Application Of FeCl3 Research Articles

Sustainable treatment of different high-strength cheese whey wastewaters: an innovative approach for atmospheric CO2 mitigation and fertilizer production.

Raw cheese whey wastewater (CWW) has been treated by means of FeCl3 coagulation-flocculation, NaOH precipitation, and Ca(OH)2 precipitation. Three different types of CWW were considered: without cheese whey recovery (CWW0), 60% cheese whey recovery (CWW60), and 80% cheese whey recovery (CWW80). Cheese whey recovery significantly influenced the characteristics of the wastewater to be treated: organic matter, solids, turbidity, conductivity, sodium, chloride, calcium, nitrogen, potassium, and phosphorus. Initial organic load was reduced to values in the interval of 60-70%. Application of FeCl3, NaOH, or Ca(OH)2 involved additional chemical oxygen demand (COD) depletions regardless of the CWW used. Under optimum conditions, the combination of 80% cheese whey recovery and lime application led to 90% reduction in COD. Turbidity (99.8%), total suspended solids (TSS) (98-99%), oils and fats (82-96%), phosphorus (98-99%), potassium (96-97%), and total coliforms (100%) were also reduced. Sludge generated in the latter process showed excellent settling properties. This solid after filtration and natural evaporation can be used as fertilizer with limitations due to its saline nature. In an innovative, low-cost, and environmentally friendly technology, supernatant coming from the Ca(OH)2 addition was naturally neutralized in 4-6days by atmospheric CO2 absorption without reagent addition. Consequently, a final aerobic biodegradation step can be applied for effluent polishing. This technology also allows for some atmospheric CO2 mitigation. Time requirement for the natural carbonation depends on the effluent characteristics. A precipitate rich in organic matter and nutrients and depletions of solids, sodium, phosphorus, magnesium, Kjeldahl, and ammoniacal nitrogen were also achieved during the natural carbonation.

Effect of soil washing with only chelators or combining with ferric chloride on soil heavy metal removal and phytoavailability: Field experiments.

In a field experiment on multi-metal contaminated soil, we investigated the efficiency of Cd, Pb, Zn, and Cu removal by only mixture of chelators (MC) or combining with FeCl3. After washing treatment, a co-cropping system was performed for heavy metals to be extracted by Sedum alfredii and to produce safe food from Zea mays. We analyzed the concentration of heavy metals in groundwater to evaluate the leashing risk of soil washing with FeCl3 and MC. Results showed that addition of FeCl3 was favorable to the removal of heavy metals in the topsoil. Metal leaching occurred mainly in rain season during the first co-cropping. The removal rates of Cd, Zn, Pb, and Cu in topsoil were 28%, 53%, 41%, and 21% with washing by FeCl3+MC after first harvest. The application of FeCl3 reduced the yield of S. alfredii and increased the metals concentration of Z. mays in first harvest. However, after amending soil, the metals concentration of Z. mays in FeCl3+MC treatment were similar to that only washing by MC. The grains and shoots of Z. mays were safe for use in feed production. Soil washing did not worsen groundwater contamination during the study period. But the concentration of Cd in groundwater was higher than the limit value of Standard concentrations for Groundwater IV. This study suggests that soil washing using FeCl3 and MC for the remediation of multi-metal contaminated soil is potential feasibility. However, the subsequent measure to improve the washed soil environment for planting crop is considered.

Structural and optical characterization of hybrid ZnO/polymer core‐shell nanowires fabricated by oxidative chemical vapour deposition

AbstractPolycrystalline (pc) n‐doped ZnO layers and n‐doped ZnO nanowires are coated with the p‐doped organic poly‐mers polypyrrole and poly(3,4‐ethylenedioxythiophene) (PEDOT) by oxidative chemical vapour deposition. The structural characterization of the polypyrrole‐coated pc‐ZnO layer shows that the thickness and homogeneity of the polymer shell depends on the amount of the oxidizing agent FeCl3. Electrical measurements of the formed pn‐junction prepared with 0.1 g and 0.3 g FeCl3, show ideality factors of η = 1.7 and η = 4, representing the quality of the formed diode (ideal case η = 1), which is decreasing with increasing amount of oxidizing agent. Structural studies of the hybrid core‐shell nanowires reveal that a thin and homogeneous coating of the nanowires is achieved by using 0.1 g FeCl3. Energy dispersive X‐ray measurements confirm that chlorine is incorporated into the polymer shell. Optical characterization of the hybrid structures via photoluminescence measurements show a broadening of the exciton lines of the ZnO nanowires after polymer coating (especially for PEDOT) which is attributed to etching processes of the wire surface occurring during the application of FeCl3, whereas, the general optical properties of ZnO are not affected. (© 2016 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Discovery of a Novel, Potent, Selective and Injectable Small Molecule Inhibitor of Blood Coagulation Factor XIa, ONO-8610539: in Vitro and in Vivo Pharmacological Profiles

