Rat Platelets Research Articles

Opposite Modulatory Effects of Crataegus aronia Aqueous Extract on Platelet Aggregation in Rats.

To reveal the mechanisms behind the dual effects of Crataegus aronia (C. aronia) aqueous extract on platelet aggregation by focusing on function, regulation, expression, and signaling of platelets P2Y12 receptors. Adult male Wistar rats (120 ± 10 g) were classified as control received the vehicle, C. aronia (200 mg/kg), and C. aronia (2,000 mg/kg)-treated rats. After treatments for consecutive 7 days, hematological and molecular experiments were conducted to detect alterations in platelet aggregation, thromboxane B2 (THXB2) and intracellular reactive oxygen species (ROS) content; protein levels of P2Y12, p-Akt, cyclic adenosine monophosphate (cAMP), phosphorylated vasodilator-stimulated-phosphoprotein (p-VASP), nuclear factor κB (NF-κB), P-selectin, and etc. in platelets were determined by Western blot; mRNA expressions of P2Y12 and some inflammatory markers were determined by real-time polymerase chain reaction. At a concentration of 200 mg/kg, C. aronia inhibited platelet aggregation through multiple interconnected mechanisms including downregulation P2Y12 synthesis and expression, stimulating intracellular cAMP levels and protein levels of p-VASP, inhibiting platelets THXB2 release and protein levels of P-selectin. Also, it inhibited platelets level of ROS and of NF-κB, a major signaling pathway that stimulates the expression of P2Y12 and THXA2 synthesis. Opposite findings were seen in platelets of rats received C. aronia at a concentration of 2,000 mg/kg. Interestingly, co-administration of N-acetylcysteine prevented all hematological and molecular alterations exerted by the high dose of the extract and inhibited platelet aggregation. Oral administration of C. aronia at low dose inhibits platelet aggregation by reducing THXB2 release, expression of P-selectin and activating cAMP and Akt signaling through two major mechanisms including downregulation of P2Y12 and inhibition of ROS-induced activation of NF-κB, an effect that is observed to be in the opposite direction with its high dose.

Regulatory effect of Jiaotai Pills on central and peripheral neurotransmitters in rats with heart-kidney imbalance insomnia

To explore the pathogenesis of heart and kidney imbalance insomnia and the regulatory effect of Jiaotai Pills, in order to study the changes of central and peripheral neurotransmitters in rat. Insomnia rats with heart and kidney imbalance were induced through intraperitoneal injection with p-chlorophenylalanine(PCPA, 400 mg·kg~(-1)·d~(-1)), and the model rats were intragastrically administrated with Jiaotai Pills(3.3 g·kg~(-1)·d~(-1)) for 7 days. Nine neurotransmitters were determined by UPLC-MS/MS and principal component analysis(PCA) method in serum, urine, brain, heart, liver, kidney and adrenal gland of rats. The results showed that the ratio of 5-HT and 5-HIAA in platelet of insomnia rats was significantly lower than that in the normal group, and Jiaotai Pills had a significant up-regulatory or down-regulatory effect. Compared with the normal group, the changed neurotransmitters in blood of insomnia rats were 5-HIAA, E, NE, DA, Glu and ACH, and except ACH, the changes of 7 kinds of neurotransmitters in urine were more significant, Jiaotai Pills had a significant up-regulatory or down-regulatory effect. Compared with the normal group, all of the 8 neurotransmitters in insomnia rats except HVA were changed. Jiaotai Pills could regulate the neurotransmitters in each tissue of insomnia rats, especially in brain, liver and adrenal gland. In conclusion, insomnia is caused by not only a change of neurotransmitters in brain, but also a series of changes in peripheral tissues. It indicates that insomnia is a systematic imbalance of neurotransmitters. Jiaotai Pills not only regulates the central nervous system, but also has a certain protective effect on other organs, reflecting the multi-target and systematic effect of Jiaotai Pills in the treatment of insomnia.

