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Objectives: Research investigating the association between reproductive health and diet has predominantly focused on the antenatal and prenatal periods, as well as childbirth in women, and semen quality in men. There is a limited amount of research addressing female fertility assessed as ovarian reserve in relation to diet. The main aim of this study was to evaluate the association between dietary patterns and the parameters of ovarian reserve, such as antral follicle count (AFC), anti-Müllerian hormone (AMH), follicle-stimulating hormone (FSH), and estradiol (E2) - predictors of reproductive health in women of childbearing age., Material and Methods: Women aged 24-39 years (N = 511) were enrolled from fertility clinic in central Poland. The count of antral follicles was determined using ultrasonography (USG), FSH and E2 levels were measured using a chemilumi- nescence method and for determination of AMH level, an enzyme-linked immunosorbent assay method was employed. Diet was assessed according to food frequency questionnaire and dietary patterns were identified by factor analysis. Women were classified into 3 groups according to scores of each dietary pattern: Western, mixed, prudent., Results: Higher adherence to the prudent dietary pattern in obese women was associated with significantly higher AFC (p = 0.03) and AMH (p = 0.05) as compared to participants with the Western dietary pattern. The results were adjusted for age, BMI, smoking and duration of fertility. Increased consumption of mixed dietary pattern was not statistically significant associated with any of examined ovarian reserve parameters., Conclusions: The prudent dietary pattern is positively associated with ovarian reserve in a cohort of women seeking fertility care. Continued research in this area will provide nutritional guidance for clinicians and their patients and provide novel insight on potential modifiable lifestyle factors which can be associated with ovarian reserve. Int J Occup Med Environ Health. 2024;37(4):411-20., (This work is available in Open Access model and licensed under a CC BY-NC 3.0 PL license.)

A series of optically pure aziridine phosphines and their corresponding phosphine oxides were synthesized through established chemical methodologies. The compounds were systematically investigated for their biological properties. Notably, all synthesized compounds demonstrated moderate antibacterial activity only against the reference strain of Staphylococcus aureus . However, compounds 5 and 7 exhibited noteworthy cell viability inhibition of human cervical epithelioid carcinoma HeLa cells and endometrial adenocarcinoma Ishikawa cells. Further studies of these compounds revealed additional biological effects, including disruption of the cell membrane in high concentrations, cell cycle arrest in the S phase, and the induction of reactive oxygen species (ROS). Comparative analysis of the two classes of chiral organophosphorus derivatives of aziridines indicated that chiral phosphine oxides displayed significantly higher biological activity. Consequently, these findings suggest that chiral phosphine oxides may be potential candidates for the development of anticancer drugs. In light of the significant interest in preparations whose structure is based on a three-membered aziridine ring in terms of potential anticancer therapy, this research fits into the current research trend and should constitute a valuable addition to the current state of knowledge and the existing library of aziridine derivatives with anticancer properties.

The platelet-derived microparticles (PMPs) have been connected with tumor progression and metastatic dissemination. PMPs infiltrate solid tumors and transfer platelet-derived cargo to cancer cells. The functional roles of PMPs in cancer progression are still poorly understood. PMPs, incorporated by colorectal cancer (CRC) cells, were shown to upregulate the expression and activity of matrix metalloproteases (MMPs). To investigate the impact of PMPs on the invasive potential of CRC, we established the protocol of dequenched gelatin (DG), fluorescein conjugate assay. The procedure confirms the activity of two gelatinases, namely, MMP2 and MMP9, that digest denatured collagen (gelatin). This "step-by-step" protocol, with notes and comments implemented to human CRC lines with different phenotypes and migratory potentials, should be sufficient to obtain representative and elegant results., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Metastasis is the main cause of death in patients with colorectal cancer (CRC). Apart from platelets, platelet-derived microparticles (PMPs) are also considered important factors that can modify the activity of cancer cells. PMPs are incorporated by cancer cells and can also serve as intracellular signalling vesicles. PMPs are believed to affect cancer cells by upregulating their invasiveness. To date, there is no evidence that such a mechanism occurs in colorectal cancer. It has been shown that platelets can stimulate metalloproteases (MMPs) expression and activity via the p38MAPK pathway in CRC cells, leading to their elevated migratory potential. This study aimed to investigate the impact of PMPs on the invasive potential of CRC cells of various phenotypes via the MMP-2, MMP-9 and p38MAPK axis., Methods: We used various CRC cell lines, including the epithelial-like HT29 and the mesenchymal-like SW480 and SW620. Confocal imaging was applied to study PMP incorporation into CRC cells. The presence of surface receptors on CRC cells after PMP uptake was evaluated by flow cytometry. Transwell and scratch wound-healing assays were used to evaluate cell migration. The level of C-X-C chemokine receptor type 4 (CXCR4), MMP-2, and MMP-9 and the phosphorylation of ERK1/2 and p38MAPK were measured by western blot. MMP activity was determined using gelatine-degradation assays, while MMP release was evaluated by ELISA., Results: We found that CRC cells could incorporate PMPs in a time-dependent manner. Moreover, PMPs could transfer platelet-specific integrins and stimulate the expression of integrins already present on tested cell lines. While mesenchymal-like cells expressed less CXCR4 than epithelial-like CRC cells, PMP uptake did not increase its intensity. No significant changes in CXCR4 level either on the surface or inside CRC cells were noticed. Levels of cellular and released MMP-2 and MMP-9 were elevated in all tested CRC cell lines after PMP uptake. PMPs increased the phosphorylation of p38MAPK but not that of ERK1/2. Inhibition of p38MAPK phosphorylation reduced the PMP-induced elevated level and release of MMP-2 and MMP-9 as well as MMP-dependent cell migration in all cell lines., Conclusions: We conclude that PMPs can fuse into both epithelial-like and mesenchymal-like CRC cells and increase their invasive potential by inducing the expression and release of MMP-2 and MMP-9 via the p38MAPK pathway, whereas CXCR4-related cell motility or the ERK1/2 pathway appears to not be affected by PMPs. Video Abstract., (© 2023. The Author(s).)