Introduction: Congenital human blood coagulation factor XI (FXI) deficiency is associated with bleeding that is less severe than that observed in patients with other coagulation factor deficiencies, whereas severe FXI deficiency confers the decreased risk of deep vein thrombosis. FXI knockout mice show a reduction in venous thrombus formation with no bleeding. Therefore, FXI is considered to be a promising drug target for treatment and prevention of venous thromboembolism without increasing bleeding risk. Recently, we discovered a novel, potent, selective and injectable small molecule inhibitor of activated FXI (FXIa), ONO-IG-012, and evaluated the in vitro and in vivo pharmacological profiles of the compound.Methods: In anin vitro study, inhibitory effects of ONO-IG-012 on enzyme activities of human FXIa, other blood coagulation factors, and fibrinolytic factors were evaluated. Anticoagulant effects of ONO-IG-012 were also evaluated in human and rabbit plasma. In in vivo studies, the antithrombotic and hemorrhagic effects of ONO-IG-012 were compared to those of enoxaparin in rabbit models of deep venous thrombosis and femur hemorrhage. In the thrombosis model, under ketamine and xylazine anesthesia, inferior vena cava was isolated and partially ligated to reduce blood flow. The vein at the distal site was wrapped with filter paper saturated with ferric chloride (FeCl3) solution for 15 minutes to induce endothelial injury and subsequent thrombus formation. Sixty minutes after the application of FeCl3, the thrombus wet weight was measured. In the bleeding model, a puncture wound was made into the medullary canal at the epiphysis of femur using a drill under isoflurane anesthesia and mechanical ventilation. Blood was continuously collected with absorbent cotton for one hour, and the blood loss volume was calculated from its specific gravity. Intravenous administration of ONO-IG-012 or enoxaparin was initiated as a loading dose an hour before applying FeCl3 or producing a puncture wound, followed by their maintenance dose infusion. Blood was collected to measure APTT and PT just before the administration of the compounds and applying FeCl3 or producing a puncture wound.Results: ONO-IG-012 competitively inhibited human FXIa with a Ki value of 0.0019 μmol/L. Although ONO-IG-012 moderately inhibited human plasma kallikrein with a Ki value of 0.15 μmol/L, it had little effect on other human blood coagulation factors, and fibrinolytic factors [thrombin, FVIIa, FIXa, FXa, FXIIa, tPA, urokinase, and plasmin (Ki value >100 μmol/L)]. ONO-IG-012 prolonged APTT, and the concentration required to double the APTT was 0.098 μmol/L in human plasma and 0.30 μmol/L in rabbit plasma. However, prolongation of PT was not observed even at 33 μmol/L. ONO-IG-012 inhibited thrombus formation even at a dose as low as 0.1 mg/kg/h and achieved maximum antithrombotic effect at greater than or equal to 0.3 mg/kg/h. The ex vivo APTT was increased from baseline by 1.7 ± 0.0-fold at 0.1 mg/kg/h, by 2.8 ± 0.3-fold at 0.3 mg/kg/h, and by 5.4 ± 0.4-fold at 1 mg/kg/h, while PT showed no changes at any of the concentrations tested. Enoxaparin also inhibited thrombus formation at a dose of 10 IU/kg/h, and the antithrombotic effect at 30 IU/kg/h was comparable to that of ONO-IG-012 at 0.3 mg/kg/h (−87% vs. −80%). ONO-IG-012 did not affect the blood loss volume at all even at 10 mg/kg/h, which is 33-fold higher than the dose showing maximum antithrombotic effect (0.3 mg/kg/h). At 10 mg/kg/h, the blood loss volume was 0.7 ± 0.1 mL which is not statistically significant as compared to 1.2 ± 0.3 mL in the vehicle group, and the APTT ratio was 9.0 ± 1.1-fold. In contrast, enoxaparin increased the blood loss volume dose-dependently with the values of 4.5 ± 1.8 mL (not statistically significant) at 10 IU/kg/h, 8.4 ± 2.2 mL (P <0.01) at 30 IU/kg/h, and 23.1 ± 5.0 mL (P <0.001) at 100 IU/kg/h.Conclusions: ONO-IG-012 demonstrated a competitive, highly selective and potent inhibitory effect on FXIa among proteases involved in blood coagulation or fibrinolysis and a potent anticoagulant effect on APTT. ONO-IG-012 did not affect the blood loss volume at all even at 33-fold higher dose than the dose showing the maximum antithrombotic effect comparable to enoxaparin. ONO-IG-012 is expected to be a novel potent anticoagulant without an increased risk of bleeding for the treatment and prevention of venous thromboembolism. DisclosuresNo relevant conflicts of interest to declare.

