed the acid-released fucose residues included both α- and β-fucose conformation, containing 2,4-di-O-sulfated fucose (Fuc2,4S), 3,4-di-O-sulfated fucose (Fus3,4S), 3-O-sulfated fucose (Fuc3S), and 4-O-sulfated fucose (Fuc4S), with a mole ratio of 6:26:22:46, respectively. Their difference to the native fCS-Pg in the sulfation pattern of the backbone could be due to selective removal of the sulfated groups during the release of the fucose branches by acid. For the acid-resistant part, the sulfation patterns of fucose were similar to the native polysaccharides, but the signal strength of Fuc3,4S was obviously decreased, whereas Fuc2,4S was kept unchanged, indicating Fuc3,4S is more liable to acid. The anticoagulant activities of the acid-resistant part were tested in vitro, and the results showed that partial degradation could result in significant reduction of anticoagulant activity, which could help to lower the risk of bleeding when developing fCS as an antithrombotic drug.

Potential antithrombotic activity detected in amaranth proteins and its hydrolysates

Amaranth protein isolate and fractions were obtained and subjected to proteolysis in order to evaluate its potential antithrombotic activity. The proteins were first hydrolyzed with alcalase (pH 10, 37 °C) and then with trypsin (pH 8, 37 °C). The samples were characterized physicochemically and antithrombotic activity was evaluated using clotting tests (PT, TT and APTT) and the microplates assay. The fractions compared to the hydrolysates exhibited different electrophoretic profiles (tricine-SDSPAGE) and gel filtration chromatograms, evidencing the presence of different molecular species. The hydrolysis improved in every sample the bioactivity detected, excepting for the glutelin fraction, which exhibited the highest antithrombotic activity, significantly superior (p < 0.05) compared to the other fractions and the isolate. This behavior was observed in the two assays that analyzed the common path of the coagulation cascade at similar concentrations: TT (81.0 ± 8.5 s with a control of 19.5 ± 0.7 s) and microplate test (IC50 80 μg/mL), indicating a possible mechanism of action that involves the thrombin activity or the polymerization of fibrin monomers. The glutelin fraction showed a potential capacity to inhibit coagulation, appearing as a promising ingredient to formulate functional foods.

Preparation of the antithrombotic and antimicrobial coating through layer-by-layer self-assembly of nattokinase-nanosilver complex and polyethylenimine

The bifunctional coating with antithrombotic and antimicrobial activity was developed using nattokinase (NK) and nanosilver (AgNPs). Firstly, the adsorption interactions between NK and AgNPs were confirmed, and the composite particles of NK–AgNPs were prepared by adsorption of NK with AgNPs. At 5FU/mL of NK concentration, the saturation adsorption capacity reached 24.35FU/mg AgNPs with a high activity recovery of 97%, and adsorption by AgNPs also enhanced the heat stability and anticoagulant effect of NK. Based on the electrostatic force driven layer-by-layer self-assembly, the NK–AgNPs were further assembled with polyethylenimine (PEI) to form coating. UV–vis analysis showed that the self-assembly process was regular, and atom force microscopy analysis indicated that NK–AgNPs were uniformly embedded into the coating. The NK–AgNPs–PEI composite coating showed potent antithrombotic activity and antibacterial activity. This study developed a novel strategy to construct the bifunctional coating with antithrombotic and antimicrobial properties, and the coating material showed promising potential to be applied in the medical device.

SEPARATION AND PURIFICATION OF ANTIOXIDANT GLYCOGLYCEROLIPIDS FROM FRUIT OF CUCURBITA MOSCHATA BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY COUPLED WITH PREPARATIVE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Glyceroglycolipids, widely distributed in edible plants, possess potent antithrombotic, antiviral, antitumor, and anti-inflammatory activities. In this paper, a simple method based upon high-speed counter-current chromatography (HSCCC) coupled with preparative high-performance liquid chromatography (Pre-HPLC) was developed for the separation and purification of antioxidant glycoglycerolipids from the fruits of Cucurbita moschata (pumpkin). HSCCC separation was performed on a two-phase solvent system composed of dichloromethane–light petroleum (60–90°C)–ethanol–H2O (6:2:4:4, v/v). Semi-purified peak fractions from HSCCC separation were further purified by Pre-HPLC to yield six glycoglycerolipid monomers with purities of over 98%. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), 1H-NMR, and 13C-NMR. Their antioxidant activities in vitro were also evaluated through 1,1-diphenyl-2-picrylhydrazyl assays. Tetrasaccharide-containing glycoglycerolipids exhibited stronger antioxidant activity compared to that of butylated hydroxytoluene, and it can be assumed to be potentially useful as a safe antioxidant in food processing industries. The results indicate that the method is simple, fast, and efficient.

