There is evidence for the involvement of peroxisome proliferator-activated receptors (PPARs) in pain, cognition, and anxiety. However, their role in pain–fear interactions is unknown. The amygdala plays a key role in pain, conditioned fear, and fear-conditioned analgesia (FCA). We investigated the effects of intra-basolateral amygdala (BLA) administration of PPARα, PPARβ/δ, and PPARγ antagonists on nociceptive behaviour, FCA, and conditioned fear in the presence or absence of nociceptive tone. Male Sprague-Dawley (SD) rats received footshock (FC) or no footshock (NFC) in a conditioning arena. Twenty-three and a half hours later, rats received an intraplantar injection of formalin or saline and, 15 min later, intra-BLA microinjections of vehicle, PPARα (GW6471) PPARβ/δ (GSK0660), or PPARγ (GW9662) antagonists before arena re-exposure. Pain and fear-related behaviour were assessed, and neurotransmitters/endocannabinoids measured post-mortem. Intra-BLA administration of PPARα or PPARγ antagonists potentiated freezing in the presence of nociceptive tone. Blockade of all PPAR subtypes in the BLA increased freezing and BLA dopamine levels in NFC rats in the absence of nociceptive tone. Administration of intra-BLA PPARα and PPARγ antagonists increased levels of dopamine in the BLA compared with the vehicle-treated counterparts. In conclusion, PPARα and PPARγ in the BLA play a role in the expression or extinction of conditioned fear in the presence or absence of nociceptive tone. Peroxisome-proliferator activated receptors (PPARs) are transcription factors and part of the nuclear hormone superfamily of receptors. There are three described isoforms: PPARα, PPARβ/δ, and PPARγ . EndogenThe amygdala is part of the limbic system and plays a key role in emotional responses including anxiety and fear . The basc-fos expression in the BLA  or treatment × conditioning , on nociceptive behaviour  = 0.296, p > 0.05], or treatment × conditioning  on formalin-induced paw oedema  = 34.508, p < 0.001]  = 38.350, * p < 0.001), and fear conditioning × time  on freezing duration ($p < 0.05) and at 0–3 min for FC GW9662 vs. FC Vehicle (#p < 0.05)  = 1.591, p > 0.05], time × treatment , time × conditioning , and time × conditioning × treatment   = 0.251, p > 0.05], treatment × conditioning [F  = 1.42]; 2 (7) = 5.685, p > 0.05]; p > 0.05], fear conditioning , and treatment × conditioning , fear conditioning , and treatment × conditioning ; The effects of fear-conditioning and intra-BLA administration of PPAR antagonists on motor behaviour were also assessed . NeitherIn order to further explore the neurochemical mechanisms underlying the effect of PPAR antagonism on fear memory, the levels of neurotransmitters  in the right  and left  BLA were measured in all groups .2 (15) = 84.814, p < 0.001) in serotonin levels in the BLA (p < 0.05). When each side was analysed separately, Kruskal–Wallis showed a significant difference among the groups in the left , but not in the right  side in serotonin levels in the BLA. However, post hoc pairwise comparisons with Dunn’s test did not reach statistical significance. The Kruskal–Wallis test revealed a significant difference between groups (χ2 (15) = 90.526, p < 0.001) in dopamine levels in the BLA (p < 0.05). When each side was analysed separately, Kruskal–Wallis did not show any significant difference between groups in the right  or in the left  sides in dopamine levels in the BLA.Kruskal–Wallis comparisons revealed a significant difference between groups (χ the BLA C. Post h the BLA D. Post h2 (15) = 20.669, p > 0.05) in GABA levels in the BLA (2 (7) = 6.288, p > 0.05] or in the left  sides in the GABA levels in the BLA. Kruskal–Wallis comparisons revealed a significant difference between groups (χ2 (15) = 39.443, p < 0.01) in glutamate levels in the BLA (2 (7) = 5.432, p > 0.05] or in the left  sides in glutamate levels in the BLA.The Kruskal–Wallis test did not show any significant difference between groups  in the BLA, and checked for possible effects of PPAR antagonism and intraplantar formalin injection on their levels.p < 0.001] and fear conditioning  on PEA levels in the BLA  = 0.669, p > 0.05], treatment × side , conditioning × side , and treatment × conditioning × side  on PEA levels. When each side was analysed separately, two-way ANOVA did not show any significant effect of treatment, conditioning, or their interaction on either the left or right BLA.Three-way ANOVA revealed an effect of side  or in the left  sides in the AEA levels in the BLA.Kruskal–Wallis comparisons did not show any significant difference between groups (χ the BLA B. When e2 (15) = 31.454, p < 0.01) in OEA levels in the BLA (2 (7) = 6.672, p > 0.05] or in the left  sides in the OEA levels in the BLA.Kruskal–Wallis comparisons revealed a significant difference between groups  = 4.241, p > 0.05] of rats that received an intraplantar injection of saline into the right hind paw  = 0.5191, p > 0.05], or treatment × conditioning  on paw diameter  = 18.037, p = 0.012] (p < 0.05). The treatment with GW6471 and GSK0660 in NFC rats also increased freezing duration . The treatment with GW9662 in NFC rats narrowly failed to reach statistical significance . These drugs had no significant effects on FC rats.Kruskal–Wallis revealed a significant difference between groups on the total duration of freezing [χ= 0.012] . Post ho2 (7) = 23.49, p < 0.01]  = 0.133, p > 0.05], treatment × conditioning ; p > 0.05], fear conditioning , treatment × conditioning ; p > 0.05], fear conditioning , and treatment × conditioning  on rearing duration  = 0.256, p > 0.05] on rearing duration.Two-way ANOVA showed an effect of treatment  and in the left  side. Because the right side almost reached statistical difference, an analysis considering the different fear conditioning groups was carried out. When we further analysed the fear conditioning groups, the Kruskal–Wallis test revealed a significant difference between groups in the NFC rats in the right  but not in the left  side. Post hoc analysis with Dunn’s test indicated that NFC rats treated with GW6471 had increased dopamine levels compared to the NFC Vehicle-treated ones (#p < 0.05). The test also indicated a strong trend for increased levels of dopamine in the NFC GW9662-treated rats compared to the NFC vehicle-treated (p = 0.0584). The Kruskal–Wallis test did not reveal any significant differences between groups in FC rats neither in the right  nor in the left  side.The Kruskal–Wallis test revealed a significant difference between groups (χe levels D in the 2 (15) = 25.622, p < 0.05) in the GABA levels (2 (7) =14.483, p < 0.05] but not in the left  side. However, post hoc analysis with Dunn’s test did not show any significant between-group differences in the GABA levels.Kruskal–Wallis test also revealed a significant difference between groups (χA levels A in the 2 (15) = 15.856, p > 0.05; 2 (15) = 22.532, p > 0.05; 2 (7) = 6.458, p > 0.05] or in the left  side. Similarly, when each side was analysed separately, Kruskal–Wallis did not reveal a significant difference in serotonin levels among groups in the right  or in the left  side.Kruskal–Wallis test did not show any significant difference between groups in glutamate  in the BLA, and checked for possible effects of PPAR antagonism in the absence of nociceptive tone on their levels.2 (15) = 32.124, p < 0.05) in the PEA levels  = 7.163, p < 0.05), and the interaction of treatment × fear conditioning  on the PEA levels in the right BLA. Post hoc analysis with Dunn’s test indicated that the FC GW6471-treated rats had decreased levels of PEA compared to the FC Vehicle treated rats in the right BLA ($p < 0.05). Two-way ANOVA did not reveal any significant effect of treatment , fear conditioning , and the interaction of treatment × fear conditioning  on the PEA levels in the left BLA.Kruskal–Wallis comparisons revealed a significant difference between groups (χA levels A in the 2 (15) = 47.410, p < 0.05) in the AEA levels (2 (7) =14.798, p < 0.05] but not in the left  side. However, post hoc analysis with Dunn’s test did not show any significant changes in the AEA levels in the Right BLA.Kruskal–Wallis comparisons revealed a significant difference between groups (χA levels B in the 2 (15) = 32.456, p < 0.05) in the OEA levels (2 (7) = 21.988, p < 0.01) but not in the left (χ2 (7) = 6.350, p > 0.05) BLA. Post hoc analysis with Dunn’s did not reveal any significant differences between groups in the OEA levels in the right BLA.Kruskal–Wallis comparisons revealed a significant difference between groups  on the levels of neurotransmitters, endocannabinoids, and NAEs.Samples from the NFC and FC vehicle-treated groups from both experiments were re-run on LC-MS/MS and re-analysed together in order to compare the possible effects of the presence of a nociceptive inflammatory tone  ap = 0.002]; glutamate ; serotonin ; and dopamine ; #p < 0.05; #p < 0.05; p < 0.05, compared to their left counterparts) in the dopamine levels  for all four neurotransmitters analysed  = 10.730, e levels D. When tap = 0.046]; OEA: ; p = 0.009] on the PEA levels in the right BLA ($p < 0.05). There were no significant differences in the levels of OEA when sides were analysed separately  was seen for two of the endogenous ligands at the PPARs analysed  = 4.191, ight BLA A. Post h2 (7) = 35.131, p < 0.05) in the AEA levels (p < 0.05). When each side was analysed separately, Kruskal–Wallis did not reveal any significant differences between groups in the right  or in the left  side.Kruskal–Wallis comparisons revealed a significant difference between groups . Administration of GW6471, a PPARα antagonist, directly into the BLA prolonged freezing duration in FC rats in the presence of formalin-evoked nociceptive tone and increased freezing duration in NFC rats in the absence of nociceptive tone. The administration of a PPARγ antagonist, GW9662, into the BLA enhanced freezing expression in the first part of the trial in the presence, but not in the absence, of nociceptive tone. Thus, endogenous PPAR signalling through PPARγ expressed in the BLA may act to attenuate or extinguish conditioned fear behaviour. Likewise, PPAR signalling through PPARα expressed in the BLA seems to be involved in the recall of fear-related memories, with its blockade resulting in potentiation of fear conditioned behaviour in the first part of the trial. Importantly, these effects were only seen in the presence of formalin-evoked nociceptive tone; they were not observed in rats that received intraplantar saline injection. The effects of PPARα and PPARγ blockade on freezing expression were associated with increased levels of dopamine in the right BLA. In the absence of nociceptive tone, the administration of the three antagonists increased freezing duration in NFC rats. These results suggest a modulatory role for PPARs in innate anxiety, but not in conditioned fear, in the absence of nociceptive tone. The intra-BLA injection of PPAR antagonists did not alter nociceptive behaviour or locomotor activity in either NFC or FC rats, irrespective of the nociceptive status. These results suggest that PPAR signalling in the BLA does not modulate pain or FCA. Taken together, these results demonstrate a differential effect of the PPAR signalling system on fear and/or anxiety in the presence versus absence of acute inflammatory pain.Extinction