Evidence for vivianite formation and its contribution to long-term phosphorus retention in a recent lake sediment: a novel analytical approach

Abstract. Vivianite, Fe3(PO4)2 · 8 H2O, is a ferrous iron phosphate mineral which forms in waterlogged soils and sediments. The phosphorus (P) bound in its crystal lattice is considered to be immobilised because vivianite is stable under anoxic, reducing, sedimentary conditions. Thus, vivianite formation can make a major contribution to P retention during early diagenesis. Much remains unknown about vivianite in sediments, because technical challenges have rendered direct identification and quantification difficult. To identify vivianite and assess its significance for P burial during early diagenesis we studied the consequences of a 1992/1993 in-lake application of FeCl3 and Fe(OH)3 aimed at restoring Lake Groß-Glienicke (Berlin, Germany). In a novel approach, we firstly applied a heavy-liquid separation to the iron-rich surface sediments which allowed direct identification of vivianite by X-ray diffraction in the high-density (ρ &gt; 2.3 g cm−3) sediment fraction. Secondly, we assessed the contribution of vivianite to P retention, combining results from chemical digestion with magnetic susceptibility data derived from magnetic hysteresis measurements. Scanning electron microscopy revealed that the dark blue spherical vivianite nodules were 40–180 μm in diameter, and formed of platy- and needle-shaped crystal aggregates. Although equilibrium calculations indicated supersaturation of vivianite throughout the upper 30 cm of the sediment, the vivianite deposits were homogeneously distributed within, and restricted to, the upper 23 cm only. Thus, supersaturated pore water alone cannot serve as a reliable predictor for the in situ formation of vivianite. In Lake Groß -Glienicke, vivianite formation continues to be triggered by the artificial iron amendment more than 20 yr ago, significantly contributing to P retention in surface sediments.

Anti-thrombotic Effects of Modified using a FeCl-induced Carotid Arterial Thrombosis Model

Objectives: The aim of this study was to examine the antithrombotic effects of the four herbal ingredients (Mume Fructus, MF; Santali Albi Lignum, SAL; Amomi Tsao-Ko Fructus, ATF; and Amomi Fructus, AF) of modified Jeho-tang (MJHT) in a ferric chloride (FeCl₃)-induced carotid arterial thrombosis model. Methods: Thirty minutes prior to a 35% FeCl₃ application, Sprague-Dawley (SD) rats were injected with saline, MF, SAL, ATF or AF (100 mg/kg, intraperitoneal injection), respectively. The effect of the MJHT ingredients was examined for time to occlusion (TTO) and thrombus weight (TW) in a FeCl₃-induced thrombosis model. Histological analysis was performed to examine the effect of the MJHT ingredients on collagen fiber damage using hematoxylin & eosin and Masson’s trichrome staining. Results: Compared with vehicle treatment, MF, SAL and ATF treatment delayed TTO (vehicle, 8.11 ± 0.60 min; MF, 16.67 ± 1.03 min; SAL, 17.50 ± 1.52 min and ATF, 13.33 ± 1.21 min; P Conclusions: Altogether, these results are the first evidence that the MJHT ingredients MF, SAL and ATF have the ability to prevent vascular damage and thrombus formation in FeCl₃-induced carotid arterial thrombosis.

Troubleshooting the rabbit ferric chloride-induced arterial model of thrombosis to assess in vivo efficacy of antithrombotic drugs

IntroductionThe FeCl3-induced arterial model of thrombosis is one of the most widely used animal models to assess arterial efficacy of new antithrombotic drug candidates. This model is well-established in rodents but in a less extent in the rabbit. In this work, we present a methodology for a rabbit FeCl3-induced arterial model of thrombosis derived from our troubleshooting which allows the generation of reliable efficacy data for new antithrombotic drug candidates. MethodsRabbits were administered with heparin 4.5U/kg/min, argatroban 10μg/kg/min or saline by intravenous infusion. The blood flow was monitored using a Doppler flow probe. The time from the application of FeCl3 to the recorded zero blood flow was defined as the time to occlusion, with a maximum recording time of 60min post-FeCl3 application. After 30min of infusion, thrombosis was induced by wrapping a FeCl3-saturated filter paper around the carotid artery caudal to the flow probe. Animals were subject to exclusion criteria based on the visual aspect of the artery FeCl3-induced injury and based on changes in blood flow upon FeCl3 application. ResultsFollowing the application of FeCl3, a mean time to occlusion for saline, heparin and argatroban of 24.3±1.8, 52.5±4.8 and 53.5±4.5min was obtained, respectively. Mean time to occlusion for heparin and argatroban administered groups was significantly different when compared to the saline-treated group (p<0.05). These results for the test compounds represent approximately 80% of the maximum possible prolongation. DiscussionThe rabbit FeCl3-induced arterial model of thrombosis presented in this paper derived from our troubleshooting is sensitive and reproducible for the generation of accurate and reliable efficacy data in the assessment of new antithrombotic agents in preclinical drug development.

Efficacy of Baxter's Longer Acting rFVIII (BAX 855) in Mouse Models of Hemophilia A