Toxinological characterization of venom from Leptodeira annulata (Banded cat-eyed snake; Dipsadidae, Imantodini)

We investigated the histology of Duvernoy’s venom gland and the biochemical and biological activities of Leptodeira annulata snake venom. The venom gland had a lobular organization, with secretory tubules formed by serous epithelial cells surrounding each lobular duct. The latter drained into a common lobular duct and subsequently into a central cistern. In contrast, the supralabial gland was mucous in nature. SDS-PAGE revealed a profile of venom components that differed from pitviper (Bothrops spp.) venoms. RP-HPLC also revealed greater complexity of this venom compared to Bothrops venoms. The venom had no esterase, l-amino acid oxidase or thrombin-like activity, but was proteolytic towards elastin-Congo red, fibrin, fibrinogen, gelatin and hide powder azure. The venom showed strong α-fibrinogenase and fibrinolytic activities and reduced the rate and extent of plasma recalcification. The proteolytic activity was inhibited by EDTA and 1,10-phenanthroline (metalloproteinase inhibitors) but not by AEBSF and PMSF (serine proteinase inhibitors). The venom had phospholipase A2 (PLA2) activity that was inhibited by varespladib. The venom cross-reacted with antivenoms to lancehead (Bothrops spp.), coralsnake (Micrurus spp.) and rattlesnake (Crotalus durissus terrificus) venoms. The venom did not aggregate rat platelets or inhibit collagen-induced aggregation, but partially inhibited thrombin-induced aggregation. The venom was hemorrhagic (inhibited by EDTA) and increased the vascular permeability (inhibited by varespladib) in rat dorsal skin. In gastrocnemius muscle, the venom caused myonecrosis and increased serum creatine kinase concentrations. In conclusion, L. annulata venom has various enzymatic and biological activities, with the local effects being mediated primarily by metalloproteinases and PLA2.

INTERACTION OF DEXTRIN CAPPED CADMIUM SULFIDE QUANTUM DOTS WITH BLOOD COMPONENTS: COAGULATION AND HEMOLYSIS STUDIES

Evaluate at interaction of dextrin capped cadmium sulfide quantum dots with blood components. The intravenous administration of quantum dots (QDs) has been widely reported as a promising alternative for delivery of drugs to specific cells. Therefore, previous studies have pointed out the importance of studying of, at least, factors like coagulation effect; and their hemolytic character when nanoparticles are intended to be administered for intravenous injection. The hemolytic activity and platelets aggregation of the nanoparticles was tested on isolated rat erythrocytes and platelets. Atomic force microscopy (AFM) facilitates the real‐time studies of blood cells and the morphological, structural, optical properties of the CdS‐dex QDs. In this work a methodology for erythrocytes and platelets study by platelet aggregation, hemolysis and AFM was developed. The results show that CdS‐Dex/QDs caused platelet aggregation at concentration‐dependent manner, this effect being greater at 1000 ug/mL, where the increase was up to 57% (p<0.05). The AFM measurements establish a more direct correlation between the surface topography of platelets with the surface nanostructure and reveal that modified the morphology of the platelets from the concentration of 0.01 ug/mL. The CdS‐Dex/QDs do not cause lysis of erythrocytes significantly, although they modified their morphology, suggesting that they can cause eriptosis. The CdS‐Dex/QDs, caused effects in oval and biconcave morphology of the erythrocytes, making it round and of edges not defined, semi‐flat and with cracks in its surface and lacking central depression in a concentration‐dependent manner and observed in its surface some artifacts that may correspond to agglomerated NPs. We found that CdS‐dex/QDs caused aggregation of rats platelets in vitro; however; there were both quantitative and qualitative differences depends to various concentrations.