Enhanced Pharmacokinetics and Anti-Tumor Efficacy of PEGylated Liposomal Rhaponticin and Plasma Protein Binding Ability of Rhaponticin

Rhaponticin (RA) extracted from many medicinal plants has been demonstrated to possess a variety of pharmacological activities including potent antitumor, antitumor-promoting, antithrombotic, antioxidant and vasorelaxant activities. However, its poor solubility and bioavailability restricted the clinical application greatly. In this work, surface modification of liposome with polyethylene glycol (PEG) was developed with the purpose of improving the pharmacokinetics and anti-tumor efficacy of RA. PEGylated liposomal RA (PEGL-RA) was prepared by dry-film method. Its mean particle size was 89.3 +/- 8.7 nm, mean zeta potential was -4.1 mV and encapsulation efficiency was 67.4 +/- 6.4%. Moreover, the results of pharmacokinetic analysis showed that the time of maximum plasma concentration (Tmax) of PEGL-RA was about 4.5 times higher than that of free RA after oral administration due to the lower distribution into the gastrointestinal tract. The mean T1/2 value of PEGL-RA and free RA were 350.12 +/- 0.87 min and 180.02 +/- 1.96 min, respectively. The AUC0-->infinity ratio of PEGL-RA to free RA was about 2.41-fold (93.23 microg/mLmin/40.81 microg/mLmin). Plasma protein binding ability of RA was also studied. The results showed that RA with 33.6 microg/mL concentrations in human plasma and HSA achieved the percent of bound 39.23 +/- 3.78% and 22.28 +/- 2.34%, respectively. In the in vivo studies utilizing solid tumor-bearing rat, it was confirmed that PEGL-RA delivered remarkably larger amount of RA to tumor tissue and provided more significant anti-tumor activity than free RA. Furthermore, the cytotoxicity and fluorescence microscopic studies showed higher intracellular uptake of PEGL-RA than that of RA. In conclusion, PEGylated liposome was an effective delivery formulation for RA to increase the pharmacokinetics and therapeutic efficacy.

Two new compounds from Crataegus pinnatifida and their antithrombotic activities

One new sesquiterpene, (1α,4aβ,8aα)-1-isopropanol-4a-methyl-8-methylenedecahydronaphthalene (1), with one new phenylpropanoid, threo-2-(4-hydroxy-3,5-dimethoxyphenyl)-3-(4-hydroxy-3-methoxyphenyl)-3-ethoxypropan-1-ol (2), along with four known phenylpropanoids were isolated from Crataegus pinnatifida. The structures of compounds 1 and 2 were elucidated on the basis of 1D, 2D NMR analyses, and HR-ESI-MS. The antithrombotic activity in vitro of all isolates was assayed, and only compound 1 exhibited potent antithrombotic activity by inhibiting platelet aggregation in rat plasma by 81.4% at 1 mg/ml.

Low molecular weight dual inhibitors of factor Xa and fibrinogen binding to GPIIb/IIIa with highly overlapped pharmacophores

Dual antithrombotic agents acting as anticoagulants and aggregation inhibitors could have substantial advantages over currently prescribed combinations of antithrombotic drugs. Herein, we report compounds with moderate inhibitory activity for factor Xa and fibrinogen GPIIb/IIIa binding (both in the micromolar range). These compounds resulted from our efforts to merge the pharmacophores of selective factor Xa inhibitor rivaroxaban with a mimic of the Arg-Gly-Asp (RGD) sequence of fibrinogen to obtain designed multiple ligands with potential antithrombotic activity. Resulting from this study, a structurally novel class of submicromolar fibrinogen GPIIb/IIIa binding inhibitor bearing 1,2,4-oxadiazol-5(4H)-one moiety is also described.

P1 and P2' site mutations convert protease nexin-2 from a factor XIa inhibitor to a plasmin inhibitor

The kunitz protease inhibitor domain of PN2 (PN2KPI) is a potent and specific inhibitor (K(i) 0.5-2 nM) of factor XIa (FXIa) and inhibits cerebrovascular thrombosis in mice. To determine whether the antithrombotic properties of PN2KPI arise from its FXIa-inhibitory activity, we have now prepared mutant forms of PN2KPI. Mutations at the P1 (Arg(15)) site in combination with P2' (Met(17)) mutations profoundly affect inhibition of FXIa, plasmin, kallikrein, factor Xa and thrombin. The mutant proteins PN2KPI-R(15)K, -M(17)K, -R(15)K,M(17)K and -R(15)K,M(17)R lost inhibitory activity against FXIa (K(i) 34, 94, 3081 and 707 nM, respectively) and kallikrein (no inhibition) and gained inhibitory activity against plasmin (K(i) 108, 7, 8 and 8 nM, respectively). The intravenous administration of rPN2KPI into mice dramatically decreased thrombus formation in a murine model of FeCl(3)-induced carotid injury, whereas rPN2KPI-R(15)K,M(17)K failed to inhibit thrombus formation. Molecular modelling studies showed that fine structural variations explain the observed functional differences in FXIa and plasmin inhibition. PN2KPI has potent antithrombotic activity due to its specific FXIa anticoagulant activity, whereas PN2KPI-R(15)K,M(17)K and PN2KPI-R(15)K,M(17)R have potent antifibrinolytic (antiplasmin) activity without anticoagulant or antithrombotic activity.

