Drug Repurposing for Therapeutic Discovery against Human Metapneumovirus Infection.

Human metapneumovirus (HMPV) is recognized as an important cause of pneumonia in infants, in the elderly, and in immunocompromised individuals worldwide. The absence of an antiviral treatment or vaccine strategy against HMPV infection creates a high burden on the global health care system. Drug repurposing has become increasingly attractive for the treatment of emerging and endemic diseases as it requires less research and development costs than traditional drug discovery. In this study, we developed an in vitro medium-throughput screening assay that allows for the identification of novel anti-HMPV drugs candidates. Out of ~2,400 compounds, we identified 11 candidates with a dose-dependent inhibitory activity against HMPV infection. Additionally, we further described the mode of action of five anti-HMPV candidates with low in vitro cytotoxicity. Two entry inhibitors, Evans Blue and aurintricarboxylic acid, and three post-entry inhibitors, mycophenolic acid, mycophenolate mofetil, and 2,3,4-trihydroxybenzaldehyde, were identified. Among them, the mycophenolic acid series displayed the highest levels of inhibition, due to the blockade of intracellular guanosine synthesis. Importantly, MPA has significant potential for drug repurposing as inhibitory levels are achieved below the approved human oral dose. Our drug-repurposing strategy proved to be useful for the rapid discovery of novel hit candidates to treat HMPV infection and provide promising novel templates for drug design.

Aurintricarboxylic acid protects isoproterenol induced left ventricular hypertrophy by modulating TWEAK signaling.

BACKGROUND: Cardiac hypertrophy is regarded as a compensation mechanism to overcome the increased workload. Aurintricarboxylic acid (ATA) is a derivative of quinomethanes and a selective inhibitor of TWEAK/Fn14 pathway. In this study, we investigated the effect of ATA on isoproterenol (ISO)-induced pathological cardiac hypertrophy. METHODS: Cardiac hypertrophy in H9C2 cells was induced using ISO 20 µM dissolved in PBS. H9C2 cells were treated with ATA (5 µM, 10 µM, 20 µM) followed by ISO stimulation for 24 h. Male SD rats were injected ISO (5 mg/kg/day, s.c) for 21 days and followed by treatment with ATA (10 mg/kg, i.p.) for 14 days. Cardiac functions were assessed. After sacrifice, hearts were subjected to histopathological and western blot analysis. RESULTS: In in-vitro results, upon ATA treatment, ICC results showed significant decrease in TWEAK and ANP expression. In in-vivo results, echocardiography showed significant restoration of cardiac function in ATA treated rats. Histopathological analysis showed a significant decrease in left ventricular wall thickness, cardiomyocytes width and reduced fibrosis in ATA treated rats. Western blotting showed decreased expression of the cardiac hypertrophy maker ANP, inflammatory markers including TWEAK and apoptotic markers after ATA treatment. CONCLUSION: These findings suggested that the TWEAK/Fn14 pathway could be a potential target for therapeutic exploration in ISO induced cardiac hypertrophy. ATA, as an inhibitor of this pathway, exerted significant cardioprotective effect against ISO-induced cardiac hypertrophy in rats.

Identification of SARS-CoV-2 Receptor Binding Inhibitors by In Vitro Screening of Drug Libraries.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) global pandemic. The first step of viral infection is cell attachment, which is mediated by the binding of the SARS-CoV-2 receptor binding domain (RBD), part of the virus spike protein, to human angiotensin-converting enzyme 2 (ACE2). Therefore, drug repurposing to discover RBD-ACE2 binding inhibitors may provide a rapid and safe approach for COVID-19 therapy. Here, we describe the development of an in vitro RBD-ACE2 binding assay and its application to identify inhibitors of the interaction of the SARS-CoV-2 RBD to ACE2 by the high-throughput screening of two compound libraries (LOPAC®1280 and DiscoveryProbeTM). Three compounds, heparin sodium, aurintricarboxylic acid (ATA), and ellagic acid, were found to exert an effective binding inhibition, with IC50 values ranging from 0.6 to 5.5 µg/mL. A plaque reduction assay in Vero E6 cells infected with a SARS-CoV-2 surrogate virus confirmed the inhibition efficacy of heparin sodium and ATA. Molecular docking analysis located potential binding sites of these compounds in the RBD. In light of these findings, the screening system described herein can be applied to other drug libraries to discover potent SARS-CoV-2 inhibitors.