Nutritional Study of Processed Amygdalus communis L. Sesamum indicum and Bertholletia excelsa Nuts on Two Weeks Old Wistar Rats

The study evaluated the nutritional benefits of processed Almond nuts, Sesame seeds and Brazil nut on two weeks old Wistar rats. The rats were weighed and arranged into seven groups and feed for 4 weeks. Group one (control) was fed with commercial rat feed and distilled water. Group two was fed with a diet formulated from Brazil nuts and distilled water. Group three was fed with a diet formulated from almond seeds and distilled water only while Group four was fed with a diet formulated from sesame seeds. Group five was fed with diet from sesame + almond seeds while Group six was fed with a diet formulated from Brazil +almond nuts. Group seven was fed with a diet formulated from Brazil + Almond nuts +Sesame seeds. Biochemical and haematological analyses were carried out applying standard methods and procedures. Results for proximate analysis indicated that Brazil nut contains 4.03% Ash, 7.61% crude moisture, 43.74% crude lipid, 12.82% crude protein, 8.7% crude fibre and 28.1% carbohydrate. Plasma enzyme activities of rats fed with Brazil nuts were: ALT (22.96±1.95U/l), AST (66.49±3.33 U/l), ALP (64.18±2.76 (U/l) and GGT (6.89±1.69 (U/l). Total protein, albumin, and total bilirubin concentrations were 53.56±2.03 g/dl, 32.13±1.21 g/dl and 3.82±0.20 g/dl respectively. Total cholesterol, triglyceride, LDL and HDL concentrations were 2.83±0.42 mmol/l, 1.35±0.02 mmol/l, 4.10±0.19 mmol/l, 4.72±0.13 mmol/l respectively. The PCV, Hb, WBC, RBC, Platelet and MCV concentrations of rats fed with Brazil nuts only were 48.20±1.79%, 15.02±0.65 g/dl, 7.06±0.44 X103/mm3, 7.28±0.23 X106/mm3, 195.00±5.79 X103/ml, and 6.63±0.43 X10-7fl respectively which were significantly (p<0.05) different from the control values. The result is suggestive that they can be incorporated as a supplementary diet for Wistar rats. However, the results obtained from rats feed with almond and Brazil nuts were more significant when compared with sesame seeds.

The Effect of Plectranthus amboinicus Lour Spreng Ethanolic Extract on Relative Organ, Body Weights Changes, and Hematology Profile in Wistar Rats Treated with 7,12Dimethylbenz(a)anthracene

This study aims to examine the protective properties of ethanol extract Plectranthus amboinicus (EEP) leaves on body weight gain, the relative weight of liver, kidney, lung, spleen, thymus and haematological profile of rats induced by 7,12Dimethylbenz(a)anthracene (DMBA). Used 25 female rats, divided into five groups namely NC (given Carboxymethyl cellulose (CMC) 1%), PC (given DMBA 20 mg/kg body weight), T1, T2, and T3 were each given DMBA 20 mg/kg body weight once every four days for 32 days and EEP 175, 350, and 700 mg/kg body weight given every day from day 33 to 59. Both the DMBA and EEP are given orally using a gastric gavage. On day 60, rats were killed by neck dislocation, bood collected in EDTA tubes for hematological analysis, rats are dissected to obtain liver, kidney, lung, thymus, and spleen organs. Data were analyzed with one way Analisys of varians (ANOVA). The results of this study indicate that EEP in T1, T2 and T3 treatments has no effect on weight loss compared to PC. There was no effect of EEP on the relative weight of the liver, kidneys, spleen, thymus and lungs. EEP increased the number of erythrocytes, leukocytes and platelets in rats that had DMBA

Ectonucleoside Triphosphate Diphosphohydrolase-1/CD39 Affects the Response to ADP of Female Rat Platelets.

There is evidence that an imbalance of extracellular purine levels may be associated with increased cardiovascular risk. Platelets play a pivotal role in vascular homeostasis and thrombosis and are important source of purine nucleotides and nucleosides. Hydrolysis of nucleotides ATP and ADP is regulated by two ectonucleotidases, triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5’-nucleotidase (ecto-5’-NT/CD73). CD39 enzyme is expressed on the endothelium, circulating blood cells, and smooth muscle cells; there is evidence that changes in CD39 expression and activity affects the potential thrombogenic of a tissue. Gender difference in the cardiovascular risk has been extensively observed; however, while the age-dependent difference in the prevalence of cardiovascular events between men and women has been attributed to the loss of the protective effect of estrogens in the postmenopausal period, the physiological mechanism behind gender disparity is still unclear. Here, we evaluated comparatively male and female rat platelet reactivity and considered the possible role of CD39 at the basis of difference observed. We found a reduced in vitro response to ADP (1–30 µM) of female compared to male platelets, associated to increased platelet CD39 expression and activity. Platelet response to ADP was strongly increased by incubation (10 min) with the CD39 inhibitor, ARL67156 (100 µM), while male platelet response was unaffected. Rat treatment with clopidogrel (30 mg/kg, per os) inhibited ex vivo platelet aggregation. Bleeding time was prolonged in female compared to male. Taken together, our results suggest that platelet ATPase and ADPase activity might be a reliable predictor of platelet reactivity.