Background: Neuromedin U (NMU) was identified as one of the hub genes closely related to colorectal cancer (CRC) progression and was recently shown to be a motility inducer in CRC cells. Its autocrine signalling through specific receptors increases cancer cell migration and invasiveness. Because of insufficient knowledge concerning NMU accessibility and action in the tumour microenvironment, its role in CRC remains poorly understood and its potential as a therapeutic target is still difficult to define., Methods: NMU expression in CRC tissue was detected by IHC. Data from The Cancer Genome Atlas were used to analyse gene expression in CRC. mRNA and protein expression was detected by real-time PCR, immunoblotting or immunofluorescence staining and analysed using confocal microscopy or flow cytometry. Proteome Profiler was used to detect changes in the profiles of cytokines released by cells constituting tumour microenvironment after NMU treatment. NMU receptor activity was monitored by detecting ERK1/2 activation. Transwell cell migration, wound healing assay and microtube formation assay were used to evaluate the effects of NMU on the migration of cancer cells, human macrophages and endothelial cells., Results: Our current study showed increased NMU levels in human CRC when compared to normal adjacent tissue. We detected a correlation between high NMUR1 expression and shorter overall survival of patients with CRC. We identified NMUR1 expression on macrophages, endothelial cells, platelets, and NMUR1 presence in platelet microparticles. We confirmed ERK1/2 activation by treatment of macrophages and endothelial cells with NMU, which induced pro-metastatic phenotypes of analysed cells and changed their secretome. Finally, we showed that NMU-stimulated macrophages increased the migratory potential of CRC cells., Conclusions: We propose that NMU is involved in the modulation and promotion of the pro-metastatic tumour microenvironment in CRC through the activation of cancer cells and other tumour niche cells, macrophages and endothelial cells. Video abstract., (© 2022. The Author(s).)

Blood platelet dysfunctions are strongly involved in the development of the micro- and macrovascular complications in diabetes mellitus (DM). However, the molecular causes of abnormal platelet activation in DM remain unclear. Experimental data suggests that platelet mitochondria can regulate the prothrombotic phenotype of platelets, and changes in these organelles may influence platelet activation and modify platelet responses to stimulation. The present study evaluates the impact of DM on mitochondrial respiratory parameters and blood platelet activation/reactivity in a rat model of experimental diabetes following 1, 2.5 and 5 months of streptozotocin (STZ)-induced diabetes. Moreover, a mild inhibition of the mitochondrial respiratory chain with the use of metformin under in vitro and in vivo conditions was tested as a method to reduce platelet activation and reactivity. The platelets were studied with a combination of flow cytometry and advanced respirometry. Our results indicate that prolonged exposure of blood platelets to high concentrations of glucose, as in diabetes, can result in elevated blood platelet mitochondrial respiration; this may be an effect of cell adaptation to the high availability of energy substrates. However, as these alterations occur later than the changes in platelet activation/reactivity, they may not constitute the major reason for abnormal platelet functioning in DM. Moreover, metformin was not able to inhibit platelet activation and reactivity under in vitro conditions despite causing a decrease in mitochondrial respiration. This indicates that the beneficial effect of metformin on the coagulation system observed in vivo can be related to other mechanisms than via the inhibition of platelet activation.