Effect of Rosolic acid on endothelial dysfunction under ER stress in pancreatic microenvironment.

Endothelial cell (EC) dysfunction is the underlying cause for the development of several pathologies, and the interdependency between the pancreatic ß-cells and ECs has been established in the pathophysiology of diabetes. ECs release several factors that govern the expression of genes involved in the proliferation, physiology, and survival of the ß-cells. Of the known factors that collapse this intricately balanced system, endothelial dysfunction is the crucial condition that manifests as the causative factor for micro and macrovascular diseases. Our earlier studies demonstrated that activation of nuclear factor erythroid-related factor (Nrf2) renders protection to the ECs experiencing ER stress. In this study, using a co-culture system, the crosstalk between pancreatic cells under ER stress and ECs and the effect of a novel Nrf2 activator Rosolic Acid (RA), on the crosstalk was investigated. ECs pre-treated with different concentrations RA and co-cultured with thapsigargin-induced ER stressed pancreatic ß-cells showed increased levels of Nrf2 and its downstream targets such as heme oxygenase-1 (HO-1) and NADPH-quinone oxidoreductase-1 (NQO-1), and reduction of ER stress evinced by the decreased levels of glucose-regulated protein (GRP) 78 and C/ERB homologous protein (CHOP). The sensitization of ECs using RA, offered protection to pancreatic cells against ER stress as displayed by increased intracellular insulin and upregulated expression of cell survival and proliferative genes BCl2 and PDX-1. In addition, RA treatment resulted in elevated levels of various angiogenic factors, while inflammatory (TNF-α and IL-1ß) and apoptotic markers (CXCL10 and CCL2) decreased. RA treatment normalized the levels of 115 proteins of the 277, which were differentially regulated as revealed by proteomic studies of ER stressed pancreatic ß-cells in co-culture conditions. These findings clearly indicate the role of small molecule activators of Nrf2 not only in restoring the functioning of pancreatic cells but also in increasing the cell mass. Further, the study impinges on the strategies that can be developed to balance the pancreatic microenvironment, leading to the restoration of ß-cell mass and their normophysiology in diabetic patients.

Complement Inhibition Attenuates Early Erythrolysis in the Hematoma and Brain Injury in Aged Rats.

Background and Purpose- Early erythrolysis in the hematoma contributes to brain injury after intracerebral hemorrhage (ICH). This study investigated the effects of N-acetylheparin, a complement inhibitor, and aurin tricarboxylic acid, a membrane attack complex inhibitor, on early erythrolysis, brain iron deposition, and brain injury in aged rats. Methods- There were 3 parts in the study. First, aged (18 months old) male Fischer 344 rats had an ICH. The time course of erythrolysis in the hematoma was determined by T2* weighted magnetic resonance imaging, and the expression of CD163 was examined. Second, aged rats had an ICH with N-acetylheparin or vehicle. Rats were euthanized at days 1, 3, and 28 after magnetic resonance imaging (T2-, T2*-weighted, and T2* array) and behavioral tests. Brains were used for immunohistochemistry. Third, aged rats had an ICH with avaurin tricarboxylic acid or vehicle. The rats had magnetic resonance imaging and behavioral tests and were euthanized at day 3. Brains were used for immunohistochemistry. Results- Early erythrolysis occurred within the clot in aged F344 rats. There were increased numbers of CD163-positive cells after ICH. Almost all perihematomal CD163-positive cells were microglia/macrophages, while positive neurons were found more distant from the hematoma. Coinjection of N-acetylheparin attenuated erythrolysis, iron accumulation, CD163 expression, microglia activation, brain swelling, and neuronal death in the acute phase, as well as reducing brain atrophy and neurological deficits in the chronic phase. Coinjection of aurin tricarboxylic acid also reduced erythrolysis and ICH-induced brain injury. Conclusions- Inhibiting complement activation resulted in less erythrolysis and brain injury after ICH.