is defined as a learned inhibition of the retrieval of previously acquired memories. Many studies have demonstrated that PPAR signalling plays a role in mnemonic formation ,49,50,51Recent studies have pointed to a possible role of PPARs in anxiety and cognition. Our recent work has shown that the administration of a PPARα antagonist exacerbated inflammatory pain-related impairment of spatial memory in rats . YoussefThe results suggest that PPAR signalling in the BLA does not mediate or modulate formalin-evoked nociceptive behaviour. As previously mentioned, other studies have demonstrated effects of PPAR agonists on pain-related behaviour ,61,62,63FCA is a potent suppression of nociceptive responses upon exposure to a fearful stimulus. Our current study investigated the effects of intra-BLA administration of PPAR antagonists on FCA. FCA has been previously shown to be associated with increased levels of AEA, an endocannabinoid that also binds to PPARs, in the BLA  and a st1 receptors in the BNST  enhanced nociceptive responses in female, but not male rats, suggesting that the dopaminergic system in the BNST may exert sexually dimorphic effects on pain , PPARα  or PPARγ receptor  and mouse monoclonal antibody to β-actin  diluted in 5% milk/0.05% TBST overnight at 4 °C. Post incubation period, the membrane was washed in washing buffer (0.1% TBST) for 3 × 10 min washes. After washing, membranes were then incubated in secondary antibody solution containing IR-Dye goat anti-mouse (k700) and goat anti-rabbit or donkey anti-goat (k800)  diluted 1:10,000 in 1% milk/0.1% TBST for 1 h. Five × 5 min washing steps were then performed with washing buffer (0.1% TBST) and one final 5 min wash in distilled water. Blots were scanned on a LI-COR Odyssey imager. IR band intensities for PPAR receptor protein expression (~52/55 kDa) for each sample were generated automatically using the background subtraction method of the LI-COR Image Studio Ver. 2.0 imaging software. Two distinct bands were observed for PPARγ  lysis buffer , according to the instructions of the manufacturer. Tissue was homogenised in 353.5 µL of lysis buffer (RA1) containing β-mercaptoethanol  for 3–5 s using an automated homogeniser . Homogenates were kept on ice until transferred to a Nucleospin filter (violet ring) and centrifuged at 11,000× g for 1 min to reduce viscosity and clear the lysate. The lysates were then treated with 350 µL of 70% molecular grade ethanol  and transferred to a Nucleospin RNA column (light blue ring) and centrifuged at 11,000× g for 30 s to bind the RNA to the membrane. The membrane column was then desalted by adding 350 µL of membrane desalting buffer (MDB) and centrifuging at 11,000× g for 1 min to dry the membrane. Samples were then treated with 10 μL rDNase and left for 15 min at room temperature to remove any DNA. Samples were then serially washed using washing buffers  and RNA was eluted in 30 µL of RNAase-free water . Nanodrop technology  was used to measure the concentration, purity, and integrity of the RNA. RNA concentration was determined by measuring the optical density (OD) at 260 nm. The integrity and purity were determined by measuring the ratios of OD260/OD280 and OD230/OD280, respectively, where a ratio of approximately 1.8–2.0 was deemed indicative of RNA of good quality and purity. All RNA samples were within the acceptable range for both integrity and purity. Samples were equalised to the same concentration of RNA (35 ng/μL) using RNase free water . Equalised samples were then stored at −80 °C until reverse transcribed. Equal amounts of total RNA (10 ng/μL) were reverse transcribed into cDNA as follows: two master mixes were made up, as shown below in Punched brain tissues from BLA of naïve male Sprague-Dawley rats were analysed by quantitative real-time PCR (RT-qPCR). RT-qPCR was carried out as described previously . RNA was extracted from BLA tissue (BLA: 2.04 mg ± 0.2 mg) using the Macherey-Nagel NucleoSpin p < 0.05. Data are expressed as group means ± standard error of the mean (S.E.M.) when parametric and as median with interquartile range and min/max when non-parametric.The SPSS 21.0 statistical package was used to analyse the data. Normality was assessed using thee Shapiro–Wilk test and homogeneity of variance was checked using Levene’s test. Behavioural data were analysed using two-factor analysis of variance (two-way ANOVA), with factors being fear-conditioning and treatment, or analysis of variance with repeated measures (repeated measures ANOVA) when appropriate . Neurochemical data were analysed using three-factor analysis of variance (three-way ANOVA), with factors being fear conditioning, treatment, and side . Post hoc pairwise comparisons were made with Student Newman–Keuls test when appropriate. If data were found to be non-parametric, three transformation protocols were applied, in this order: square root of the data values, log of the data values, and ranking of the data values. Additionally, we checked whether the highest standard deviation was less than or equal to two times the smallest standard deviation for the particular dataset being analysed . If data


Peganum harmala seed alkaloid fraction. The ζ-potential of the synthesized Pt NPs, Pd NPs and Pt–Pd NPs were −11.2 ± 0.5, −9.7 ±1.2, and −12.7 ± 2.1 mV; respectively. Transmission electron microscopy (TEM) revealed the formation of spherical-shaped nanoparticles with smooth margins. The mean diameters of the synthesized Pt NPs, Pd NPs, and Pt–Pd NPs were determined using TEM analysis and were found to be 20.3 ± 1.9, 22.5 ± 5.7, and 33.5 ± 5.4 nm, respectively. The nanoparticles’ bioreduction was confirmed by ultraviolet–visible (UV–vis) spectroscopy, X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy, and their organic contents were determined by thermal gravimetric analysis (TGA). The Pt–Pd NPs mixture showed more pronounced antioxidant activity of 843.0 ± 60 μM Trolox equivalent (TE)/mg NPs compared to the individual Pt NPs (277.3 ± 13.5 μM TE/mg NPs) and Pd NPs (167.6 ± 4.8 μM TE/mg NPs). Furthermore, the Pt–Pd NPs exhibited significant cytotoxic activities against lung cancer (A549) and breast adenocarcinoma (MCF-7) cells, IC50 of 8.8 and 3.6 µg/mL, respectively; as compared to Pt NPs  and Pd NPs . Moreover, molecular docking studies were conducted to explore the possible anticancer and antioxidant mechanisms of the biogenic nanoparticles. Pt NPs, Pd NPs, and their mixture showed inhibitory activity against cysteine proteinase, which supports their high antitumor activity, but moderate antioxidant activity. In conclusion, Pd-Pt NPs mixture prepared using harmala seed alkaloid fraction showed potential as effective antineoplastic agents.This study reports a facile and eco-friendly method for the green synthesis of platinum and palladium nanoparticles (Pt NPs and Pd NPs) using  Dioscorea bulbifera. The produced bimetallic NPs demonstrated a significant antineoplastic activity (74%) against human cervical (HeLa) cancer cells compared to individual Pd NPs (33%) and Pt NPs (12.6%) [Recently, noble metals nanoparticles (NNPs), including platinum and palladium nanoparticles , have gained attention as promising tools in cancer therapy and biomedical applications ,2,3. Pt  (12.6%) .Cacumen platycladi leaf extract [Terminalia chebula aqueous fruit extract for the green synthesis of spherical Ag–Pd NPs with an average particle size of 20 nm zeta potential of −14.4 mV. The bimetallic Ag–Pd NPs showed noticeable anticancer activity against A549 cancer cells with IC50 of 48.45 µg/mL [Various chemical approaches have been used for fabrication of Pt NPs and Pd NPs, however, they commonly use harsh reagents and synthetic capping agents ,5,10,11.45 µg/mL . Most ofPeganum harmala L., belonging to Zygophyllaceae family, grows naturally in uncultivated zones in the Middle East and North Africa [P. harmala is enriched in therapeutically active alkaloids, including β-carboline and quinazoline alkaloids , jointly known as harmala alkaloids [P. harmala contains oxygenated monoterpenes (such as eugenol), anthraquinones, flavonoids, and polysaccharides which are responsible for the bioreduction of different metal ions [P. harmala possess a broad spectrum of biological molecules which have antimicrobial, antioxidant, and anticancer activities [P. harmala extracts has been reported [h Africa . The seelkaloids . Also, ttal ions ,18. The tivities ,19. Greereported ,21,22.Peganum harmala seed extract. The greenly synthesized nanoparticles were investigated for cytotoxic and antioxidant activities. In silico molecular modeling studies were conducted to explore their possible modes of action as anticancer and antioxidant agents , Dulbecco’s Modified Eagle’s Medium (DMEM) for sulforhodamine B (SRB) assay, and Tris(hydroxymethyl)aminomethane were purchased from Lonza, .Platinum (IV) chloride and palladium (II) acetate were obtained from BLD Pharmatech Co., Limited, Cincinnati, OH, USA. TPTZ  and FeClPeganum harmala L. were obtained from the local Egyptian market. A voucher specimen was deposited (18.1.17) at the Department of Pharmacognosy Herbarium, Faculty of Pharmacy, Cairo University .Dried mature seeds of P. harmala seeds were ground and then extracted by soaking overnight in 70% ethanol at room temperature. The ethanolic extract was filtered and evaporated under reduced pressure yielding a dark reddish-brown viscous residue which was dissolved in 5% HCl, filtered, and partitioned using dichloromethane. The aqueous layer was adjusted to pH 9 using NH4OH and then fractionated with dichloromethane. The dichloromethane layer was washed with water and evaporated under reduced pressure at 40 °C, yielding a reddish-brown powder  which was stored at room temperature in dark glass bottles [ bottles .P. harmala seed alkaloid fraction powder (0.2 g) was dissolved in 10 mL deionized water by magnetic stirring at 80 °C for 90 min followed by centrifugation at 4 °C and 12,000 rpm for 5 min. The yellow-colored solution was then filtered through Whatman no. 40 filter paper. Ten mL of the solution were added to 90 mL of an aqueous solution containing 1 mM Pt (IV) chloride or 1 mM Pd (II) acetate followed by magnetic stirring for 24 h at 60 °C. Color change from yellow to dark brown indicated the formation of Pt NPs and Pd NPs. A mixture of Pt and Pd NPs was also prepared by adding 10 mL of the alkaloid solution to 90 mL of an aqueous solution containing 1 mM of both Pt (IV) chloride and Pd (II) acetate. The reaction conditions were the same as above. The prepared nanoparticles were collected by centrifugation  for 30 min at 4 °C. The pellet was washed with a mixture of ethanol and deionized water and then freeze-dried. UV–vis spectrophotometric tracking for the local surface Plasmon resonance (LSPR) of the greenly synthesized nanoparticles was conducted on a CARY 500 UV–vis–NIR Scan dual-beam spectrophotometer . The ζ-potential of the synthesized nanoparticles was studied using a laser Doppler velocimetry employing Zetasizer Nano ZS  in a clear disposable folded capillary cell . v/v) with deionized water and stained with 2% aqueous phosphotungstic acid. This mixture was placed and dried over a carbon-coated copper 200 mesh grid, imagined, and photographed. The nanoparticles’ morphology and diameter size were examined using transmission electron microscopy (TEM) employing a JEOL-JEM 2100 