Abstract 4350Factor VIII (FVIII) is a critical component of the intrinsic coagulation pathway. FVIII concentrates are used in patients with hemophilia A to provide a hemostatic FVIII level sufficient to treat and prevent bleeding episodes. Multiple prophylactic administrations per week are necessary to maintain a FVIII level of at least 1% of normal to effectively prevent or reduce spontaneous bleeding episodes. A longer acting FVIII concentrate would reduce the frequency of infusions. Baxter and Nektar have developed a longer-acting PEGylated variant (BAX 855) of Baxter's recombinant FVIII (ADVATE process). The aim of the presented studies was to evaluate the efficacy of BAX 855 in two primary pharmacodynamics in hemophilia A mice: a tail-tip bleeding model (TTBM) and a carotid occlusion model (COM).BAX 855 was tested at a dose of 200 IU/kg rFVIII. ADVATE™ served as the active reference item, and formulation buffer as the negative control item. Mice received intravenous treatment with either BAX 855 or ADVATE™ 12–40h prior to the experiment. Buffer was administered 5–15min prior to the experiment.Animals were anesthetized using ketamine and xylazine. In the TTBM (n=16) the tip of the tail was cut off and total blood loss [mg] was assessed over 60 minutes. In the COM study (n=10) the left carotid artery was exposed and the endothelium was denuded by topical application of FeCl3. Time to occlusion [min] was assessed.Buffer-treated animals had a median blood loss of 951 mg in the TTBM and did not show vessel occlusion within 30 min in the COM. In the TTBM, a prophylactic effect of ADVATE™ could be observed for up to 18 h, leading to a reduction in blood loss to 121 mg after this treatment interval. Prophylactic efficacy after treatment with BAX 855 could be observed for up to 40 h with a median blood loss of 73 mg after 30 h and 436 mg after 40 h. In the COM, treatment with ADVATE™ 12 h before the experiment shortened the time to occlusion to 3.8 min. With BAX 855, a similar efficacy could be observed for up to 24 h after administration (5.2 min).In summary, the results of our studies show that BAX 855 is efficacious in these two mouse models of hemophilia A and that the efficacy of BAX 855 is prolonged compared with an unmodified rFVIII. Furthermore, the intravenous injection of BAX 855 was well tolerated in all animals across all treatment groups without any signs of acute toxicity. Disclosures:Schiviz:Baxter Innovations GmbH: Employment. Barbara:Baxter Innovations GmbH: Employment. Lawo:Baxter Innovations GmbH: Employment. Ehrlich:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Turecek:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment. Hoellriegl:Baxter Innovations GmbH: Employment.

Infusion of Recombinant Full-Length ADAMTS13 and Carboxyl-Terminal Truncated Variant Alters Composition of Ferric Chloride-Induced Arterial Thrombi in Adamts13−/− mice

Abstract 2313Proteolytic cleavage of ultra large von Willebrand factor (ULVWF) released from endothelial cells by ADAMTS13 metalloprotease is critical for maintaining normal hemostasis. However, the effect of infusing ADAMTS13 on thrombus composition remains poorly understood. In this study, we determined the morphology and composition of thrombi formed in carotid arteries after topical application of FeCl3 in Adamts13−/− mice receiving PBS, recombinant human full-length ADAMTS13 (FL) and carboxyl-terminal truncated variant after spacer domain (S), using scanning electron microscopy and quantitative image analysis. We showed that in Adamts13−/− mice 5 min after FeCl3 injury, formed arterial thrombi were comprised ∼39% platelets, ∼26% red blood cells, and ∼35% fibrin. The arterial thrombi in these mice were structurally deformed. An infusion of recombinant FL (final concentration of 10 nM) significantly reduced the accumulation of platelets (∼18%) but increased the fibrin network (57%) without affecting the composition of red blood cells (∼25%) in the arterial thrombi formed at the same time point after FeCl3 injury. Similar effects on the morphology and composition of FeCl3-induced arterial thrombi were observed after infusion of recombinant S (10 nM) into Adamts13−/− mice. Kinetic analysis showed that there was a decrease in platelet accumulation over the time of 30 min during thrombus formation with a slight increase in accumulation of red blood cells and formation of fibrin in Adamts13−/− mice. But, the infusion of recombinant FL and S into Adamts13−/− mice restored the kinetics of platelet/red blood cell accumulation and fibrin formation to those observed in wild-type mice. Our findings, revealing the apparent difference in thrombus composition, provide novel insight into the mechanism of ADAMTS13 function in vivo, which may shed more light on the pathogenesis of thrombotic thrombocytopenic purpura and other arterial thrombotic disorders associated with deficiency of plasma ADAMTS13 activity. Disclosures:No relevant conflicts of interest to declare.

Comparison of antithrombotic efficacy between edoxaban, a direct factor Xa inhibitor, and fondaparinux, an indirect factor Xa inhibitor under low and high shear rates

Edoxaban is an oral, direct factor Xa (FXa) inhibitor under late-phase clinical development. This study compared the antithrombotic efficacy of edoxaban with that of an indirect FXa inhibitor, fondaparinux, in in vivo venous and arterial thrombosis models and in ex vivo perfusion chamber thrombosis model under low and high shear rates in rats. Venous and arterial thrombi were induced by platinum wire insertion into the inferior vena cava and by application of FeCl₃ to the carotid artery, respectively. The perfusion chamber thrombus was formed by blood perfusion into a collagen-coated capillary at 150 s⁻¹ (low shear rate) and 1,600 s⁻¹ (high shear rate). Effective doses of edoxaban that reduced thrombus formation by 50% (ED₅₀) in venous and arterial thrombosis models were 0.076 and 0.093 mg/kg/h, respectively. In contrast, ED₅₀ of fondaparinux in the arterial thrombosis model (>10 mg/kg/h) was markedly higher compared to ED₅₀ in the venous thrombosis model (0.021 mg/kg/h). In the perfusion chamber thrombosis model, the ratio of ED₅₀ under high shear rate (1.13 mg/kg/h) to that under low shear rate (0.63 mg/kg/h) for edoxaban was 1.9, whereas that for fondaparinux was more than 66. While the efficacy of fondaparinux markedly decreased in arterial thrombosis and in a high-shear state, edoxaban exerted consistent antithrombotic effects regardless of flow conditions. These results suggest that shear rate is a key factor in different antithrombotic effects between edoxaban and fondaparinux.