The Ex Vivo Treatment of Donor T Cells with Cosalane, an HIV Therapeutic and Small-Molecule Antagonist of CC-Chemokine Receptor 7, Separates Acute Graft-versus-Host Disease from Graft-versus-Leukemia Responses in Murine Hematopoietic Stem Cell Transplantation Models.

Despite recent advances in therapy, allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative option for a range of high-risk hematologic malignancies. However, acute graft-versus-host disease (aGVHD) continues to limit the long-term success of HSCT, and new therapies are still needed. We previously demonstrated that aGVHD depends on the ability of donor conventional T cells (Tcons) to express the lymph node trafficking receptor, CC-Chemokine Receptor 7 (CCR7). Consequently, we examined the ability of cosalane, a recently identified CCR7 small-molecule antagonist, to attenuate aGVHD in mouse HSCT model systems. Here we show that the systemic administration of cosalane to transplant recipients after allogeneic HSCT did not prevent aGVHD. However, we were able to significantly reduce aGVHD by briefly incubating donor Tcons with cosalane ex vivo before transplantation. Cosalane did not result in Tcon toxicity and did not affect their activation or expansion. Instead, cosalane prevented donor Tcon trafficking into host secondary lymphoid tissues very early after transplantation and limited their subsequent accumulation within the liver and colon. Cosalane did not appear to impair the intrinsic ability of donor Tcons to produce inflammatory cytokines. Furthermore, cosalane-treated Tcons retained their graft-versus-leukemia (GVL) potential and rejected a murine P815 inoculum after transplantation. Collectively, our data indicate that a brief application of cosalane to donor Tcons before HSCT significantly reduces aGVHD in relevant preclinical models while generally sparing beneficial GVL effects, and that cosalane might represent a viable new approach for aGVHD prophylaxis.

Salutary effect of aurintricarboxylic acid on endotoxin- and sepsis-induced changes in muscle protein synthesis and inflammation.

Small molecule nonpeptidyl molecules are potentially attractive drug candidates as adjunct therapies in the treatment of sepsis-induced metabolic complications. As such, the current study investigates the use of aurintricarboxylic acid (ATA), which stimulates insulinlike growth factor 1 receptor and AKT signaling, for its ability to ameliorate the protein metabolic effects of endotoxin (lipopolysaccharide [LPS]) + interferon γ (IFN-γ) in C2C12 myotubes and sepsis in skeletal muscle. Aurintricarboxylic acid dose- and time-dependently increases mTOR (mammalian or mechanistic target of rapamycin)-dependent protein synthesis. Pretreatment with ATA prevents the LPS/IFN-γ-induced decrease in protein synthesis at least in part by maintaining mTOR kinase activity, whereas posttreatment with ATA is able to increase protein synthesis when added up to 6 h after LPS/IFN-γ. Aurintricarboxylic acid also reverses the amino acid resistance, which is detected in response to nutrient deprivation. Conversely, ATA decreases the basal rate of protein degradation and prevents the LPS/IFN-γ increase in proteolysis, and the latter change is associated reduced atrogin 1 and MuRF1 mRNA. The ability of ATA to antagonize LPS/IFN-γ-induced changes in protein metabolism was associated with its ability to prevent the increases in interleukin 6 and nitric oxide synthase 2 and decreases in insulinlike growth factor 1. In vivo studies indicate ATA acutely increases skeletal muscle, but not cardiac, protein synthesis and attenuates the loss of lean body mass over 5 days. These data suggest ATA and other small molecule agonists of endogenous anabolic hormones may prove beneficial in treating sepsis by decreasing the inflammatory response and improving muscle protein balance.