Enhanced Pharmacokinetics and Anti-Tumor Efficacy of PEGylated Liposomal Rhaponticin and Plasma Protein Binding Ability of Rhaponticin

Rhaponticin (RA) extracted from many medicinal plants has been demonstrated to possess a variety of pharmacological activities including potent antitumor, antitumor-promoting, antithrombotic, antioxidant and vasorelaxant activities. However, its poor solubility and bioavailability restricted the clinical application greatly. In this work, surface modification of liposome with polyethylene glycol (PEG) was developed with the purpose of improving the pharmacokinetics and anti-tumor efficacy of RA. PEGylated liposomal RA (PEGL-RA) was prepared by dry-film method. Its mean particle size was 89.3 +/- 8.7 nm, mean zeta potential was -4.1 mV and encapsulation efficiency was 67.4 +/- 6.4%. Moreover, the results of pharmacokinetic analysis showed that the time of maximum plasma concentration (Tmax) of PEGL-RA was about 4.5 times higher than that of free RA after oral administration due to the lower distribution into the gastrointestinal tract. The mean T1/2 value of PEGL-RA and free RA were 350.12 +/- 0.87 min and 180.02 +/- 1.96 min, respectively. The AUC0-->infinity ratio of PEGL-RA to free RA was about 2.41-fold (93.23 microg/mLmin/40.81 microg/mLmin). Plasma protein binding ability of RA was also studied. The results showed that RA with 33.6 microg/mL concentrations in human plasma and HSA achieved the percent of bound 39.23 +/- 3.78% and 22.28 +/- 2.34%, respectively. In the in vivo studies utilizing solid tumor-bearing rat, it was confirmed that PEGL-RA delivered remarkably larger amount of RA to tumor tissue and provided more significant anti-tumor activity than free RA. Furthermore, the cytotoxicity and fluorescence microscopic studies showed higher intracellular uptake of PEGL-RA than that of RA. In conclusion, PEGylated liposome was an effective delivery formulation for RA to increase the pharmacokinetics and therapeutic efficacy.

Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

AbstractEP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (kon > 107M−1s−1), a high affinity (KI = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (KD = 30nM) and activated the serpin to rapidly (kass ∼ 106M−1s−1) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.

Heparin-induced Leukocytosis Requires 6-O-Sulfation and Is Caused by Blockade of Selectin- and CXCL12 Protein-mediated Leukocyte Trafficking in Mice

Leukocytosis refers to an increase in leukocyte count above the normal range in the blood and is a common laboratory finding in patients. In many cases, the mechanisms underlying leukocytosis are not known. In this study, we examined the effects, the structural determinants, and the underlying mechanisms of heparin-induced leukocytosis, a side effect occurring in 0.44% of patients receiving heparin. We observed that heparin induced both lymphocytosis and neutrophilia, and the effects required heparin to be 6-O-sulfated but did not require its anticoagulant activity. Cell mobilization studies revealed that the lymphocytosis was attributable to a combination of blockage of lymphocyte homing and the release of thymocytes from the thymus, whereas the neutrophilia was caused primarily by neutrophil release from the bone marrow and demargination in the vasculature. Mechanistic studies revealed that heparin inhibits L- and P-selectin, as well as the chemokine CXCL12, leading to leukocytosis. Heparin is known to require 6-O-sulfate to inhibit L- and P-selectin function, and in this study we observed that 6-O-sulfate is required for its interaction with CXCL12. We conclude that heparin-induced leukocytosis requires glucosamine 6-O-sulfation and is caused by blockade of L-selectin-, P-selectin-, and CXCL12-mediated leukocyte trafficking.

Evaluation of acute and subchronic toxicity of a non-anticoagulant, but antithrombotic algal heterofucan from the Spatoglossum schröederi in Wistar rats

Fucan is a term used to denominate a family of sulfated polysaccharides rich in L-fucose. The brown alga Spatoglossum schröederi, Dictyotaceae, synthesizes three heterofucans named A, B, and C. Fucan A is a non-anticoagulant heterofucan which possesses potent antithrombotic (in vivo) and antiproliferative (in vitro) activities. However, its toxicity in vivo has not been determined. The present study examined the acute and subchronic toxicity of the fucan A in Wistar rats after subcutaneous administration. After that, the animals were killed and examined. The results showed in the acute study that fucan A did not cause general adverse effects and mortality in the concentrations 0, 20, 100, 1000, and 2000 µg/g body weight per rat for seven days. Regarding the subchronic study, the data showed that the fucan A did not cause any change in hematological and biochemistry parameters, as well as in the morphology, and in the size of the rat's organs analyzed at a concentration of 20 µg/g body weight per rat during a 62-day period. In conclusion, this study indicates this heterofucan is a compound with potential pharmacological value that has no toxicity in vivo.