electron microscope operating at 160 kV. A 50 µL aliquot of the nanoparticles was diluted in the ratio of 1:2  spectroscopy performed on a FTIR- 8400s . Samples were first compressed with KBr into disks, scanned, and spectra recorded in the range of 500–4000 cm−1. The thermal stability of the nanoparticles was studied using a Q50 TGA Thermogravimetric Analyzer, USA. The lyophilized powder was placed in the platinum pan and heated from 0 to 400 °C at a heating rate of 10 °C/min under a nitrogen atmosphere.X-ray diffraction (XRD) analysis was performed to study the crystalline and elemental states of the prepared metallic nanoparticles. It was conducted in the range of 5° to 90°  at a generator voltage of 40 kV and current of 30 mA employing the Bruker D8 DISCOVER Family. The functional groups of Ten serial dilutions  of Trolox stock solution  were prepared. Pt NPs, Pd NPs, and Pt–Pd NPs were prepared at a concentration of 0.68 mg/mL methanol.3, in a ratio of 10:1:1 v/v/v; respectively. TPTZ was then mixed with 10 µL of the sample in a 96-well plate (n = 6) and incubated at room temperature for 30 min in the dark. The resulting blue color was measured using a microplate reader FluoStar Omega . The ability of the prepared nanoparticles to reduce ferric ions was expressed as a μM Trolox equivalent (TE)/mg sample using the linear regression equation extracted from the linear dose-response curve of Trolox. Data are represented as means ± standard deviation (SD).The ferric-reducing antioxidant power (FRAP) assay relies on the antioxidant’s capability to reduce ferric ions into ferrous ions in the presence of TPTZ forming ferrous-TPTZ complex (intense blue color). The assay was conducted in microplates according to the method described by Benzi et al. with few modifications . Brieflyv/v) CO2 at 37 °C.Breast adenocarcinoma cells (MCF-7) and lung cancer cells (A-549) were obtained from the American Type Culture Collection  and maintained in DMEM medium supplemented with streptomycin (100 mg/mL), penicillin (100 units/mL), and 10% heat-inactivated fetal bovine serum. Cells were incubated in 5% (3 cells) were seeded in 96-well plates. Cells were incubated for 24 h at 37 °C and 7% CO2 in DMEM medium. Cells were treated with aliquots of 100 μL DMEM containing containing different concentrations of Pt (IV) ions, Pd (II) ions, Pt–Pd ions, Pt NPs, Pd NPs, and Pt–Pd NPs . After 72 h, media were discarded, and 10% TCA (150 μL) was added to each well and incubated for 1 h at 4 °C then washed several times with distilled water. The SRB solution  was added to cells and incubated for 10 min in dark at room temperature. Finally, plates were washed with 1% acetic acid (3×) and allowed to dry overnight. TRIS  was added to dissolve the protein-bound SRB stain and absorbance measured at 540 nm . The IC50 (in µg/mL) was computed from concentration-response curves by Sigma Plot software, version 12.0 , using an E-max model equation. All experiments were conducted in triplicates, and data are demonstrated as mean ± standard deviation.Different concentrations of Pt (IV) ions, Pd (II) ions, Pt–Pd ions, Pt NPs, Pd NPs, and Pt–Pd NPs were added to breast adenocarcinoma cells (MCF-7) and lung cancer cells (A-549) and anticancer activities assessed using SRB assay ,25,26. ABased on the anticancer activities obtained from the in vitro studies, molecular modeling studies were performed to gain an insight into the mechanistic actions of the prepared biogenic nanoparticles as anticancer agents.+2 was downloaded from pdb; code: 6DQY. Minimization and docking procedures were carried out using MOE v.2010, together with dynamic simulations and both 2D and 3D resulted from interactions visualization. Hydrogens were added to downloaded proteins, and “automatically connect and type” was applied for all atoms and bonds. The receptor was selected, and fixing potential was carried out. Then, the sequential unneeded chains and ligands were deleted while pocket and complexed ligands were identified. Although the interaction between nanoparticles and enzyme proteins still lacks the adequate forcefield that could capture such binding, we tried to build a simple model of the three prepared metal nanoparticles  following similar procedures reported in the literature [Active metals and metal complexes have been widely reported to inhibit several significant enzymes, including thioredoxin reductase (TrxR), cysteine proteinases, kinases, and glutathione transferase, which made them beneficial for cancer treatment . The expterature . The strP. harmala seed alkaloid fraction was tracked visually and using UV–Vis spectroscopy. Initially, the formation of the nanoparticles was confirmed qualitatively by the gradual color change from pale yellow into black, brown, and dark brown colors in the case of Pt NPs, Pd NPs, and Pt–Pd NPs, respectively. The formation of the greenly synthesized NPs was further confirmed by UV–Vis spectroscopy (The bioreduction of Pt(IV) chloride, Pd(II) acetate, and Pt–Pd salt solutions to biogenic Pt NPs, Pd NPs, and Pt–Pd NPs mediated by troscopy . SignifiTransmission electron microscopy (TEM) analysis was used to determine the particle morphology diameter of the greenly synthesized nanoparticles. As depicted in The ζ-potential of the biogenic Pt NPs, Pd NPs, and Pt–Pd NPs was investigated using laser Doppler velocimetry and demonstrated an outstanding negative surface charge of −11.2 ± 0.5, −9.7 ± 1.2, and −12.7 ± 2.1 mV. The negative charge of nanoparticles’ surface prevents particle aggregation.P. harmala seed alkaloid fraction and Pt–Pd NPs were compared , 1602.6 cm−1 (C=O asymmetry stretching), 1430.9 cm−1 (O–H bending) and 1047.2 cm−1 (C–O stretching). The presence of three characteristic peaks of P. harmala seed alkaloid fraction in the FTIR spectrum of Pt–Pd nanoparticles may suggest that molecules in the seed extract coated the nanoparticles. However, the peaks’ intensities at 3438.6 cm−1 (–OH stretching), 1430.9 cm−1 (O–H bending), and 1430.9 cm−1 (C–O stretching) were significantly reduced, indicating a lower concentration of P. harmala seed alkaloid fraction, possibly indicating the rearrangement and deprotonation of these groups which may be involved in the bioreduction and stabilization processes [−1 (attributed to the carbonyl group) was significantly diminished in the FTIR spectrum of Pt–Pd NPs, which may suggest conjugation of nanoparticles with the P. harmala seed alkaloid fraction via carbonyl groups [The FTIR spectra of Pt NPs and Pd NPs were found to be similar to that of the Pt–Pd NPs. Thus, the FT-IR spectra of compared . Four mal groups ,18. The P. harmala seed alkaloid fraction coating the Pt–Pd NPs reduced their weight by 44.24%. The TGA thermogram of Pt–Pd NPs is illustrated in P. harmala seed alkaloid fraction has almost quadrupled their antioxidant activities. Our findings are in good agreement with previous studies that reported remarkable antioxidant activity of the Pt–Pd NPs [The antioxidant activities of the prepared metallic nanoparticles compared to harmala alkaloid fraction  were assessed by FRAP assay. The results were expressed with reference to Trolox standard as μM Trolox equivalent (TE)/mg samples. The FRAP values of harmala alkaloid fraction, Pt NPs, Pd NPs, and Pt–Pd NPs were 33.91 ± 5.82, 277.3 ± 13.5, 167.6 ± 4.8, and 843 ± 60 μM TE/mg NPs, respectively, . Notablyt–Pd NPs .50 in μg/mL, computed by Sigma plot) of the biogenic metallic nanoparticles against both cell lines are presented in 50 of 8.8 and 3.6 µg/mL, respectively; compared to either Pt NPs  and Pd NPs . This is attributed to the synergistic effect of both transition metals. The synthesized Pt NPs showed higher cytotoxic effects against both A549 and MCF-7 cancer cell lines than Pd NPs. Previous studies showed that the biogenic Pt NPs exert their cytotoxic effects via the induction of apoptosis and cell cycle arrest [50 of 23 and 9.5 µg/mL, respectively) [Agaricus bisporus (mushroom) fungi were found to be biocompatible with red blood cells [Padina gymnospora were found to be biocompatible to red blood cells with no detected hemolytic activity [Maytenus royleanus, were reported to exhibit anticancer activity against A549 cancer cells and low cytotoxicity against normal cells [The cytotoxic activities of Pt NPs, Pd NPs, and Pt–Pd NPs compared to the individual metals ions were evaluated on lung cancer cells A549) and breast adenocarcinoma cells (MCF-7) employing SRB assay. The green synthesized metallic nanoparticles exhibited significant in vitro cytotoxic activities compared to the individual metal ions. The cytotoxic activities (ICe arrest . Additioe arrest . Moreovectively) . Synthetctively) ,43. On tctively) . Some st9 and breod cells . Also, Pactivity . Pt NPs al cells . These fStaphylococcus Aureus V8 was downloaded from pdb; 1CV8 [+2 for a fair comparison to the candidate nanoparticles. After preparing the enzyme to be ready for docking, the dynamic nanoparticle models were set using MOEv.2010 and the resulted models were reference (Au+2NPs) and tested metals . Dynamic models were based on most of the nanoparticles in the reduced forms and only a small percentage in charged forms except for the Pt model where the charged forms constituted most of the model. Molecular docking studies were also performed between the prepared enzymes and each conformer obtained at equilibrium from the prepared four models using MOEv.2010. The results are presented in +2NP displayed a score of interaction energy of the best-docked pose = −17.07 kcal/mol showing ligand exposure to the key amino acid Arg 86, as shown in 0NP showed a higher score than reference = −22.4 kcal/mol, the interaction mode was contrarily presenting ligand exposure to Arg 113. On the other hand, Pt+2NP showed both higher score = −26.19 kcal/mol and enhanced mode of interaction with two amino acids ligand exposure; Asn 85 and Arg 86, thus being more embedded in the pocket of the active site as shown in 0-Pt+2 mixture displayed the highest score = −30.29 kcal/mol and enhanced interaction similar to that of Pt+2NP where Pd0 was closer to Asn 85 and Pt+2 distanced from Pd0 by 6.75 Å, was closer to Arg 86. These findings correlated to those obtained from the in vitro cell viability assays conducted on both cancer cell lines. Thus, in vitro cysteine proteinase inhibition assay may be performed in the future to evaluate the anticancer activities of platinum-based drugs.The crystal structure of staphopain cysteine proteinase from db; 1CV8 ; its actdb; 1CV8 , thus Au+2 complexed with SOD as a reference. The crystal structure of superoxide dismutase from Trichoderma reesei  was downloaded from the protein data bank. Four dynamic simulated models were built; reference agonist Mn+2NPs, Pd0NPs, Pt+2NPs, and Pd0-Pt+2 mixture using MOE v.20120. Alpha triangle docking was applied between the prepared superoxide dismutase, and the four built dynamic models. The results  protects organisms against potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide . Hence,  results  show thaPeganum harmala seed extract fraction. The average diameters of the green synthesized spherical Pt NPs, Pd NPs, and Pt–Pd NPs were 20.3 ± 1.9, 22.5 ± 5.7, and 33.5 ± 5.4 nm, respectively. The ζ-potential of the Pt NPs, Pd NPs, and Pt–Pd NPs were −11.2 ± 0.5, −9.7 ± 1.2, and −12.7 ± 2.1 mV, respectively. The metal nanoparticles’ bioreduction was confirmed by UV–vis spectroscopy, XRD, FTIR, and their organic contents determined by TGA analysis. The Pt–Pd NPs showed more pronounced antioxidant activity of 843 ± 60 μM TE/mg NPs compared to the individual Pt NPs (277.3 ± 13.5 μM TE/mg NPs) and Pd NPs (167.6 ± 4.8 μM TE/mg NPs). Furthermore, the Pt–Pd NPs exhibited significant cytotoxic activities against lung cancer and breast adenocarcinoma cells with IC50 of 8.8 and 3.6 µg/mL, respectively, compared to the individual Pt NPs  and Pd NPs  and compared to carboplatin . The in silico studies were carried out using MOEv.2010, where dynamic simulation models were built up to mimic the prepared nanoparticles’ kinetics. Docking studies showed that Pt and Pd metals exhibited promising activity against the selected cysteine proteinase enzyme and less activity against superoxide dismutase as compared to other metal references under the same conditions. Docking results proposed a possible mechanism of action of the antitumor activity of the biogenic metals, which is supported by the in vitro assay results, via inhibition of a critical proteolytic enzyme. In conclusion, Pt–Pd NPs capped with biological entities from alkaloid-rich harmala seed extract could be used as promising safe antitumor agents. This study will revive interest in Pt-based cancer therapeutics synthesized by green approaches as potentially effective and safe cancer therapy candidates.This work reports a simple environmentally friendly method for the green synthesis of Pt NPs, Pd NPs, and Pt–Pd NPs employing alkaloid-rich