Aurintricarboxylic acid ameliorates experimental autoimmune encephalomyelitis by blocking chemokine-mediated pathogenic cell migration and infiltration.

Multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), are autoimmune diseases characterized by the immune-mediated demyelination and neurodegeneration of the CNS. Overactivation of CD4(+) T cells, especially the Th1 and Th17 subpopulations, is thought to be the direct cause of this disease. Aurintricarboxylic acid (ATA), an inhibitor of protein-nucleic acid interaction, has been reported to block with the JAK/STAT signaling pathway that is critical for Th cell differentiation. In this study, we discovered that ATA treatment significantly reduces the clinical score of EAE, but it does not directly inhibit the differentiation of Th1 and Th17 cells in vitro. ATA was found to block the chemotaxis and accumulation of dendritic cells in the spleen of EAE mice before the onset of the disease and to reduce the percentage of Th1 and Th17 cells in the spleen. Further study revealed that ATA also blocks the infiltration of pathogenic T cells into the CNS and blocks the onset of passive EAE. ATA was found to inhibit the functions of many chemokine receptors. By blocking chemokine-mediated migration of dendritic cells and pathogenic T cells, ATA alleviates the pathogenesis of EAE and might be used to treat autoimmune diseases, including multiple sclerosis.

Lack of efficacy of aurintricarboxylic acid and ethacrynic acid against vaccinia virus respiratory infections in mice.

BACKGROUND: Aurintricarboxylic acid (ATA) and ethacrynic acid (ECA) have been reported to exhibit antiviral activity against vaccinia virus infections in cell culture by inhibiting early and late gene transcription, respectively. The purpose of this work was to determine if these inhibitors would effectively treat vaccinia virus infections in mice, which has not previously been studied. METHODS: ECA was investigated by cell culture plaque reduction assay for the inhibition of cowpox and vaccinia virus infections to clarify issues regarding its potency and selectivity. Mice infected intranasally with vaccinia virus were treated by intraperitoneal route twice daily for 5 days with ATA (10 and 30 mg/kg/day) and ECA (15 and 30 mg/kg/day) or once daily for 2 days with cidofovir (100 mg/kg/day). RESULTS: ECA caused 50% inhibition of virus plaque formation at 20-79 muM in four cultured cell lines, with 50% cytotoxicity at 84-173 muM, giving low (1.3-4.2) selectivity index values. Preliminary toxicity tests in uninfected mice indicated that ATA and ECA were both overtly toxic at 100 mg/kg/day. No protection from mortality was afforded by treatment of vaccinia virus infections with ATA or ECA, but 100% survival was achieved in the cidofovir group. ATA- and ECA-treated mice died significantly sooner than placebo-treated animals, indicating that these compounds exacerbated the infection. CONCLUSIONS: Both ATA and ECA lack antiviral potency and selectivity in cell culture. The compounds were ineffective in treating mice at intraperitoneal doses of

Characterization of aurintricarboxylic acid as a potent hepatitis C virus replicase inhibitor.