A rat model of severe VWD by elimination of the VWF gene using CRISPR/Cas9

BackgroundVon Willebrand Disease (VWD) is the most common inherited bleeding disorder, caused by quantitative and qualitative changes in von Willebrand factor (VWF). The biology of VWD, studied in canine, porcine, and murine models, differ in species‐specific biology of VWF and the amenability to experimental manipulations such as phlebotomy. The factor VIII (FVIII) levels in these models are higher than in humans with type 3 VWD, suggesting functional differences between FVIII and VWF.ObjectivesTo develop a VWF knock out (VWF–/–) rat by excision of all 52 exons of the VWF locus. MethodsThe entire VWF gene was eliminated in Sprague‐Dawley (Crl:SD) rats via CRISPR/Cas9‐mediated gene editing. VWF antigen (VWF:Ag), VWF propeptide, and VWF collagen IV binding (VWF:CB4) levels were determined by ELISA assays and FVIII chromogenic activity (FVIII:C) levels by chromogenic FVIII assays. Lateral tail veins were transected to measure bleeding time. VWF–/– rats were infused with FVIII–/– rat platelet poor plasma (PPP) to determine response of plasma FVIII. ResultsBreeding of VWF ± rats yielded VWF–/– offspring at normal Mendelian ratios. VWF:Ag, VWF propeptide, VWF:CB4, and FVIII:C plasma levels were undetectable in VWF–/– rats. VWF–/– rats bled longer and more than VWF+/– and VWF+/+ rats when challenged. Transfusion of FVIII‐deficient platelet‐poor plasma induced a rapid rise in endogenous FVIII:C in VWF–/– rats. ConclusionThis rat model of severe VWD due to elimination of the entire VWF gene recapitulates the severe secondary deficiency of FVIII seen in human type 3 VWD and facilitates the study of VWF and FVIII and their interactions.

Macrophage Depletion Mitigates Platelet Aggregate Formation in Splenic Marginal Zone and Alleviates LPS-Associated Thrombocytopenia in Rats.

Sepsis is often accompanied with thrombocytopenia partly due to platelet sequestration in the lung and liver. The spleen can store up to one-third of circulating platelets and can also significantly affect platelet transfusion outcomes by accumulating platelets. However, in sepsis, it is not clear whether there are platelet changes in the spleen which could contribute to sepsis-associated thrombocytopenia and also influence platelet transfusion outcomes. By using confocal microscopy, we examined endogenous rat platelets and infused human platelets in the spleen of severe combined immune deficient Rag2 KO rats which were injected intraperitoneally with lipopolysaccharide (LPS). LPS-injected Rag2 KO rats developed sepsis as indicated by increased TNFa, IL-6, IL-1b, and IL-10 levels and thrombocytopenia. Large platelet aggregates were observed in the spleen with majority located in the marginal zone and closely associated with CD169+ macrophages. Depletion of macrophages by clodrosome resulted in reduction of LPS-induced cytokine generation and alleviated LPS-induced thrombocytopenia. Macrophage depletion also remarkedly diminished large platelet aggregate formation in the splenic marginal zone but had less effect on those in red pulp. Infusion of human platelets into LPS-injected rats failed to raise platelet counts in the peripheral blood. In LPS-injected rat spleen, human platelets interacted with aggregated rat platelets in the marginal zone. In contrast, human platelets infused into control rats were located outside of splenic marginal zone. This study provides morphological evidence of platelet aggregates in the splenic marginal zone in sepsis which can interact with infused platelets and thus can contribute to platelet infusion refractoriness in sepsis. It indicates that macrophages play an important role in LPS-associated thrombocytopenia. It also suggests that CD169+ macrophages support platelet aggregate formation in the splenic marginal zone.