Research Article: The Effect of Ferric Chloride Addition for Phosphorus Removal on Ultraviolet Radiation Disinfection of Wastewater

In light of the potential need for disinfection and nutrient removal at the Metropolitan Water Reclamation District of Greater Chicago's water reclamation plants (WRPs), the inhibitory effects of ferric chloride (FeCl3) on ultraviolet (UV) radiation disinfection was examined. Results of a 2007 preliminary study indicated that a 2-log reduction in fecal coliform (FC) concentrations for each WRP secondary effluent was achieved with a UV dose of 10 mJ/cm2 except for the John E. Egan (Egan) WRP, where FeCl3 addition in the aeration basin for phosphorus removal was being employed. A subsequent study examined the effect of Egan unfiltered (secondary) and filtered (final) effluent on UV disinfection in 2008 during FeCl3 application and 2009 upon cessation of FeCl3 application: FeCl3 addition and filtration had little effect on UV transmittance (UVT). However, with FeCl3 addition, secondary and final effluent had higher total iron and lower total phosphorus relative to the respective secondary and final effluent without FeCl3 addition. Final effluent with and without FeCl3 addition showed lower total suspended solids concentrations relative to secondary effluent. Collimated beam test results indicated that a higher UV dose was needed to achieve the desired log reductions with FeCl3 addition and secondary effluent. Therefore, filtration of the effluent may be required if FeCl3 addition for nutrient removal is used at WRPs that employ UV disinfection.

Discovery of a New Signaling Complex Based on Spinophilin That Regulates Platelet Activation In Vitro and In Vivo

AbstractAbstract 161Platelets play an essential role in hemostasis, but excessive platelet responses to vascular injury or disease can be catastrophic. We have recently reported that members of the RGS protein family can modulate platelet responses to injury by limiting the duration of G protein-dependent signaling initiated by platelet agonists. Here we show that platelets contain a previously-unrecognized regulatory complex comprised of the 130 kDa scaffold protein, spinophilin (SPL) with at least two RGS proteins (RGS10 and RGS18) and the protein tyrosine phosphatase, SHP-1. In resting platelets, this complex is phosphorylated on spinophilin residues Y398 and Y483. Mutating both tyrosines to phenylalanine inhibits the binding of SHP-1 to spinophilin. Platelet activation by thrombin or thromboxane A2, but not ADP or collagen, stimulates a transient increase in spinophilin-associated SHP-1 tyrosine phosphatase activity and causes dephosphorylation and decay of the SPL/RGS/SHP-1 complex. Conversely, blocking SHP-1 phosphatase activity in platelets or omitting SHP-1 in transfected CHO cells inhibits dephosphorylation of spinophilin and prevents dissociation of the SPL/RGS/SHP-1 complex. While we have shown previously that inhibiting interactions between G proteins and RGS proteins produces a gain of function in platelets, knocking out spinophilin in mice inhibits platelet aggregation. This aggregation defect does not occur with all agonists, but is selective for those that are able to trigger decay and dephosphorylation of the SPL/RGS/SHP-1 complex. In addition to inhibiting platelet aggregation in vitro, the spinophilin knockout delays carotid artery occlusion in vivo following application of FeCl3 and reduces platelet accumulation following laser injury in cremaster muscle arterioles. Underlying these effects of the knockout is a decrease in Rap1 activation, an event that supports integrin activation, and attenuation of the cAMP increase otherwise caused by endothelial PGI2. Collectively, these observations show for the first time that a regulatory complex based on spinophilin helps to regulate platelet responses to injury and suggest that it does this by sequestering RGS proteins in resting platelets and releasing them after activation begins. Dissociation of the SPL/RGS complex is regulated by an agonist-induced increase in the activity of SHP-1 associated with spinophilin. Disclosures:No relevant conflicts of interest to declare.

Endothelial Injury and Thrombus Formation In the Ferric Chloride Murine Model of Hemostasis