BACKGROUND: Hepatitis C virus (HCV) NS5B is an essential component of the viral replication machinery and an important target for antiviral intervention. Aurintricarboxylic acid (ATA), a broad-spectrum antiviral agent, was evaluated and characterized for its anti-NS5B activity in vitro and in HCV replicon cells. METHODS: Recombinant NS5B, HCV replicase and Huh-7 cells harbouring the subgenomic HCV replicon of genotype 1b were employed for biochemical and mechanistic investigations. RESULTS: Analysis of ATA activity in vitro yielded equipotent inhibition of recombinant NS5B and HCV replicase in the submicromolar range (50% inhibition concentration [IC(50)] approximately 150 nM). Biochemical and mechanistic studies revealed a bimodal mechanism of ATA inhibition with characteristics of pyrophosphate mimics and non-nucleoside inhibitors. Molecular modelling and competition displacement studies were consistent with these parameters, suggesting that ATA might bind to the benzothiadiazine allosteric pocket 3 of NS5B or at its catalytic centre. Kinetic studies revealed a mixed mode of ATA inhibition with respect to both RNA and UTP substrates. Under single-cycle assay conditions, ATA inhibited HCV NS5B initiation and elongation from pre-bound RNA, but with > or =fivefold decreased potency compared with continuous polymerization conditions. The IC(50) value of ATA for the native replicase complex was 145 nM. In HCV replicon cells, ATA treatment ablated HCV RNA replication (50% effective concentration =75 nM) with concomitant decrease in NS5B expression and no apparent cytotoxic effects. CONCLUSIONS: This study identified ATA as a potent anti-NS5B inhibitor and suggests that its unique mode of action might be exploited for structural refinement and development of novel anti-NS5B agents.

In vitro and in vivo activity of aurintricarboxylic acid preparations against Cryptosporidium parvum.

OBJECTIVES: The aim of this study was to assess the effect of commercial aurintricarboxylic acid (ATA) against Cryptosporidium parvum. METHODS: The anticryptosporidial effect of ATA was evaluated in vitro using cell culture and double fluorogenic staining, and in vivo in experimentally infected neonatal C57BL/6 mice. Mice were orally treated for 9 consecutive days starting on the day of infection with daily ATA doses of 50 and 100 micromol/kg. Paromomycin (100 mg/kg) was used as a positive control. RESULTS: In both in vitro models, ATA at concentrations of 100 and 10 micromol/L completely inhibited sporozoites within 10 and 60 min, respectively. Viability of oocysts exposed to 100 micromol/L and assessed by flow cytometry and in cell culture was reduced by 65% and 61%, respectively. The treatment of neonatal mice with a daily ATA dose of 100 micromol/kg led to 97-99% inhibition of infection without any observable negative effects on the animals. In comparison, the mean reduction of infection for paromomycin was 79-84%. CONCLUSIONS: ATA exerted high anticryptosporidial activity and should be considered for further study.

Inhibition of myocardial apoptosis reduces infarct size and improves regional contractile dysfunction during reperfusion.

OBJECTIVE: Myocardial apoptosis is primarily triggered during reperfusion (R) through various mechanisms that may involve endonuclease to cleavage genomic DNA in the internucleosomal linker regions. However, the relative contribution of myocardial apoptosis to development of myocardial injury during R remains unknown. In the present study, we examined whether inhibition of apoptosis with aurintricarboxylic acid (ATA), an endonuclease inhibitor, during R reduces infarct size and improves regional contractile function. METHODS AND RESULTS: In two groups of chronically-instrumented dogs, 1 h of left anterior descending (LAD) coronary occlusion was followed by 24 h of R with infusion of saline (control, n=8) or ATA (1 mg/kg/h, n=8) into the left atrium starting 5 min before R and continuing for 2 h. ATA significantly reduced apoptotic cells (TUNEL staining) in the peri-necrotic myocardium (12+/-1%* vs. 36+/-4%), consistent with the absence of DNA laddering. To confirm inhibition of apoptosis with ATA, densitometrically, Bcl-2 (% of normal myocardium) was significantly increased vs. control (102+/-12* vs. 68+/-9) and Bax as well as the activated caspase-3 were significantly reduced vs. control (108+/-17* vs. 194+/-42 and -29+/-4* vs. 174+/-43, respectively). ATA significantly improved segmental shortening (3.3+/-1.2* vs. -1.8+/-0.7%) and segmental work (79.3+/-11.3* vs. 7.1+/-5.8 mmHg/mm) in area at risk myocardium, and reduced infarct size (TTC staining, 27+/-0.2* vs. 37+/-0.5%), confirmed by lower plasma creatine kinase activity. In addition, myocardial blood flow (0.9+/-0.1* vs. 0.4+/-0.1 ml/min/g) and endothelial-dependent maximal vascular relaxation (119+/-6* vs. 49+/-8%) were significantly improved. Myeloperoxidase activity in area at risk myocardium, a marker for neutrophil accumulation, was also significantly reduced (17+/-4* vs. 138+/-28 Delta Abs/min). CONCLUSIONS: These data suggest that the inhibition of apoptosis during R is associated with a reduction in infarction, improvement in regional contractile and vascular endothelial functions as well as augmentation in myocardial blood flow. *P<0.05 vs. control group.