Effects of Glycosaminoglycan from Urechis Unicinctus on the P2Y12 Receptor Signaling Pathway in Rat Platelets

This study aims to investigate the effects of glycosaminoglycan (GAG) from Urechis unicinctus on the P2Y12 receptor signaling pathway in rat platelets. The concentrations of cyclic adenosine monophosphate (cAMP), thromboxane B2 (TXB2) and glycoprotein IIb/IIIa (GPIIb/IIIa) in rat platelets were determined using enzyme-linked immunosorbent assay (ELISA) kits. The phosphorylation levels of protein kinase A (PKA) and vasodilator-stimulated phosphoprotein (VASP) in rat platelets were detected through Western blot. The expression of P2Y12 gene in rat platelets was analyzed via real-time fluorescence quantitative PCR and reverse-transcription PCR. It was observed that GAG significantly increased the cAMP content (plessthan 0.05, plessthan 0.01) and decreased the TXB2 and GPIIb/IIIa concentrations (plessthan 0.05,plessthan 0.01) in rat platelets. GAG significantly enhanced the phosphorylation levels of PKA and VASP in rat platelets plessthan 0.01) and had a synergistic effect with the P2Y12 receptor blocker. GAG significantly reduced the expression level of P2Y12 gene in rat platelets (plessthan 0.01). We speculated that GAG from U. unicinctus inhibited platelet aggregation in rats through the P2Y12 receptor signaling pathway.

Vepoloxamer Enhances Fibrinolysis of tPA (Tissue-Type Plasminogen Activator) on Acute Ischemic Stroke.

Background and Purpose- Thrombolytic treatment of acute ischemic stroke with tPA (tissue-type plasminogen activator) is hampered by its narrow therapeutic window and potential hemorrhagic complication. Vepoloxamer is a nonionic surfactant that exerts potent hemorheologic and antithrombotic properties in various thrombotic diseases. The current study investigated the effect of vepoloxamer on tPA treatment in a rat model of embolic stroke. Methods- Male Wistar rats subjected to embolic middle cerebral artery occlusion were treated with the combination of vepoloxamer and tPA, vepoloxamer alone, tPA alone, or saline initiated 4 hours after middle cerebral artery occlusion. Results- Monotherapy with tPA did not reduce infarct volume, and adversely potentiated microvascular thrombosis and vascular leakage compared with the saline treatment. Vepoloxamer monotherapy reduced infarct volume by 25% and improved brain perfusion. However, the combination treatment with vepoloxamer and tPA significantly reduced infarct volume by 32% and improved neurological function, without increasing the incidence of gross hemorrhage. Compared with vepoloxamer alone, the combination treatment with vepoloxamer and tPA robustly reduced secondary thrombosis and tPA-augmented microvascular leakage and further improved brain perfusion, which was associated with substantial reductions of serum active PAI-1 (plasminogen activator inhibitor-1) level and tPA-upregulated PAI-1 in the ischemic brain. Mechanistically, exosomes derived from platelets of ischemic rats treated with tPA-augmented cerebral endothelial barrier permeability and elevated protein levels of PAI-1 and TF (tissue factor) in the endothelial cells, whereas exosomes derived from platelets of rats subjected to the combination treatment with vepoloxamer and tPA diminished endothelial permeability augmented by tPA and fibrin and reduced PAI-1 and TF levels in the endothelial cells. Conclusions- The combination treatment with vepoloxamer and tPA exerts potent thrombolytic effects in rats subjected to acute ischemic stroke. Vepoloxamer reduces tPA-aggravated prothrombotic effect of platelet-derived exosomes on cerebral endothelial cells, which may contribute to the therapeutic effect of the combination treatment.

Platelet Chemiluminescence during Physical Exercise of Various Intensity.