Uric acid (UA) transporters mediate the uptake and outflow of UA, and are greatly involved in the control of UA concentrations. Glucose transporter 9 (GLUT9), one of the UA transporters, has been confirmed to be expressed in human umbilical vein endothelial cells (HUVECs). This study aimed to characterize GLUT9’s effect on intracellular UA accumulation in HUVECs in a high-UA environment and to explore the mechanism of cellular dysfunction.HUVECs were treated with UA to establish a model of cellular dysfunction. Then, UA uptake, GLUT9 expression and endothelial nitric oxide synthase (eNOS) and reactive oxygen species (ROS) amounts were measured. UA uptake was concentration- and time-dependent, and UA treatment significantly reduced nitric oxide (NO) levels and eNOS activity. UA also upregulated pro-inflammatory molecules and GLUT9, and increased intracellular ROS amounts in HUVECs. GLUT9 knockdown reduced UA uptake and ROS content, but antioxidant treatment did not reduce GLUT9 expression. To assess the function of JAK2/STAT3 signaling, HUVECs were treated with UA, and the phosphorylation levels of JAK2, STAT3, IL-6 and SOCS3 were increased by a high concentration of UA. In addition, GLUT9 knockdown reduced the phosphorylation of JAK2/STAT3 intermediates and increased p-eNOS amounts.GLUT9 mediated the effects of high UA levels on HUVECs by increasing the cellular uptake of UA, activating JAK2/STAT3 signaling, and reduced the production of active eNOS and NO in HUVECs.The online version contains supplementary material available at 10.1007/s11033-021-06840-w. Uric acid (UA) constitutes the end-product of primates’ metabolic reactions transforming purine nucleosides. Although at normal concentrations, UA plays antioxidative roles in humans, epidemiological evidence indicates abnormally high UA concentrations are associated with cardiovascular abnormalities, such as hypertension, coronary heart disease, and atherosclerosis –4. HowevSerum UA concentration mainly depends on the dynamic balance between UA production and its excretion. UA transporters, including URAT1, GLUT9 and breast cancer resistance protein (BCRP), mediate UA uptake and outflow, greatly regulating UA concentrations in the body . It was 2. UA  was dissolved in serum-free F12-K medium, incubated at 37 °C with constant shaking, and filtered using 0.22-μm syringe filters  [HUVECs provided by the China Center for Type Culture Collection  underwent culture in F12-K basal medium supplemented with 10% FBS, ECGS and 0.1 mg/ml heparin sodium at 37 °C under 5% COre, USA) . The UA HUVECs were cultured in 96-well plates and administered diverse amounts of UA  at ~ 80% confluence. Cell Counting Kit-8  was utilized to assess cell viability as directed by the manufacturer.UA uptake assays were performed in serum-free F12-K medium containing various amounts of UA . A Uric Acid Assay Kit (Sigma) was then utilized for the measurement of UA amounts in the cell culture supernatant after incubation for 24 h and 48 h, as directed by the manufacturer. Next, cell lysis was performed after cell counting with a hemocytometer. UA uptake was quantified by normalizing UA concentration to the cell count.NO release was measured in the cell culture supernatant using a Total Nitric Oxide Assay Kit , as directed by the manufacturer. This was achieved by measuring nitrite and nitrate amounts with the Griess reagent. Absorbance was measured on a microplate reader at 520 nm after color development.The concentration of eNOS in the cell culture supernatant was assessed with a Human eNOS ELISA kit (Beyotime Biotech), as directed by the manufacturer. Briefly, eNOS in the supernatant was bound by an anti-eNOS antibody in an ELISA plate, and then horseradish peroxidase-labeled avidin and the developing agent were added in sequence. Absorbance at 520 nm was used to calculate eNOS concentration.4 cells/ml, stimulated with UA. The culture medium was replaced with DCFH-DA diluted in serum-free medium to 10 µM, followed by a 20-min incubation. After three washes with serum-free medium, fluorescence was immediately assessed on a Leica fluorescence microscope , with excitation and emission at 488 nm and 525 nm, respectively.Intracellular ROS concentration was assessed with DCFH-DA (Sigma). The cells underwent seeding in 35-mm plates at 4 × 10RNA extraction utilized Transzol reagent , and RNA purity was determined by absorbance measurements at 260 and 280 nm. Reverse transcription was carried out with a Reverse Transcriptase kit (TransGen Biotech), and transcript abundance was quantitated on a 7500 Real-Time PCR System  using the SYBR Green detection system (TransGen Biotech).Gapdh mRNA amounts. The 2−ΔΔCt method was utilized for data analysis. The primers used are listed in Table S1.Target gene expression was normalized to Lysates were prepared from HUVECs in the RIPA buffer supplemented with protease and phosphatase inhibitors . The BCA kit (Thermo Fisher Scientific) was utilized for protein quantitation. Equal amounts of total protein underwent separation by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE), followed by electro-transfer unto PVDF membranes . Upon blocking with 5% skim milk (2 h at ambient), successive incubations were carried out with primary (overnight at 4 °C) and secondary (50 min at ambient) antibodies. After washing, detection of immunoreactive bands utilized the enhanced chemiluminescence reagent (Thermo Fisher Scientific). ImageJ was used for imaging and quantitation. Target protein expression was normalized to that of GAPDH. The utilized antibodies, provided by Abcam (UK), Proteintech (USA) or Cell Signaling (USA), were directed against GLUT9 (1:1000), eNOS (1:1000), phosphorylated (p)-eNOS (1:1000), total (t)-JAK2 (1:1500), p-JAK2 (1:1000), t-STAT3 (1:1500), p-STAT3 (1:1000), IL-6 (1:2000), SOCS3 (1:2000) and GAPDH .HUVECs underwent fixation with 4% formalin in PBS and permeabilization with 0.3% Triton X-100 for 10 min. Upon blocking with 3% BSA for 30 min, incubation was carried out with mouse anti-p-eNOS (1:500) and rabbit anti-GLUT9 (1:500) antibodies, respectively, overnight at 4 °C in a humidified environment. After washing, the cells were covered with secondary antibodies and incubated at ambient for 50 min. DAPI counterstaining was performed, and cells were finally examined under a Leica fluorescence microscope (Leica).N-acetylcysteine , an antioxidant, was supplemented to some wells for determining the impact of oxidative stress on GLUT9 expression.Small interfering (si)RNAs targeting SLC2A9 (GLUT9), shown in Table S2, were designed and produced by GenePharma (China). A 20-μM siRNA stock solution was prepared as directed by the manufacturer. Then, the transfection complex was prepared by mixing Opti-MEM , siRNA stock solution (5 µl) and siRNA-Mate transfection reagent . HUVECs were transfected using these transfection complexes with siRNA concentration of 50 nM in a 6-well plate at 37 °C for 24 h. Next, UA was supplemented to appropriate wells, and cells were subsequently collected to measure transfection efficiency by RT-PCR and immunoblot. t-test and one-way ANOVA for normally distributed data. Otherwise, a non-parametric test was applied. P < 0.05 was deemed to reflect statistical significance.SPSS 22.0  was utilized for data analysis. Data are mean ± SD. Group pairs and multiple groups were compared by Student’s UA at 5, 10 and 15 mg/dl had no clear effect on cell survival, but decreased survival was detected at a UA concentration of 20 mg/dl   Fig. D. Next, 05) Fig. E. While 05) Fig. F, G.Fig.JAK2/STAT3 signaling activation is known to worsen cellular oxidative stress. Therefore, we aimed to determine whether UA increases intracellular ROS content by inducing JAK2/STAT3 signaling, subsequently leading to endothelial cell dysfunction and inflammation. The results showed that UA treatment had no effects on JAK2 and STAT3 mRNA amounts, but SOCS3 was significantly upregulated by UA at concentrations of 10, 15 and 20 mg/dl Fig. A–C. FurtMultiple mechanisms explaining UA’s role in endothelial dysfunction and cardiovascular diseases have been suggested. It was previously demonstrated UA increases calcium amounts in the mitochondria, which leads to greater production of mitochondrial superoxide, mitochondrial dysfunction, ROS generation , 14, lowHyperuricemia was associated with lower plasma membrane expression of the UA efflux transporter BCRP, which leads to decreased UA efflux and intracellular UA accumulation . In addiNO release by endothelial cells plays a critical role in vascular homeostasis , 18. In To explore the mechanism underpinning this effect, we examined GLUT9’s role. GLUT9’s effect on oxidative stress was determined by treating HUVECs with siRNA-GLUT9 and the antioxidant NAC, respectively. The results showed that siRNA and NAC treatment reduced intracellular ROS content, and GLUT9 expression was not inhibited when oxidative stress was relieved by NAC administration. Therefore, we can speculate that overexpression of GLUT9 would increase the accumulation of UA in cells and stimulate oxidative stress, which has never been addressed previously.Inflammation is critical in initiating and sustaining vascular injury. Previous studies have shown that UA causes inflammation in endothelial cells, which manifests as upregulation and enhanced release of pro-inflammatory molecules, including IL-6, THF-α, IL-1β and MCP-1 . HoweverHigh UA levels induce endothelial cell dysfunction, which is characterized by decreased eNOS activity and NO production. These effects require GLUT9, which mediates the accumulation of UA in cells, which in turn activates the JAK2/STAT3 pathway, inducing inflammation and oxidative stress.Supplementary file1 (PDF 102 kb)Supplementary file2 (PDF 80 kb)Below is the link to the electronic supplementary material.


IntroductionNonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, with a global prevalence of 20%-40%. Approximately 40%-60% of patients with type 2 diabetes mellitus (DM2) experience NAFLD; out of which 20%-40% cases may have higher severity. Due to the scarcity of available reports from the eastern part of India, we aimed to evaluate the effects of dapagliflozin, a sodium-glucose cotransporter-2 inhibitor used in these types of cases.Material and methodsThe study included consecutive patients with DM2 and NAFLD, treated with dapagliflozin at 10 mg daily for six months. All patients underwent detailed anthropometric, biochemical, abdominal ultrasonography, and transient elastography studies at baseline and after therapy as well as a comparative analysis.Results2 at baseline to 26.21 ± 1.51 kg/m2 after the therapy. The patients’ transaminitis, dyslipidemia, and glycemic status significantly improved over the course of the therapy. We also observed significant (p < 0.05) improvement in hepatic steatosis by the end of the treatment. Although transient elastography by FibroScan-measured hepatic fibrosis score  significantly decreased from 6.95 ± 1.42 to 6 ± 1.44 kPa, hepatic fibrosis did not improve significantly (p ≥ 0.05) following therapy.In the 100 patients included in our study, the male patients outnumbered the female patients  and the mean age at presentation was 44.11 ± 8.24 years. The mean body mass index significantly decreased over the course of the therapy, from 27.31± 1.87 kg/mConclusionAlthough dapagliflozin improved body mass index, transaminitis, dyslipidemia, glycemic status, and hepatic steatosis, it had a minimal effect on hepatic fibrosis. Nonalcoholic fatty liver disease (NAFLD) is currently the most prevalent chronic liver disease, with a global burden of 17 % to 46% of chronic liver disease cases ,3. PatieAs NAFLD typically has a very long asymptomatic course and may silently lead to hepatic steatohepatitis and fibrosis, early diagnosis by noninvasive methods and implementation of appropriate therapy is the need of the hour . AlthougGiven that the early diagnosis of NAFLD is an essential precautionary measure, the disease should be thoroughly scrutinized in susceptible patients, with available conventional diagnostic modalities such as ultrasonography elastography, and noninvasive fibrosis markers. Transient elastography by FibroScan typically evaluates hepatic steatosis by quantifying the controlled attenuation parameter (CAP) and hepatic fibrosis by estimating liver stiffness (LS), which can accurately predict the thepatic fibrosis stage . An inva In view of the paucity of data on the role of dapagliflozin in cases of DM2 with NAFLD in the eastern part of India, we aimed to analyze the variable effects of dapagliflozin in this specific population group.This prospective, single-center, open-label, uncontrolled, interventional, multidisciplinary cohort study was conducted in the Department of ‘General Medicine’ and ‘Gastroenterology and Hepatobiliary Sciences’ of the Institute of Medical Sciences and SUM Hospital, Bhubaneswar, Odisha, India. The study included and prospectively evaluated consecutive patients with DM2 and NAFLD (who attended the outdoor clinic) of the above-mentioned department between January 2020 and June 2020.By subjecting the patients to appropriate investigations, NAFLD was diagnosed on the basis of ultrasound evidence of hepatic steatosis, after ruling out other causative factors of hepatic steatosis, such as significant alcohol consumption (daily intake >-20g for men and >-10g for women), negative hepatitis B and C viral serology, autoimmune hepatitis, hepatic storage disorders, such as hemochromatosis and Wilson’s disease, the consumption of suspicious drugs, and other diseases that might be associated with hepatic steatosis. DM2 was diagnosed based on fasting blood glucose level ≥126 mg/dL and postprandial or 2-h postprandial blood glucose levels and random blood glucose levels of 200 mg/dL on at least two occasions . The patWe calculated the noninvasive fibrosis markers for NAFLD according to the following formulas. For the NAFLD fibrosis score = the equation was -1.675 + 0.037 × age(years) +0.094 × body mass index (BMI (kg/m2) + 1.13 × impaired fasting glucose/diabetes  + 0.99 × aspartate aminotransferase (AST)/alanine transaminase (ALT) ratio -0.013 × platelet count (109/ L) -0.66 × albumin (g/dL) For the FIB- 4 index, the equation was (age {years} × AST {units/L}/platelet count (×109/L) ×  to quantify the hepatic fibrosis in our study. Hepatic steatosis was graded into three levels as per abdominal ultrasound findings as follows: (1) mild, minimally increased hepatic parenchymal echogenicity with a normal-looking diaphragm and portal vein wall; (2) moderate, moderately increased hepatic echogenicity with a minimally-blurred portal vein wall and the diaphragm; and (3) severe, markedly increased hepatic echogenicity with a very poorly visualized portal vein wall, diaphragm, and posterior part of the right liver lobe . Transie Statistical analysisThe statistical analysis was performed using SPSS software version 23 . We calculated the sample size to be 71 for this interventional study on the basis of NAFLD prevalence in the community; which was around 25-30% and expected post-therapy drop in ALT level by 10% for the power of the study to be 80% and alpha to be 0.05. The results are presented as mean ± standard deviation or frequency in percentages. We compared the normally distributed quantitative and categorical variables at baseline and after therapy, using paired Student’s t-test and chi-square test, respectively. P values<-0.05 were considered significant in our study.One hundred consecutive patients with DM2 and fatty liver were evaluated prospectively in this study. Males outnumbered the females  in our study. Mean age of the study population at baseline was 44.11 ± 8.24 years. Mean BMI decreased from 27.31 ± 1.87 kg/m2 to 26.21 ± 1.51 kg/m2 (p=0.0001) following six months of dapagliflozin therapy. The mean total platelet count (TPC) and serum bilirubin at baseline were 200,100±54,810/µL and 1.24 ± 0.34 mg/dL respectively. The mean serum liver enzymes at baseline in IU/L were as follows: SGOT - 55.88 ± 20.92, SGPT - 64.69 ± 22.73 and GGT - 67.54 ± 17.72. Mean high-density lipoprotein cholesterol (HDL-C) and triglyceride (TG) level at baseline were 43.52 ± 10.19 mg/dL, and 172 ± 21.41 mg/dL respectively. The mean serum albumin level at baseline was 3.72 ± 0.46 g/L. Mean FibroScan CAP score at baseline and after six months of therapy was 273.11 ± 17.51 dB/m and 251.16 ± 11.7 dB/m, respectively (p = 0.0001). Table This study is first of its kind to comprehensively evaluate the effects of the SGLT2 inhibitor dapagliflozin on both hepatic steatosis and fibrosis using ultrasonography and transient elastography study and by analyzing the results of various noninvasive hepatic fibrosis scales in patients with DM2 and NAFLD. Most of the patients in our study were male and around 40 to 50 years old. We found significant improvement in various liver enzyme biochemical parameters after dapagliflozin therapy, as similarly reported in previous studies -21. EvenLimitationsThis study has some limitations, which were as follows: open-label, non-randomized and uncontrolled study design; lack of long-term therapy and post-therapy follow-up and nonperformance of liver biopsy for histological confirmation, which is considered as the gold standard for NAFLD assessment. However, robust evidence suggested that transient elastography by FibroScan study is equally efficacious as liver biopsy study to assess liver fibrosis . We alsoStrength of the studyPossibly, this study is the first of its kind to vividly and comprehensively assess various noninvasive fibrosis biomarkers for NAFLD, along with a comparative assessment of the various biochemical parameters, anthropometric variables, abdominal ultrasonography, and simultaneous FibroScan study, both at baseline and after completing the therapy.Although this study was a short-term, non-randomized, interventional uncontrolled study, we could observe significant improvement in hepatic steatosis following dapagliflozin therapy, as reported by both ultrasonography and FibroScan assessment. Dapagliflozin is possibly non-antifibrotic in nature, as we failed to observe significant improvement in hepatic fibrosis with its use in our study. However, our study findings should be validated in the future by long-term, randomized, well-controlled interventional trials for better judgment of its use in the diabetic NAFLD population.