Multiple inhibition of platelet activation by aurintricarboxylic acid prevents vascular stenosis after endothelial injury in hamster carotid artery.

Activated platelets are instrumental in restenosis due to their role in thrombus formation. Aurintricarboxylic acid (ATA) has been reported to prevent platelet activation by inhibiting von Willebrand factor binding to platelet glycoprotein (GP)Ib. We investigated the effects of ATA in vitro and in vivo in hamsters. ATA inhibited the in vitro platelet aggregation induced by ADP, botrocetin and thrombin, but not by collagen. The IC50 values during the ex vivo platelet aggregation by ADP, botrocetin and thrombin were 8.2 +/- 1.8 microM, 0.9 +/- 0.4 microg/ml and 2.4 +/- 0.8 unit/ml, respectively. The platelet retention time to collagen-coated beads of hamster blood samples was inhibited by ATA (0.1, 0.3 and 1.0 mg/kg per hour) in a dose-dependent manner. Continuous administration of ATA (0, 0.1, 0.3, 1.0, 3.0 and 10.0 mg/kg per h) via an infusion pump produced dose-dependent antithrombotic effects: the time to occlude the carotid artery after vascular injury with a modified catheter was prolonged. Only when infused at doses of 3.0 and 10.0 mg/kg per hour, bleeding times were significantly prolonged. The continuous treatment with ATA (1.0 mg/kg per h) using a 2ML1 Alzet infusion pump for 2 weeks, resulted in a decrease in neointimal area by 22.2 +/- 6.8% when measured 2 weeks after injury induction. DNA synthesis using DDT1MF2 hamster SMCs was decreased by ATA in a dose-dependent manner. ATA reduced the number of platelets adhering on the injured area, as detected by electron microscopy. These results indicated that treatment with ATA inhibited platelet adhesion but also SMC proliferation. These observations may explain the effect of ATA on neointima formation.

Inhibition of von Willebrand factor binding to platelet GP Ib by a fractionated aurintricarboxylic acid prevents restenosis after vascular injury in hamster carotid artery.

BACKGROUND: Aurintricarboxylic acid (ATA) prevents von Willebrand factor binding to platelet glycoprotein (GP) Ib, with higher-molecular-weight ATA more effective than the lower-molecular-weight compound. We investigated the effects of high-molecular-weight ATA (Mr=7500), obtained by fractionating commercial ATA, in the injured hamster carotid artery. METHODS AND RESULTS: Platelet aggregation was induced in vitro with ADP (2.5 micromol/L) or botrocetin (5 microg/mL) in hamster platelet-rich plasma. IC50 values were 348.6+/-22.4 and 8.2+/-3.2 microg/mL, respectively. The endothelium of hamster carotid artery was denuded with a modified catheter. Continuous administration of high-molecular-weight ATA (10, 30, and 100 microg x kg(-1) x h(-1)) with an infusion pump produced antithrombotic effects in a dose-dependent manner, as evaluated by prolongation of time to occlusion. Neointima formation was observed 2 weeks after catheterization, and proliferating smooth muscle cells (SMCs) were identified by the thymidine analogue 5-bromo-2-deoxyuridine (BrdU). Continuous treatment with the compound (100 microg x kg(-1) x h(-1)) with a 2ML1 Alzet infusion pump resulted in a reduction of neointimal area by 38.0+/-8.8% and decreased the BrdU index on days 1 and 7 significantly. DNA synthesis in DDT1MF2 hamster SMCs was also decreased by the compound in a dose-dependent manner. In histological observation, the process of endothelial healing was improved by this treatment with the compound. CONCLUSIONS: Inhibition of platelet adhesion by von Willebrand factor binding to platelet GP Ib by high-molecular-weight ATA results in the prevention of thrombus formation and the suppression of neointima lesion. In addition, high-molecular-weight ATA has an inhibitory effect on SMC proliferation. This inhibition of both platelet adhesion and SMC proliferation markedly reduced vascular stenosis.