Generation of ROS (including free radicals) in rat platelets during physical exercises of different intensities was evaluated by the chemiluminescent method. The acute or chronic submaximal exercises increased the platelet chemiluminescence by 2.5-3.0 times. In the control, platelet activation with physiological aggregant ADP enhanced their chemiluminescence by 7.5 times. Addition of ADP to platelets isolated from animals subjected to exercise of any intensity increased chemiluminescence by 4-5 times. In a trained organism, functional reserve of the platelets is preserved during long-term physical exercise of any intensity, but is exhausted after acute physical exercise. Thus, intensive physical exercises enhanced the hemostatic potential of the blood, which is associated with the risk of thrombohemorrhagic complications, especially in acute or submaximal long-term exercises.

Inhibitory effects of hydrogen on in vitro platelet activation and in vivo prevention of thrombosis formation

AimsHydrogen (H2) has antioxidant effects. The pharmacologic function of H2 in platelets is not yet clear. Therefore, in this study we sought to investigate the inhibitory effects of H2 on in vitro platelet activation and in vivo prevention of thrombus formation. Main methodsAfter platelets were incubated with H2-rich saline (HRS), platelet adhesion in whole human blood was assessed in fibrinogen-coated perfusion chambers, while rat platelet aggregation induced by ADP, collagen and H2O2 was detected through light transmission aggregometry. The level of P-selectin, thromboxane B2, nitric oxide (NO), malondialdehyde, reactive oxygen species (ROS), cGMP, extracellular signal-regulated kinases 1 and 2 (p-ERK1/2), and fibrinogen binding to platelets were evaluated in vitro. Besides, the in vivo effects were examined in arterio-venous shunt thrombosis, FeCl3-induced artery thrombus formation, and tail bleeding time in mice and rats. Key findingsHRS prolonged tail bleeding time in mice and rats, decreased thrombus weight and prolonged the time to occlusion in rat and mouse thrombosis models in vivo and inhibited platelet adhesion as well as aggregation in vitro. Additionally, HRS decreased P-selectin expression, release of thromboxane B2, ROS, and fibrinogen binding, but enhanced NO levels in H2O2-exposed platelets. HRS also decreased malondialdehyde levels in plasma of the rat arterial thrombosis or H2O2-exposed platelet model. Moreover, HRS increased cGMP level, decreased p-ERK1/2 (diminished with KT5823) in the platelets stimulated by H2O2. SignificanceThese results suggest that H2 has antithrombotic effects, which may be due to its antioxidant property and subsequent inhibition of platelet activation via NO/cGMP/PKG/ERK pathway.

Recognition of specific sialoglycan structures by oral streptococci impacts the severity of endocardial infection.

Streptococcus gordonii and Streptococcus sanguinis are primary colonizers of the tooth surface. Although generally non-pathogenic in the oral environment, they are a frequent cause of infective endocarditis. Both streptococcal species express a serine-rich repeat surface adhesin that mediates attachment to sialylated glycans on mucin-like glycoproteins, but the specific sialoglycan structures recognized can vary from strain to strain. Previous studies have shown that sialoglycan binding is clearly important for aortic valve infections caused by some S. gordonii, but this process did not contribute to the virulence of a strain of S. sanguinis. However, these streptococci can bind to different subsets of sialoglycan structures. Here we generated isogenic strains of S. gordonii that differ only in the type and range of sialoglycan structures to which they adhere and examined whether this rendered them more or less virulent in a rat model of endocarditis. The findings indicate that the recognition of specific sialoglycans can either enhance or diminish pathogenicity. Binding to sialyllactosamine reduces the initial colonization of mechanically-damaged aortic valves, whereas binding to the closely-related trisaccharide sialyl T-antigen promotes higher bacterial densities in valve tissue 72 hours later. A surprising finding was that the initial attachment of streptococci to aortic valves was inversely proportional to the affinity of each strain for platelets, suggesting that binding to platelets circulating in the blood may divert bacteria away from the endocardial surface. Importantly, we found that human and rat platelet GPIbα (the major receptor for S. gordonii and S. sanguinis on platelets) display similar O-glycan structures, comprised mainly of a di-sialylated core 2 hexasaccharide, although the rat GPIbα has a more heterogenous composition of modified sialic acids. The combined results suggest that streptococcal interaction with a minor O-glycan on GPIbα may be more important than the over-all affinity for GPIbα for pathogenic effects.