Deinagkistrodon acutus) venom, and evaluated its interaction with GPIbα by surface plasmon resonance and in silico modeling. We demonstrated that anfibatide interferds with both VWF and thrombin binding, inhibited ristocetin/botrocetin- and low-dose thrombin-induced human platelet aggregation, and decreased thrombus volume and stability in blood flowing over collagen. In a single-center, randomized, and open-label phase I clinical trial, anfibatide was administered intravenously to 94 healthy volunteers either as a single dose bolus, or a bolus followed by a constant rate infusion of anfibatide for 24 h. Anfibatide inhibited VWF-mediated platelet aggregation without significantly altering bleeding time or coagulation. The inhibitory effects disappeared within 8 h after drug withdrawal. No thrombocytopenia or anti-anfibatide antibodies were detected, and no serious adverse events or allergic reactions were observed during the studies. Therefore, anfibatide was well-tolerated among healthy subjects. Interestingly, anfibatide exhibited pharmacologic effects in vivo at concentrations thousand-fold lower than in vitro, a phenomenon which deserves further investigation.The interaction of platelet GPIbα with von Willebrand factor (VWF) is essential to initiate platelet adhesion and thrombosis, particularly under high shear stress conditions. However, no drug targeting GPIbα has been developed for clinical practice. Here we characterized anfibatide, a GPIbα antagonist purified from snake (Trial registration: Clinicaltrials.gov NCT01588132. The interaction between the platelet receptor glycoprotein (GP) Ibα and von Willebrand factor (VWF) initiates platelet adhesion, particularly under high shear stress7. Following this initial tethering, platelets become activated allowing integrin αIIbβ3 (GPIIbIIIa) to bind fibrinogen and other ligands, including VWF, mediating platelet aggregation and the formation of a hemostatic plug to stop bleeding or an occluding endovascular thrombus, causing diseases10. Notwithstanding the essential αIIbβ3 contribution to thrombogenesis, the GPIbα-VWF interaction remains critical for pathological endovascular growth of occlusive thrombi at sites of arterial stenosis where blood flows with wall shear rates that may exceed 40,000 s−1, corresponding to shear stresses exceeding 1600 Pa11.Platelet adhesion and aggregation at sites of vascular injury are key events in the arrest of bleeding, but also contribute to vascular thrombosis, such as in the atherosclerotic coronary or cerebral arteries, causing heart attack and stroke, the leading causes of morbidity and mortality worldwide15. Several αIIbβ3 inhibitors, such as abciximab, eptifibatide, and tirofiban, have been in use for over 20 years16. They are effective anti-platelet and antithrombotic agents, but may induce thrombocytopenia and severe bleeding in some patients18. To date, no drug targeting GPIbα has been successfully developed, despite favorable arguments supporting such an approach21, as outlined above, in particular, the likely selective advantage of a GPIbα antagonist in conditions involving arterial thrombosis.Currently, aspirin, clopidogrel, and αIIbβ3 antagonists are the primary anti-thrombotic agents used in the long-term treatment of cardiovascular thrombotic disorders23. Recently, studies have focused on agkisacucetin (generic name: anfibatide)24 and agkisacutacin27, two GPIb-binding snaclecs purified from the venom of Deinagkistrodon acutus  with different sequences listed in the UniProt database . Anfibatide and agkisacutacin, like other snaclecs, consist of α- and β-subunits that are disulfide-linked, forming αβ heterodimers. Interestingly, anfibatide contains an unpaired Cys residue, as seen in the crystal structure24, which is unusual for snaclecs and may contribute to its unique function through disulfide-interactions with other snaclec subunits or targeted proteins28. Anfibatide also contains no Ca2+ binding loops24, consistent with the lack of interaction with the Vitamin K-dependent carboxylation/gamma-carboxyglutamic (GLA) domains of coagulation factors.Snaclecs (snake C-type lectins) are a subset of non-enzymatic proteins isolated from venom of different snakes and have been evaluated for their therapeutic potential, including as antithrombotic agents for decades; however, none have yet successfully entered clinical practiceTrimeresurus flavoviridis venom30, but with no effect on the activated partial thromboplastin time (aPTT)25. One year later, the same group reported a protein identified with the same name (agkisacutacin) that completely inhibited ristocetin and thrombin (but not collagen or ADP)-induced platelet aggregation, and had no fibrinolytic or anticoagulant activity26. The discrepancies between the two reports have not yet been adequately explained; interestingly, however, the properties reported in the latter study closely correspond to the results we obtained with anfibatide21. Subsequently, agkisacutacin was reported to prolong prothrombin time (PT) and aPTT, and decrease platelet aggregation induced by collagen, ADP, and thrombin while demonstrating both antithrombotic and thrombolytic activities26. After further purification, agkisacutacin was later found to bind platelet GPIbα as well as coagulation factors IX and X, and inhibit GPIbα-VWF dependent ristocetin-induced platelet aggregation27. Thus, although anfibatide and agkisacutacin share the properties of binding to and inhibiting VWF-mediated functions of GPIbα, only anfibatide shows exclusive selectivity for platelet GPIbα.Agkisacutacin was initially identified as a fibrinolytic protein with high sequence homology to coagulation factor IX/X-binding protein (BP) derived from Deinagkistrodon acutus venom; agkicetin C and akitonin, which inhibit platelet function, and agkaggregin, which induces platelet activation. Flavocetin-A and echicetin, isolated from Habu snake venom, are two snaclecs that also inhibit VWF access to GPIbα; however, they can both support and inhibit platelet aggregation31. Akitonin and jararaca GPIb-BP, isolated from Bothrops jararaca, are GPIbα antagonists that decrease platelet aggregation in vitro, but their in vivo efficacy and bleeding diathesis have not been reported33.At least three additional GPIbα-binding snaclecs have been isolated from , with mouse models of thrombogenesis21. We showed that anfibatide specifically inhibits the GPIbα-VWF interaction as well as platelet functions known to depend on it, including ferric chloride- and laser-induced thrombus formation in mesenteric and cremaster muscle arterioles34. Such findings suggest that anfibatide has the potential of acting as a GPIbα selective antithrombotic agent in humans, as we briefly reported in 2013 annual meeting of American Society of Hematology35. Since the GPIbα-VWF interaction is strictly required for thrombogenesis at pathological levels of shear stress, anfibatide may offer an improved risk/benefit ratio compared to αIIbβ3 and VWF antagonists36, particularly in the management and treatment of acute coronary syndromes in which acute occlusion involves pathologically elevated shear stress.We previously evaluated properties of purified anfibatide in vitro and in vivo40. Also, when anfibatide is administered at the optimal dosage, route, and interval, it is effective in treating spontaneous and bacterial shigatoxin-induced TTP in murine models. These studies may provide the basis for further development of anfibatide for the treatment of acute TTP in humans41. In addition, the first balanced expression strain and pilot-scale production of recombinant anfibatide in P. pastoris has been reported43, aiming to solve the quality control difficulties of purifying anfibatide from raw snake venom and the limited supply of this natural resource. Further improvements of recombinant anfibatide are still under development.Since then, anfibatide has been shown as a promising candidate that could be beneficial for the treatment of ischemic stroke and has a protective effect on cerebral ischemia/reperfusion injury in animal models, ex vivo with human blood, and after injection and infusion in healthy human subjects—making anfibatide the first GPIbα antagonist tested in humans. Our data suggest that anfibatide may be a potentially safe and effective agent for antithrombotic therapy targeting platelet GPIbα.This present study evaluates the antithrombotic efficacy and safety of anfibatide in vitro43. The eluted product was further purified by size exclusion chromatography using a Sephacryl S-100 column. After the four-step purification  by anion-exchange chromatography and cation-exchange chromatography followed by immuno-affinity chromatography with a specific anti-anfibatide monoclonal antibody 1B9 fication , MALDI-T24. To elucidate the potential binding site of anfibatide on GPIbα, a three-dimensional structural model of the GPIbα-anfibatide complex was generated by in silico molecular docking based on information available in the Protein Data Bank (PDB), 1OOK44 for GPIbα, 3UBU24 for anfibatide, and superposed to the GPIbα-VWF-A1 domain (VWA) complex (PDB entry 1SQ045). These structural models show close proximity between the anfibatide and VWF-A1 binding sites on GPIbα, implying that anfibatide can act as a competitive inhibitor of the VWF-GPIbα interaction  sequence, and one with the mutation of three Tyr residues  with Phe (designated 3Y/F), preventing post translational sulfation that normally occurs at these positions and is responsible for contacting thrombin exosite II and exosite I residues46. Anfibatide bound to GPIbαN-Long WT with a KD = 21.0 ± 0.9 nM and bound to the 3Y/F mutant GPIbαN-Long with approximately fourfold lower affinity, KD = 90.5 ± 9.2 nM  that recognizes the human GPIbα sulfotyrosine region47 and itself is an inhibitor of thrombin binding  was used to evaluate anfibatide binding to a recombinant chimeric protein formed by residues − 2 to 288 of human GPIbα, followed by 133 residues of the SV40 large T antigen; this disulfide-linked dimeric molecule was designated GPIbαN-Long49.Concordant with the results of binding studies, anfibatide specifically blocked VWF-mediated ristocetin-induced platelet aggregation in human platelet-rich plasma (PRP) without interfering with aggregation induced by various doses of ADP, thrombin-receptor activating peptide (TRAP/PAR1AP), collagen, or high dose thrombin (1 U/mL); however, it delayed and decreased aggregation induced by low dose thrombin (0.1 U/mL) Fig. , which c53 in perfusion chambers coated with collagen, on which soluble VWF from blood quickly anchors. Whole blood from healthy volunteers was perfused over the collagen-coated surfaces with or without the addition of anfibatide, and imaged in real-time by fluorescence microscopy. Anfibatide markedly inhibited platelet adhesion, aggregation, and thrombus formation under high shear rate conditions  to groups of healthy volunteers either as a single dose bolus    reduce platelet aggregation but prolong cutaneous bleeding time60, possibly because VWF is a ligand for both GPIbα and αIIbβ3. In contrast to VWF knockout mice, thrombus formation was completely abolished in GPIbα knockout mice6; therefore, GPIbα ligands other than VWF may also play a role in platelet aggregation and thrombus formation. Our recent murine study supported