Retinal ischemia leads to apoptosis which is ameliorated by aurintricarboxylic acid.

Transient retinal ischemia results in a delayed cell death of the inner retinal layers. This study demonstrates that this ischemic cell death occurs, at least in part, through apoptosis. The general endonuclease inhibitor, aurintricarboxylic acid, protected rat retinal cells from ischemic cell damage when administered before the onset of ischemia and, more importantly, when administered 6 hr after the insult. Thus, the demonstration that transient retinal ischemia results in cell damage as a result of apoptosis opens new therapeutic strategies aimed at lessening retinal damage as a result of this process.

A comparative study of the antithrombotic effect of aurintricarboxylic acid on arterial thrombosis in rats and guinea pigs.

1. The antithrombotic effect of aurintricarboxylic acid (ATA) which inhibits binding of von Willebrand factor (vWF) to platelet glycoprotein lb (GPlb) receptor was evaluated in photochemically-induced thrombosis models in the femoral artery of rats and guinea-pigs. 2. ATA at a dose of 10 mg kg-1 significantly prolonged the time required for thrombotic occlusion of the artery in rats. The antithrombotic efficacy was associated with a significant inhibition of platelet retention and ex vivo botrocetin-induced platelet aggregation. 3. On the other hand, in guinea-pigs, ATA at the same dose inhibited the platelet retention and the platelet aggregation, but did not prevent thromboocclusion. 4. ATA inhibited aggregation of washed platelets from rats or guinea-pigs in response to botrocetin and thrombin in a dose-dependent manner (1-30 microM), and to the same extent. 5. ATA moderately increased activated partial thromboplastin time and bleeding time in both species. 6. These results indicate that vWF may play a role in the development of occlusive arterial thrombosis in the rat, but not in the guinea-pig. 7. The antithrombotic activity of ATA may partly arise from its inhibitory effect on thrombin, in addition to that on the vWF-GPlb pathway

The effect of aurintricarboxylic acid, an endonuclease inhibitor, on ischemia/reperfusion damage in rat retina.

Apoptosis is a form of cell death distinct from necrosis showing distinctive morphologic features and may require energy. It is under various control mechanisms and may involve an endonuclease, which cleavages genomic DNA in the internucleosomal linker regions. Previously, we reported that ischemic/reperfusion injury to rat retina induced endonuclease mediated apoptosis of retinal neurons. In this study, we examined the effect of aurintricarboxylic acid (ATA), an endonuclease inhibitor, on ischemia/reperfusion damage in rat retina in our established rat model. A single intraperitoneal injection of ATA at 2 mg/kg given immediately after 60 minutes of ischemia to the retina showed no observable effect. At 10 mg/kg, there was notable beneficial effect morphologically but not morphometrically. ATA at 100 mg/kg showed significant effect both morphologically and morphometrically. This observation is consistent with the hypothesis that endonuclease mediated apoptosis may be involved in retinal cell loss after ischemia/reperfusion insult.

Inhibition by aurintricarboxylic acid of von Willebrand factor binding to platelet GPIb, platelet retention, and thrombus formation in vivo.