AbstractAbstract 650Laboratory mice are used in hemostasis and thrombosis research in experiments ranging from the investigation of basic mechanisms of thrombus formation to the generation of preclinical data regarding potential therapeutic anti-thrombotic agents. With the common ferric chloride (FeCl3) model of vascular thrombosis, FeCl3 is applied directly to outside of the carotid artery or mesenteric vessels to induce thrombus formation. However, despite its widespread use, surprisingly little is understood regarding the mechanisms by which FeCl3 induces endothelial injury and subsequent thrombus formation, although it is believed that endothelial denudation and collagen exposure occur, and trigger the onset of thrombosis. Similarly, very little is known regarding the nature of the thrombi that are generated by this method, and importantly, whether these experimental thrombi resemble bona-fide thrombi that occur as a result of “true” vascular injury. To address these issues, we developed a scanning electron microscopy (SEM) protocol to visualize endothelial damage and thrombus formation that occur in situ. Briefly, thrombus formation is initiated by direct application of FeCl3 (or vehicle control) to the carotid artery, and subsequently at defined time points, the circulation is flushed, and then internally aldehyde-fixed. The affected section of carotid artery is removed, externally fixed, sectioned, processed for SEM, and visualized. With this procedure, we have obtained high-quality and high-magnification images of FeCl3-induced endothelial damage and thrombus formation through approximately 5 minutes of injury, in multiple samples and uninjured controls (N>100). Interestingly, we found that through these time points, FeCl3 induces little, if any, endothelial damage or subendothelial exposure. Rather, the endothelium rapidly assumes an “activated” phenotype, clearly changed from baseline, but non-denuded and intact. For verification, we found that mechanical injury of the murine carotid results in endothelial denudation which is easily identified by SEM. Perhaps more surprisingly, we also found that the first cells to adhere to the endothelium following FeCl3 application actually are red blood cells (RBCs), and not platelets. Following binding to the vascular surface, RBCs become misshapen and elongated in the direction of blood flow, or break off, leaving fragments associated with the endothelial surface. Subsequently, surface-bound RBCs and fragments bind additional RBCs and platelets from the circulation, forming large and characteristic-appearing complexes. From this point forward, large numbers of additional platelets accumulate on the RBC and platelet/RBC complexes, and the thrombus grows inward rapidly until the vessel becomes occluded. Next, to investigate formation of these potential RBC and platelet/RBC complexes with an independent system, we turned to intravital fluorescent microscopy in mesenteric venules. We found that FeCl3 rapidly induces structures consistent in size and shape with the “early” platelet/RBC complexes observed by SEM. With further time points, these small/early lesions subsequently bound more labeled platelets, and increased rapidly in size until occlusion of the vessel. As these platelet lesions grew, they also resembled in size and shape the “later” platelet/RBC complexes observed by SEM, including possession of a characteristic trailing “tail” of platelets. It is interesting that similar structures appeared to form in both the carotid artery and mesenteric venules, given the marked difference in shear stress between the two (1000-1500 s-1 vs. 100–200 s-1, respectively). To our knowledge, the existence of such platelet/RBC complexes has not been documented in any system. In summary, we have developed a technique to investigate in situ endothelial damage and thrombus formation in mice by SEM, and demonstrated that thrombus formation in the commonly-used FeCl3 murine model occurs in the absence of endothelial denudation, and appears to involve the active participation of RBCs. Further work will be necessary to confirm whether or not RBCs are necessary for thrombus formation in this model, and whether RBCs participate in any type of naturally-occurring hemostasis or thrombosis. Additionally, these results will likely have strong impact on future interpretation of experiments resulting from the use of FeCl3 in mice. Disclosures:No relevant conflicts of interest to declare.

The MBL‐complex is necessary for FeCl3‐mediated thrombosis

Occlusive thrombosis, resulting from atherosclerotic plaque rupture or restenosis plays an important role in the onset of two major causes of death in developed nations: myocardial infarction and ischemic stroke. It is well‐known that the complement and coagulation systems interact, however no studies have investigated the specific role of the lectin complement pathway in thrombus formation. Thus, the purpose of this study was to examine the role of the complement system, specifically the mannose‐binding lectin (MBL) complex, in a mouse model of ferric‐chloride (FeCl3)‐induced thrombosis. For this model, 2 strips of Whatman paper were soaked in 3.5% FeCl3 and applied to either side of the exposed right carotid artery for 3min then removed. Blood flow was measured at baseline, during FeCl3 administration, and for 30min following FeCl3 exposure using a Doppler flow probe positioned distal to the FeCl3 application. Using wild‐type (WT) and C2/fB−/− mice, we observed occlusive thrombosis of the carotid artery 10min after FeCl3 administration. In contrast, MBL−/− mice had significantly decreased FeCl3‐induced thrombosis formation compared to WT or C2/fB−/− mice (P&lt;0.05). In conclusion, our study provides evidence for a significant role for the MBL‐complex in FeCl3‐induced coagulation and may lead to the development of improved anti‐thrombotic therapies. Supported by HL56086, HL92469 and HL99130.

Role of Coagulation Factors in Cerebral Venous Sinus and Cerebral Microvascular Thrombosis

The objective of this study was to define the relative contributions of three major pro- and anti-coagulation pathways (heparin-antithrombin, protein C, and tissue factor (TF)) in the thrombogenic responses that occur in large and small vessels of the brain. Cerebral venous sinus thrombosis was induced by topical application of FeCl3 on the superior sagittal sinus, while photoactivation of fluorescein was used to induce thrombus formation in cerebral microvessels. Heparin, activated protein C (APC), and antibodies to either APC or TF were used to assess thrombogenesis in wild-type mice. Mutant mice that overexpress the endothelial protein C receptor (EPCR-tg) or with TF deficiency in Tie2-expressing endothelial cells (LTFE) were also used. Thrombus formation in the superior sagittal sinus of wild-type mice was attenuated by heparin and in EPCR-tg mice, while treatment with the APC antibodies enhanced thrombogenesis. Arteriolar thrombosis was largely unresponsive to the interventions studied. However, in cerebral venules, thrombosis was inhibited by heparin and in EPCR-tg mice. TF antibody treatment also inhibited venular thrombosis, with a similar attenuation noted in LTFE mice. Thrombin promotes while the APC pathway blunts thrombus formation in an experimental model of cerebral venous sinus thrombosis. TF involvement is more evident in cerebral microvascular thrombogenesis, with endothelial cell-associated TF mediating this response in venules, but not arterioles.