this hypothesis; we observed that anfibatide inhibited thrombus formation even in VWF knockout mice21. Furthermore, recent studies showed that GPIbα interacts with thrombin, P-selectin, Mac-1, and Thrombospondin-161. In this study, we observed that anfibatide competitively inhibited α-thrombin binding and inhibited low dose thrombin-induced platelet aggregation. Thus, anfibatide may have additional benefits for anti-thrombotic therapy beyond inhibition of the GPIbα-VWF interaction.Based on recent evidence from murine model and structural studies63. Interruption of this GPIbα outside-in signaling may reduce the level of platelet and integrin activation. Thus, the inhibition of GPIbα by anfibatide may induce thrombus dissolution, as well as prevent further thrombus growth and vessel occlusion.We demonstrated here that GPIbα antagonism by anfibatide inhibited platelet adhesion, aggregation, and thrombus formation in human blood in vitro, especially at high shear. We observed inhibition of ristocetin-induced (i.e. GPIbα-VWF-mediated) platelet aggregation in human blood when anfibatide was added to blood in vitro or administered to volunteers before blood collection. Anfibatide also inhibited low dose thrombin-induced (αIIbβ3-fibrinogen-mediated) platelet aggregation. Furthermore, anfibatide dissolved preformed thrombi in our ex vivo perfusion chamber thrombosis models. Several mechanisms may contribute to this effect of thrombolysis: first, thrombus growth and dissolution are dynamic processes and occur simultaneously under flow conditions; blocking GPIbα-VWF interaction likely prevents platelet recruitment, thus favoring thrombus dissolution over thrombus growth. Second, GPIbα binding to other ligands  may provide additional bridges between adjacent activated platelets to enhance thrombus stability; breaking these bridges may facilitate thrombus dissolution. Third, VWF-GPIbα and thrombin-GPIbα interactions deliver outside-in signaling via the GPIb-IX-V complex which activates newly recruited platelets in the thrombus and likely maintains their αIIbβ3 integrins in the active conformation required for fibrinogen- and other integrin ligand-mediated platelet aggregation64, can enhance the inhibitory effect of anfibatide on the ristocetin-induced platelet aggregation in these participants. Notably, a recent study showed that the disulfide bonds in the GPIbα ligand binding domain can be catalyzed by protein disulfide isomerase, which affects GPIbα-ligand interactions28. Interestingly, anfibatide can interfere in this process and GPIbα-ligand binding28, which may synergistically contribute to the inhibitory effect observed in vivo in the human participants.Our phase I clinical trial evaluated the pharmacodynamics, pharmacokinetics, safety, and tolerability of anfibatide. Healthy volunteers were treated with a moderate range of anfibatide doses using two different modes of administration: single dose intravenous injection, or single dose injection followed by a 24 h constant rate infusion. In PRP collected from anfibatide-treated participants (as low as 1 μg/60 kg), ristocetin-induced platelet aggregation was inhibited. This in vivo effect seems to be far more potent than our in vitro assays since the doses used in this clinical trial are several thousands times less. It is currently unknown whether other in vivo factors, as also observed during other antithrombotic agent developmentPharmacokinetics of anfibatide detected the free (i.e. platelet unbound) plasma anfibatide, which appeared as dose proportional at high doses administered in this study. In healthy volunteers receiving single dose intravenous injection at 1–3 μg/60 kg, the maximum plasma concentration was observed at 4–5 h post-dose. Following administration, anfibatide likely quickly binds to platelet GPIbα in blood circulation and was disassociated over time from the platelet surface. The peak plasma anfibatide was reached 8 h after the injection of higher doses (4–5 μg/60 kg). In addition, the apparent volume of distribution (Vd) was estimated between 3.6 and 8 L, indicating that anfibatide is mainly distributed to the intravascular fluid that reflects a high degree of blood cell or plasma protein binding. Given that anfibatide is transient in the plasma and undergoes fast disassociation, platelet dysfunction is also reversible. Notably, anfibatide-driven inhibition of ristocetin-induced platelet aggregation had a rapid recovery to baseline levels within 4–8 h after drug discontinuation, while plasma anfibatide concentrations remained at moderate to high levels. Although not fully understood, it is possible that plasma anfibatide, after disassociation from platelets may become inactive (metabolite) or may induce platelet GPIbα ectodomain shedding that likely prevents soluble GPIbα-bound anfibatide from binding to other platelets, a hypothesis that deserves future exploration. The relationship between anfibatide concentration, inhibition of the GPIb-IX-V complex, and platelet function was consistent across all doses and dosing regimens. This study suggests a fast onset, potent, and reversible antithrombotic effect of anfibatide among healthy subjects and provides the basis for assessing the non-ST Segment Elevation Myocardial Infarction (NSTEMI) and ST-segment elevation myocardial infarction (STEMI) patients undergoing percutaneous coronary intervention in the phase Ib-IIa and phase II trials.18. However, aspirin is an irreversible platelet cyclooxygenase inhibitor, which can cause severe bleeding when patients require emergency surgery65. In addition, although αIIbβ3 inhibitors represent effective anti-platelet and antithrombotic agents, they often lead to thrombocytopenia and life-threatening bleeding complications, such as cerebral, alveolar, and gastrointestinal system hemorrhages71. Similarly, prasugrel demonstrated enhanced platelet inhibition, but prevention of ischemic complications was often achieved with increased bleeding complications72. As mentioned previously, novel VWF antagonists (e.g. aptamer ARC1779 and nanobody caplacizumab) reduced platelet aggregation but also have bleeding concerns in clinical trials60. An optimal therapy for acute vascular syndromes would be targeting platelet aggregation at high shear conditions without impairing hemostasis, such as observed with anfibatide. GPIbα antagonism may, therefore, be a more attractive strategy for antihrombotic therapy.As the processes involved in hemostasis and thrombosis are similar, the quest for an antihrombotic agent that does not affect hemostasis has been challenging. Current first-line antithrombotic agents target platelet aggregation by COX-1 inhibition (aspirin), αIIbβ3 antagonists , and P2Y12 antagonists (e.g. clopidogrel and prasugrel)73. Second, the VWF-GPIbα interaction pathway may be independent of (i.e. not interfere) other pathways leading to clot formation, such as platelet activation via collagen or ADP receptors. Third, anfibatide may have no effect on VWF functions in hemostasis that are independent of GPIbα (such as VWF-β3 integrin interaction)74. Importantly, no serious adverse events, premature discontinuations due to adverse events, or deaths occurred during the study. Anfibatide did not significantly affect platelet count, and no anti-anfibatide antibodies were detected in the subjects, suggesting that anfibatide may be safe and well-tolerated in healthy individuals.Anfibatide significantly inhibited GPIbα-mediated platelet aggregation at low concentrations without impairing coagulation or prolonging bleeding time. We propose three possible reasons to explain why anfibatide did not affect hemostasis. First, VWF-GPIbα interactions predominantly exist in the presence of high shear stress found in the arterial circulation; thus, GPIbα antagonist may act specifically at sites of thrombosis, and not systemically. This spatial specificity may help to avoid the common hemorrhagic consequences of conventional anti-platelet agentsIn summary, this study is the first demonstration of the safety and efficacy of a GPIbα antagonist in healthy human subjects. Anfibatide is a promising GPIb-IX-V complex antagonist that exhibited strong anti-platelet effects, excellent reversibility, and low bleeding tendency in this clinical trial, representing a new therapeutic agent that may advance the treatment of acute coronary syndrome. This study also demonstrates the promising safety profile of anfibatide and provides the basis for the phase Ia-IIb and phase II clinical trials among NSTEMI and STEMI patients, respectively. Further studies will be required to define the optimal dosing strategy for patients with acute coronary syndrome, who often have a high intracoronary thrombus burden and may require a higher therapeutic dosage of anfibatide in order to achieve a higher anti-platelet effect.N = 6) as well as in perfusion chamber models of platelet thrombus formation (N = 12). Human platelets or blood samples were randomly allocated to experimental or control groups. The investigators performing the experiments were blinded for the study duration. The sample size was selected based on our previous publications and publications from other groups. All experimental procedures using human blood samples in vitro and ex vivo were approved by the Research Ethics Board of St. Michael’s Hospital—Unity Health Toronto, Toronto, Canada. All data were included in the analysis.This work comprises an in vitro pre-clinical study, where the interaction between anfibatide with GPIbα was evaluated by surface plasmon resonance and simulated by computer modeling, and functional properties of anfibatide were tested by platelet binding and aggregation studies  under Investigational New Drug (IND) approval number of 2005L04666 , assigned by the State Food and Drug Administration (SFDA), now known as the National Medical Products Administration (NMPA) in China. The clinical protocol and its amendments were approved by the Yijishan Hospital Review Board and the study was performed in compliance with the Declaration of Helsinki on medical research involving human subjects and the Guideline for Good Clinical Practice recommended by the NMPA.This study also includes a prospective, single-centered, randomized, open-label, and dose-escalating phase I clinical trial Trial subjects meeting the inclusion and exclusion criteria underwent randomization by statisticians according to the random number coding table generated by the SAS software. Written informed consent was obtained prior to participation for all subjects. 