Commercial aurintricarboxylic acid (ATA) was separated into molecular-weight (MW) fractions of < 210 to > 25,000, using gel permeation chromatography. Fractions with MW > 1,300 effectively inhibited both botrocetin-induced vWF and bovine vWF binding to fixed human platelets. These activities decreased with a MW > 17,000. Platelet retention for a human in vitro was reduced by ATA at 150 microM, as was that for rats ex vivo at 3 mg/kg. ATA prolonged tail transection bleeding time in rats but had only a weak effect on buccal mucosal bleeding time in dogs. ATA had no effect on platelet count but markedly prolonged PTT. ATA at 10 mg/kg exhibited antithrombotic activity and caused a marked improvement in patency status following successful thrombolysis by t-PA in electrically and copper coil-induced thrombosis models. These results suggest that specific inhibitors of the vWF-GPIb interaction such as ATA may prove useful as antithrombotic agents.

Aurin tricarboxylic acid inhibits experimental venous thrombosis.

In vitro, aurin tricarboxylic acid (ATA) inhibited ristocetin-induced human platelet agglutination in a dose-dependent manner. The IC50 value (dose which inhibits 50% of platelet agglutination) was 60 +/- 8.7 micrograms/ml. In vivo, the i.v. administration of ATA to rats reduced the thrombus formation in an arteriovenous shunt with an ED50 value of 9.0 +/- 1.6 mg/kg. In a venous thrombosis model, using a combination of a thrombogenic challenge and stasis, ATA displayed a significant, dose-dependent antithrombotic effect, the ED50 value being of 18.3 +/- 2.0 mg/kg. In an experimental model of disseminated intravascular coagulation, ATA protected mice from the lethal effect of thromboplastin-induced thromboembolism with a ED50 value of 1.1 +/- 0.15 mg/kg, being in that respect 12 times less potent than standard heparin (ED50 = 90 +/- 15 micrograms/kg). These observations therefore show that ATA is active in both arterial- or venous-type thrombosis models and suggest that von Willebrand Factor might be important not only in arterial but also in venous thrombosis.

Aurintricarboxylic acid in a canine model of coronary artery thrombosis.

Platelet thrombus formation occurs at sites of severe arterial narrowing where shear stress is elevated. Shear stress appears to induce platelet aggregation in vitro by means of initiation of von Willebrand factor binding to platelet glycoprotein Ib. Recent in vitro studies have demonstrated that aurintricarboxylic acid can inhibit shear stress-induced platelet aggregation. This effect is mediated by aurintricarboxylic acid binding to von Willebrand factor; this binding results in inhibition of von Willebrand factor interaction with glycoprotein Ib. In this study, we examined the effect of aurintricarboxylic acid on platelet-dependent cyclic flow reductions (CFRs) in a canine coronary stenosis model. In dose-response experiments, six animals received 4 mg/kg aurintricarboxylic acid by bolus infusion, followed by 1 mg/kg every 10 minutes. Total inhibition of CFRs was observed in all animals after 6.7 mg/kg aurintricarboxylic acid; CFRs could not be reinitiated by the thromboxane A2 analogue U46619. Continuous infusion of epinephrine (0.4 micrograms/kg/min) caused CFRs to return; however, 3.7 mg/kg additional aurintricarboxylic acid again induced total inhibition of CFRs. In addition, five animals received a bolus infusion of 10 mg/kg aurintricarboxylic acid, which caused total inhibition of CFRs. The average area of stenosis in the constricted vessels was 83%, and shear stress at the site of constriction averaged 350 dynes/cm2. Aurintricarboxylic acid did not alter hemodynamics, thrombin time, platelet count, or ADP/epinephrine-induced platelet aggregation. These data indicate that platelet glycoprotein Ib-von Willebrand factor interactions are important during coronary occlusion and that aurintricarboxylic acid can inhibit coronary thrombosis associated with coronary constriction.