Synthesis of Phenyl m-coumarylamide Analogs and Their Anti-platelet Aggregation Activity

Phenyl m-coumarylamide analogs were designed based on phenylpropanoid analogs such as coumaric acid, ferulic acid, and caffeic amide having anti-platelet aggregation activity. They prepared through concise synthetic method. Their anti-platelet aggregation was confirmed using ADP-induced rat platelet by aggregrometer. Compound 1b and 1d having dimethylamine and methoxy group on para-position of phenylamide ring, respectively, exhibit potent inhibitory activity (92.3 and 89.3% at 80 μg/mL). The results suggest that phenyl m-coumrylamide analogs could be used to develop potent anti-platelet aggregation agents.

In vitro and in silico evaluation of stereoselective effect of ginsenoside isomers on platelet P2Y12 receptor

BackgroundP2Y12 receptor (P2Y12R) is a newly discovered Gi-coupled ADP receptor that plays critical role in platelet function. Ginsenosides are the main constituents responsible for most of pharmacological actions of ginseng, especially cardio-cerebrovascular protective efficacy that is closely related to the influence on platelet function. Hypothesis/PurposeTo explore stereoselective effect of naturally abundant ginsenoside isomers, including the C-20 epimers of protopanaxadiol (PPD), protopanaxatriol (PPT), and their glycosides Rg2, Rg3, Rh1, Rh2 on P2Y12R in platelets. Study Design/MethodsBoth in vitro assay and in silico molecular docking study were performed to investigate the stereoselective effects. ResultsIn vitro assay using washed rat platelets revealed differential effects of ginsenoside isomers on ADP-induced platelet aggregation with the direction and degree of action varying with chemical structures. More to the point, the ginsenoside 20S-Rh2 but not its 20R-epimer was found to be the only one that could significantly promote in vitro platelets aggregation induced by ADP. The correlation analysis demonstrated that ginsenosides may have impact on P2Y12R related platelet functions through a cAMP-dependent pathway. Molecular docking stimulation further indicated that ginsenoside isomers could be potent substrate of P2Y12R with differential protein–ligand interaction that would be responsible for the stereoselective efficacy of C-20 ginsenoside epimers. Hydrogen bonding with Asp266 via the C-20 hydroxyl may provide ginsenosides with promoting effect on ADP-induced platelets aggregation, whereas interactions with Tyr105 could contribute to the promotion of inhibitory efficacy. ConclusionGinsenosides are potent P2Y12R substrate with stereoselective effects on P2Y12R-related platelet function, which result from their chemical diversity and are closely related to the different interaction ways as P2Y12R ligand.

INTERACTION OF DEXTRIN CAPPED CADMIUM SULFIDE QUANTUM DOTS WITH BLOOD COMPONENTS: COAGULATION AND HEMOLYSIS STUDIES

Evaluate at interaction of dextrin capped cadmium sulfide quantum dots with blood components. The intravenous administration of quantum dots (QDs) has been widely reported as a promising alternative for delivery of drugs to specific cells. Therefore, previous studies have pointed out the importance of studying of, at least, factors like coagulation effect; and their hemolytic character when nanoparticles are intended to be administered for intravenous injection. The hemolytic activity and platelets aggregation of the nanoparticles was tested on isolated rat erythrocytes and platelets. Atomic force microscopy (AFM) facilitates the real‐time studies of blood cells and the morphological, structural, optical properties of the CdS‐dex QDs. In this work a methodology for erythrocytes and platelets study by platelet aggregation, hemolysis and AFM was developed. The results show that CdS‐Dex/QDs caused platelet aggregation at concentration‐dependent manner, this effect being greater at 1000 ug/mL, where the increase was up to 57% (p<0.05). The AFM measurements establish a more direct correlation between the surface topography of platelets with the surface nanostructure and reveal that modified the morphology of the platelets from the concentration of 0.01 ug/mL. The CdS‐Dex/QDs do not cause lysis of erythrocytes significantly, although they modified their morphology, suggesting that they can cause eriptosis. The CdS‐Dex/QDs, caused effects in oval and biconcave morphology of the erythrocytes, making it round and of edges not defined, semi‐flat and with cracks in its surface and lacking central depression in a concentration‐dependent manner and observed in its surface some artifacts that may correspond to agglomerated NPs. We found that CdS‐dex/QDs caused aggregation of rats platelets in vitro; however; there were both quantitative and qualitative differences depends to various concentrations.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Functional Features of Platelets in Rats Fed a Standard Diet with Low Antioxidant Content During Ontogenesis