93 participants who completed the phase I study were included in the analysis . All primary data are in the 24, a three dimensional model of the GPIbα-anfibatide complex was simulated using HADDOCK software76. The coordinates used in the docking experiments were from Protein Data Bank . The figure of the GPIbα–VWF-A1 domain complex structure was created from PDB entry 1SQ045 and the figure of the GPIbα–α-thrombin complex is from PDB entry 1OOK. The figures were created using PyMOL software .Based on our X-ray crystallography data77. Briefly, human recombinant GPIbα amino fragment ) fused to 133 residues of the SV40 large T antigen with terminal Cys residues to mediate dimerization were expressed in Drosophila melanogaster S2 cells and purified from culture supernatant. A monoclonal antibody specific for the SV40 sequence (LJ-3A2) was linked to an SPR chip . Using a Biacore 3000 SPR instrument and 135 mM NaCl, 20 mM N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid  containing 0.005% Tween 20 as the running buffer, GPIbα-SV40 fusion protein was coupled to the mAb immobilized SPR chip. After GPIbα-SV40 immobilization, anfibatide solutions at varying concentrations prepared in running buffer were injected over the chip (association phase) at 75 μL/min for 3 min followed by running buffer for 20 min (dissociation phase). Binding affinities were determined by plotting the steady state SPR determined mass ratios of anfibatide to GPIbα-SV40 (pre-dissociation), against anfibatide concentration, and fitting the data to a one-site ligand binding model using the Graphpad Prism 8.0 software package (https://www.graphpad.com/scientific-software/prism/).Surface plasmon resonance (SPR) was performed to analyze the interaction between GPIbα and anfibatide as was previously done for GPIbα-VWF A1 domain interaction46. Briefly, blood from healthy human volunteers was collected in tubes containing acid-citrate-dextrose. The tubes were centrifuged at 600g for 12 min to prepare platelet-rich-plasma (PRP). The PRP was then centrifuged at 800g for 15 min in the presence of 10 μM PG E1 (Enzo Life sciences) and 0.6 U∕mL Apyrase Grade VII (Sigma Aldrich) to obtain a platelet pellet. The pellet was washed once by resuspension and centrifugation in modified Tyrode’s buffer, pH 6.5  biotin-PPACK active site blocked, BP-αFIIa, mixed with phycoerythrin-conjugated streptavidin, S-PE, recombinant human VWF A1 domain (residues 445–733 expressed in house) mixed with anti-VWF mAb conjugated with FITC or mAb LJ-Ib10 conjugated with FITC, were mixed with aliquots of the platelet suspension to a final concentration of their respective KD  and incubated at room temperature for 30 min. When indicated anfibatide, at concentrations ranging from 0 to 200 nM, was added to the platelet suspensions 5 min before the corresponding GPIbα ligand. Binding was measured by flow-cytometry, without sample dilution, using a FACS Calibur II equipped with a 488 nm argon laser and the software CellQuest for data evaluation (Becton Dickinson and Company). Results were expressed as geometric mean of the fluorescence intensity (MFI) of 10,000 events and analyzed with GraphPad Prism. Data represented as % binding relative to the MFI of the GPIbα ligands at their KD concentration in the absence of anfibatide. Inhibition constants were determined by the Cheng–Prusoff transformation79 using the Graphpad Prism 8.0 software package (https://www.graphpad.com/scientific-software/prism/).Anfibatide platelet binding experiments were adapted from previous experiments detailing FIIa platelet binding81. Platelet aggregation in PRP was induced by ristocetin (1.2 mg/mL), adenosine diphosphate , or thrombin receptor activating peptide , or collagen  , and in gel-filtered platelets was induced by human thrombin (0.1–1.0 U/mL) with or without anfibatide (6 µg/mL) using a computerized Chrono-log aggregometer , as described21.Human platelet-rich plasma (PRP) and gel-filtered platelets were prepared from sodium-citrate anti-coagulated whole blood by centrifugation as described82. Briefly, sodium-citrated control or anfibatide-treated (6 μg/mL) whole blood (340 μL) from each volunteer (N = 6) was mixed with CaCl2  and studied in parallel. Time until initial clot formation (R time) and maximum amplitude (MA), which reflects maximum strength of the platelet–fibrin clot, were calculated. Experiments were halted after clot forming parameters were calculated or after 1.5 h, whichever occurred first.To study hemostatic clot formation and strength, fresh whole blood was tested on a TEG 5000 Analyzer  as we previously describedan ex vivo perfusion chamber system, as described52. Briefly, rectangular microcapillary glass tubes (0.1 × 1 mm) were coated with Horm collagen . Anti-coagulated whole blood (heparin 15 U/mL) from healthy donors was fluorescently-labeled with DiOC6 . Then, control or anfibatide-treated (6 μg/mL) whole blood was perfused over the collagen-coated surface at shear rates of 300 s−1 and 1500 s−1 for 3 min using a syringe pump . Platelet accumulation and thrombus formation were recorded in real-time under a Zeiss Axiovert 135-inverted florescent microscope (60 ×/0.90 NA water objective). Quantitative dynamics of platelet fluorescence intensity were acquired using Intelligent Imaging Innovations SlideBook version4.1 software (https://slidebook.software.informer.com/4.1/).To measure platelet adhesion, aggregation, and thrombus formation at different shear rates, whole blood from 12 healthy volunteers was perfused over a type I collagen-coated surface using −1). Control whole blood was perfused for 4 min to form thrombi on collagen as above. Then, without interruption, perfusion was continued for an additional 4 min with control or anfibatide-treated (6 μg/mL) whole blood. Thrombus formation and subsequent thrombolysis were analyzed as above.Thrombolysis was studied at high shear flow conditions (1500 s75.Inclusion Criteria were: (1) Healthy volunteers, male and female, aged 18–28 years, (age difference in each group < 10 years); (2) Body mass index (BMI) between 19 and 24 (body weight difference in each group < 10 kg); (3) No history of heart, liver, kidney, digestive tract, nervous system and metabolic disorder, or ulcer, significant hemorrhage, without the history of drug allergy and postural hypotension; (4) No abnormalities in medical examinations; (5) Have not taken any medications within 2 weeks before the study; (6) Willing to participate in the study and give a signed informed consent form9/L; (7) Trauma history  recently; (8) Past history of unexplained syncope or convulsion; (9) History of organic or psychogenic disease or the disabled; (10) Persons who were unlikely to participate in the study (such as the infirm) in the investigator's opinion; (11) Have donated blood or experienced blood collection in other trials within 3 months75.Exclusion Criteria were: (1) History of HBV or HCV infection; (2) Addicted to smoking or alcohol; (3) Women during pregnancy, lactation or menstrual period; (4) Past history of hemoptysis, bloody stool, bleeding spots in the skin and mucous membrane, or hemorrhagic tendency ; (5) History of active bleeding ; (6) Blood platelet count less than 150 × 10Trial subjects were screened for the following inclusion and exclusion criteria.i.v. push based on a modified Fibonacci method at: 0.33, 0.66, 1, 1.5, 2, 3, 4, and 5 µg/60 kg body weight  followed by a continuous infusion at a constant rate of 0.12 μg/60 kg/h for 24 h . Finally, 1 mL of blood was incubated at 37 ºC; every 30 s the tube was tilted to observe blood fluidity until complete coagulation occurred.Blood samples were collected from subjects’ superficial elbow veins into: anticoagulant tubes  for testing platelet aggregation and blood coagulation, and dry tubes without any anticoagulant for ELISA and coagulation time assay. In Part A (Group 3–8), samples were collected at 0, 5, 15, 30 min, 1, 2, 3, 4, 5, 6, 8, 12, 24, and 48 h after drug administration. In Part B (Group 9–11), samples were collected at 0, 5 min and 1.5 h (1.5 h was for Group 9 only) after loading dose, and 1, 3, 6, 12, 18, 24, 25, 26, 28, 36, and 48 h after constant rate infusion. One tube was immediately used for hematology and blood chemistry tests. Samples were analyzed with a Stago Compact CT Blood coagulation analyzer  for thrombin time, prothrombin time, activated partial thromboplastin time, international normalized ratio, and Bleeding time for single dose groups 3–8 were measured at 0 min, 5 min, 30 min, 2 h, 4 h, and 6 h, while for multiple dose groups 9–11 were measured at 0 min, 5 min, 3 h, 12 h, 24 h, and 26 h. A blood pressure cuff on the volunteer’s upper arm was inflated to 40 mmHg. An incision making device, Surgicutt  device, was placed on the forearm and used to make a standard-sized cut  as per manufacturer’s instructions. Filter paper was used to draw off blood at 30 s intervals until bleeding stopped completely. The time when the incision was made until all bleeding stopped was measured. The bleeding time was determined to the nearest 30 s, as per manufacturer’s instructions.80. Ristocetin  was added to 350 µL PRP from anfibatide-treated volunteers in colorimetrical cylinders and incubated at 37 °C. Aggregation was analyzed using a LBY-NJ4 platelet aggregometer  and curves were read within 5 min. The laboratory used standardized protocols and instructions provided by the manufacturer.Platelet aggregation assays were performed using ristocetin , which induces GPIbα receptor-dependent platelet aggregation, as describedThe rate of inhibition of platelet aggregation at different time points was calculated as follows: % Inhibition of platelet aggregation (Time n) = (% of platelet aggregation (Time 0)−% of platelet aggregation (Time n))/% of platelet aggregation (Time 0). Note that Time 0 means before drug infusion; Time “n” means n min/h after drug infusion.Drug safety was monitored via standard clinical laboratory parameters. Adverse events were monitored during the entire course of the study through investigator inquiries, spontaneous reports, and clinical evaluations including physical examinations, vital sign measurement, electrocardiography, and clinical laboratory tests . A detailed description is provided in the Supplementary Tables Urine samples were collected and analyzed before, and 24 h after drug administration. All urine was assayed using an automatic urine analyzer SC7KBXL .To check whether anfibatide induces generation of anti-anfibatide antibodies, serum samples collected 1 month after drug administration were analyzed by ELISA at Zhaoke Pharmaceutical Limited (China) according to standard laboratory practices.Data are presented by summary statistics as follows: number of observations, arithmetic mean, and SD or SEM as indicated. For the in vitro data, ANOVA was used to test for significant differences between multiple groups; unpaired, two-tailed Student’s t-test was used to test for significant differences between 2 groups. All groups in clinical trial I were normally distributed as determined by the Kolmogorov–Smirnov test; therefore, ANOVA and Bonferroni analysis were used to test for significant differences between multiple dosing groups; unpaired, two-tailed Student’s t-test was used to test for significant differences between 2 groups. Repeated measures ANOVA was used to assess differences in aggregation after anfibatide administration. All adverse events were recorded with: type, onset, duration, severity, and drug relation. Vital signs (blood pressure and pulse rate), electrocardiogram, safety laboratory data, and antibody titers were evaluated using descriptive statistics.Some data were orally presented at the XXVI Congress of International Society of Thrombosis and Haemostasis, Amsterdam, Netherlands, July 2013; Annual Meeting of the American Society of Hematology (ASH) in New Orleans, USA, December 2013; and XXVII Congress of International Society on Thrombosis and Haemostasis, Toronto, Canada, June 20, 2015. This study has also been selected for inclusion in the press program of the 55th ASH Annual Meeting.Supplementary Information.