CD36 Mediated Reactive Oxygen Species Formation Contributes to Thrombosis.

We previously showed that mice in which the CD36 gene was deleted had significantly prolonged time to form an occlusive thrombus in response to FeCl3-induced vascular injury than wild type (WT) mice. In other studies we and others have shown that CD36 promotes reactive oxygen species (ROS) formation in murine models of cerebral and cardiac ischemia. In this study, we examined the formation and roles of ROS in FeCl3-induced carotid artery injury and thrombosis using CD36 null and WT mice. Hydroethidine, a fluorescent superoxide indicator, was injected via the jugular vein and then carotid artery injury was induced in the contralateral artery by direct topical application of FeCl3 at concentrations of 7.5% or 12.5%. Fluorescence images were monitored with intravital microscopy. FeCl3 dose dependently induced significant accumulation of ROS in the vessel wall; however, the degree was significantly less in CD36 null mice than in WT mice exposed to the lower dose of FeCl3. Direct injection of Edavarone (1 mg/Kg body weight), a free radical scavenger, significantly elongated the time to thrombosis in WT mice. Thrombosis time was also increased in the CD36 null mice, but not to the same extent as WT. We also found fewer endothelial cell-derived microparticles (CD105 positive) in plasma of CD36 null mice 3 minutes after injury with the low dose of FeCl3. Immunoblots of lysates prepared from carotid arteries with thrombi or from untreated normal carotid arteries showed that levels of peroxiredoxin 2 (Prx 2), an antioxidant enzyme known to detoxify ROS, were lower in the thrombi formed from CD36 null mice, but higher in the normal vessel wall, suggesting that an anti-oxidative system may exist in the vessel wall of CD36 null mice. We conclude that CD36 plays a role in ROS formation during thrombosis and may also sensitize the vessel to injury induced by FeCl3. High Prx 2 expression in the vessel wall in CD36 null mice may contribute to the anti-oxidative effect observed in these mice.

Effect of tiplaxtinin (PAI-039), an orally bioavailable PAI-1 antagonist, in a rat model of thrombosis.

To assess the antithrombotic and profibrinolytic effects of tiplaxtinin (PAI-039), an orally bioavailable antagonist of PAI-1, in rat models of thrombosis. Carotid artery and vena cava vascular injury was produced by application of FeCl3 and blood flow was monitored using ultrasonic technology. To assess efficacy in a thrombosis prevention paradigm, PAI-039 was administered orally 90 min before injury (1-30 mg kg(-1)). To assess efficacy in a thrombosis treatment paradigm, vascular injury and stable thrombus formation were followed 4 h later by recovery and PAI-039 administration. PAI-039 prevented carotid artery occlusion in 20, 68 and 60% of animals pretreated with 0.3, 1.0 and 3.0 mg kg(-1), respectively. Time to occlusive thrombosis was increased from 18.2 +/- 4.6 min in controls to 32.5 +/- 8.7 (P = ns), 46.1 +/- 7.0 (P < 0.05), and 41.6 +/- 11.3 min (P < 0.05) in the respective PAI-039 treatment groups. In the vena cava protocol, PAI-039 pretreatment significantly reduced thrombus weight at PAI-039 doses of 3, 10 and 30 mg kg(-1). When PAI-039 was dosed in a treatment paradigm 4 h after stable arterial and venous thrombosis, a significant reduction in thrombus weight was observed 24 h later at PAI-039 doses of 3, 10 and 30 mg kg(-1). PAI-039 (10, 30 and 100 mg kg(-1)) had no effect on platelet aggregation in response to ADP or collagen and was not associated with increased bleeding or prolonged prothrombin time. In animals bearing no vascular injury, PAI-039 had no effect on circulating, low-levels of PAI-1 activity. In contrast, circulating PAI-1 activity increased 5-fold following the induction of vascular injury, which was completely neutralized by PAI-039. PAI-039 exerts antithrombotic efficacy in rat models of arterial and venous vascular injury without effecting platelet aggregation.