Functional Features of Platelets in Rats Fed a Standard Diet with Low Antioxidant Content During Ontogenesis

Effects of water-soluble tomato concentrate on platelet aggregation

Objective: To investigate the antiplatelet aggregation effect of water-soluble tomato concentrate (WSTC) and explore the underlying molecular mechanisms. Materials and Methods: Platelet aggregometry was used to quantify rat platelet aggregation with the maximum aggregation rate in vitro and ex vivo. Then, the fibrinogen (FIB) binding assay was employed to detect the effect of WSTC on the activation of platelet integrin αIIβ3 (GP IIb/IIIa). Furthermore, Western blot was performed to assess the platelet protein levels of phosphoinositide 3-kinase 110 β (PI3K110 β), protein disulfide isomerase (PDI), platelet endothelial cell adhesion molecule 1 (PECAM-1), and β1-Tubulin. Results: WSTC inhibited adenosine diphosphate (ADP) and collagen-induced platelet aggregation in a concentration-dependent manner in vitro, at IC50values of 3.05 g/L and 8.03 g/L, respectively. Significantly reduced ex vivo ADP induced platelet aggregation was observed after oral consumption of WSTC for 4 weeks in rats; average inhibition rates were 24.42%, 21.48%, and 20.87% for 25 mg/Kg, 75 mg/Kg, and 150 mg/Kg WSTC, respectively. It appeared that WSTC had no influence on coagulation function in rats. Incubation with WSTC decreased FIB binding to GP IIb/IIIa by 17.47% and 32.29% at the concentrations of 0.6 and 6 g/L, respectively. WSTC at 0.6 and 6 g/L markedly downregulated PI3K110 β, PDI, and PECAM-1 in platelets, and upregulated β1-Tubulin, in a concentration-dependent manner. Conclusion: WSTC inhibits platelet activation through modulation of platelet skeletal stability and suppresses GP IIb/IIIa receptor-mediated platelet aggregation, likely via the PI3K signaling pathway and PDI inhibition.

PDGF-BB Induces Formation of Bridging Callus After Reconstructive Surgery of Large Bone Defect

Background:Reconstructive surgery by using allografts often conducted to manage large bone defects, either due to traumatic or non-traumatic causes. However, poor vascularization of the graft bed is still problematic. To overcome this, bone tissue engineering method has been developed that uses growth factor as an angiogenic stimulator, such as platelet derived growth factor BB (PDGF BB).Objective:This study aimed to evaluate the administration of recombinant rat Platelet Derived Growth Factor BB (rrPDGF BB) on bone healing process, showed by formation of bridging callus, Vascular Endothelial Growth Factor (VEGF), Bone Morphogenetic Protein-2 (BMP-2) and osteocalcin inmassivefresh frozen allograft post reconstructive surgery.Methods:This was a Post Test Only Control Group Design study involved 32 Wistar rats divided into two groups,i.e.treatment group (defect on right femoral bone and received fresh frozen allograft with the addition of rrPDGF BB) and control group (without addition of rrPDGF BB). Expression of VEGF, BMP-2 and osteocalcin was identified through immunohistochemistry.Results:A significantly higher expression of VEGF, BMP-2 and osteocalcin was observed in the treatment group as compared to the control group (p< 0.05). The presence of bridging callus on the fresh frozen allograft also showed to be significant (p= 0.003). Path analysis showed formation of bridging callus after administration of PDGF on allograft occur through three pathways, in which VEGF holds the most important role.Conclusion:The application of rrPDGF BB significantly enhances the formation of new bone through increased expression of VEGF, BMP-2 and osteocalcin.