Key Role of Platelet Procoagulant Activity in Tissue Factor‐and Collagen‐Dependent Thrombus Formation in Arterioles and Venules In Vivo Differential Sensitivity to Thrombin Inhibition

Blood coagulation and platelet activation are mutually dependent processes, but contribute differently to venous and arterial thrombosis. We investigated the interplay of these processes in vivo in a mouse model of arteriolar and venular thrombus formation. Thrombus formation was studied by intravital (fluorescence) microscopy after topical application of FeCl3 on mouse mesenteric microvessels. Both in arterioles and venules, the thrombus-forming process relied on tissue factor-factor VII(a) interaction, collagen exposure, and glycoprotein VI-mediated platelet activation. Arterial thrombus formation was impaired by mild thrombin inhibition or platelet inhibition, while venous thrombosis was only suppressed by strong thrombin inhibition or by mild thrombin inhibition together with platelet inhibition. Phosphatidylserine-exposing platelets were present in thrombi of both vessel types, as detected with fluorescently labeled annexin A5. Injection of annexin A5 to shield exposed phosphatidylserine abolished thrombus formation in arterioles and venules, while mutant M1234-annexin A5 was ineffective. Arterial and venous thrombus formations were only slightly affected in mice carrying the factor V Leiden mutation, suggesting insensitivity to factor Va inactivation. In this microvascular model, the formation of both arterial and venous thrombi relies on collagen-induced platelet activation and tissue factor-induced thrombin generation. Activated, phosphatidylserine-exposing platelets play a key role in thrombus growth in arterioles and venules.

In Vitro and In Vivo Characterization of a Murine Model of Suppressed Intrinsic Pathway Using a FX Variant: FXRoma.

The complex and dynamic interplay between the intrinsic and extrinsic pathways of blood coagulation is incompletely understood. The mediator of prothrombin cleavage, Factor X (FX), plays a pivotal role as part of both the extrinsic and intrinsic tenase complexes. Moreover, the existence of naturally occurring Factor X mutations that can be asymmetrically activated through one but not both of these pathways affords one strategy for analyzing the relationship of the two pathways. The Factor X Roma (FXRoma) variant, originally described in a patient with mild bleeding tendency (severe following trauma, De Stefano et al., 1988), is due to a missense mutation (Thr318←Met) in exon 8. Coagulation testing revealed markedly decreased activity (1–3% wild-type) in the intrinsic pathway as measured by aPTT, but substantially higher activity (30–50% wild-type) in the extrinsic pathway as measured by PT. We chose to generate a mouse model of FX asymmetric activation to further probe the extrinsic-intrinsic pathway physiological relationship in hemostasis and thrombosis. For this, we used both an in vitro and an in vivo approach. We first constructed and purified the mouse homolog of FXRoma (mFXRoma) as well as wild-type mFX. Using a clotting-based assay, mFXRoma exhibited intrinsic and extrinsic activity comparable to that reported for the human mutation (5% and 18%, respectively). The reduced intrinsic and extrinsic activity of mFXRoma was not due to a secretion defect, based on Western blot analysis of supernatant and cell extracts from mFXRoma and mFX stably-transfected human embryonic kidney (HEK-293) cell lines. Mice homozygous for the analogous mutation (Thr315←Met) in exon 8 of the murine FX gene were generated by using a plug-and-socket approach. This resulted in the endogenous mFX exon 8 sequence being replaced with the mutated one, thus affording gene expression under the endogenous promoter. Analysis of mFXRoma homozygous mice showed a 6.4% and 19.2% intrinsic and extrinsic activity relative to wild-type littermates, respectively, confirming our in vitro data. The reduced activity in these mice resulted in a slight reduction in levels of the thrombin-antithrombin (TAT) complex. To determine any physiological defect of this mutation on the two pathways of coagulation, we performed two hemostatic challenges of the macrocirculation (tail clip and FeCl3-induced thrombus formation). In the tail-clip assay, blood loss showed no statistical difference between wild-type (n=5) and mFXRoma (n=6) mice. In contrast, following FeCl3-induced injury on the carotid artery (larger vessel diameter that in the tail), mFXRoma mice (3/3) failed to result in vessel occlusion (up to 30 min of observation), whereas wild-type littermates showed stable vessel occlusion (3/4) within ∼6 min of FeCl3 application. Although the type of injury was different, these data suggest that an impeded intrinsic activity of FX does not appear to affect hemostasis of the macrocirculation at relatively small diameter vessels but is essential for thrombus formation in large diameter vessels, and a relatively normal extrinsic activity does not compensate for this defect. This mouse model will aid in determining the safety and efficacy of therapeutic approaches based on impeding the intrinsic pathway of coagulation.

The effects of ferric iron on voltage-gated potassium currents in molluscan neurons

In isolated neurons of Helix pomatia, a two-microelectrode voltage clamp technique was used to study the effect of Fe3+ on voltage-gated potassium currents: a low-threshold fast-inactivating current (I(A)) and a high threshold slow-inactivating current with calcium-dependent (I(C)) and calcium-independent (I(DR)) components. Extracellular application of FeCl3 rapidly, reversibly and dose-dependently reduced the amplitude of I(A), I(C) and I(DR) with IC50 values of 49, 45 and 70 microM, respectively. Complete inhibition of K+ currents was reached at 100-500 microM Fe3+. The threshold for the total slow-inactivating potassium current shifted in a positive direction by 10-30 mV in the presence of Fe3+ (50-300 microM). Our work is the first demonstration of the strong blocking effect of Fe3+ on potassium currents of neuronal membrane.