Blood platelets are considered as promising candidates as easily-accessible biomarkers of mitochondrial functioning. However, their high sensitivity to various stimulus types may potentially affect mitochondrial respiration and lead to artefactual outcomes. Therefore, it is crucial to identify the factors associated with platelet preparation that may lead to changes in mitochondrial respiration. A combination of flow cytometry and advanced respirometry was used to examine the effect of blood anticoagulants, the media used to suspend isolated platelets, respiration buffers, storage time and ADP stimulation on platelet activation and platelet mitochondria respiration. Our results clearly show that all the mentioned factors can affect platelet mitochondrial respiration. Briefly, (i) the use of EDTA as anticoagulant led to a significant increase in the dissipative component of respiration (LEAK), (ii) the use of plasma for the suspension of isolated platelets with MiR05 as a respiration buffer allows high electron transfer capacity and low platelet activation, and (iii) ADP stimulation increases physiological coupling respiration (ROUTINE). Significant associations were observed between platelet activation markers and mitochondrial respiration at different preparation steps; however, the fact that these relationships were not always apparent suggests that the method of platelet preparation may have a greater impact on mitochondrial respiration than the platelet activation itself.

Diallyl disulfide (DADS) and diallyl trisulfide (DATS) are garlic oil compounds exhibiting beneficial healthy properties including anticancer action. However, these compounds are sparingly water-soluble with a limited stability that may imply damage to blood vessels or cells after administration. Thus, their encapsulation in the oil-core nanocapsules based on a derivative of hyaluronic acid was investigated here as a way of protecting against oxidation and undesired interactions with blood and digestive track components. The nuclear magnetic resonance ( 1 H NMR) technique was used to follow the oxidation processes. It was proved that the shell of the capsule acts as a barrier limiting the sulfur oxidation, enhancing the stability of C=C bonds in DADS and DATS. Moreover, it was shown that the encapsulation inhibited the lysis of the red blood cell membrane (mainly for DADS) and interactions with serum or digestive track components. Importantly, the biological functions and anticancer activity of DADS and DATS were preserved after encapsulation. Additionally, the nanocapsule formulations affected the migration of neoplastic cells-a desirable preliminary observation concerning the inhibition of migration. The proposed route of administration of these garlic extract components would enable reaching their higher concentrations in blood, longer circulation in a bloodstream, and thus, imply a better therapeutic effect.

We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y 12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y 12 receptor antagonists-cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y 12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y 12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y 12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y 12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y 12 receptor antagonists.

Blood platelets play a crucial role in the early stages of atherosclerosis development. The process is believed to require firm adhesion of platelets to atherosclerosis-prone sites of the artery. However, little evidence exists regarding whether the blood platelets of individuals with pathological conditions associated with atherosclerosis have higher potential for adhesion. This process is to a large extent dependent on receptors present on the platelet membrane. Therefore, the aim of the presented study was to determine whether blood platelets from diabetic patients have higher capacity of adhesion under flow conditions and how diabetes affects one of the crucial platelet receptors involved in the process of adhesion-GPIIIa. The study compares the ability of platelets from non-diabetic and diabetic humans to interact with fibrinogen and von Willebrand factor, two proteins found in abundance on an inflamed endothelium, under flow conditions. The activation and reactivity of the blood platelets were also characterized by flow cytometry. Platelets from diabetic patients did not demonstrate enhanced adhesion to either studied protein, although they presented increased basal activation and responsiveness towards low concentrations of agonists. Platelets from diabetic patients were characterized by lower expression of GPIIIa, most likely due to an enhanced formation of platelet-derived microparticles PMPs, as supported by the observation of elevated concentration of this integrin and of GPIIIa-positive PMPs in plasma. We conclude that altered functionality of blood platelets in diabetes does not increase their adhesive potential. Increased glycation and decrease in the amount of GPIIIa on platelets may be partially responsible for this effect. Therefore, higher frequency of interactions of platelets with the endothelium, which is observed in animal models of diabetes, is caused by other factors. A primary cause may be a dysfunctional vascular wall., Competing Interests: The authors declare no conflict of interest.

Aging has become a significant risk factor for several diseases, including breast cancer.Platelet activation and platelet-cancer cell aggregate fractions were found to increase with tumor progression in a mouse model of breast cancer. At advanced stages of tumor development, platelets from mice with breast cancer were hyperreactive to low agonist concentrations and hyporeactive to high ones. Platelet activation and reactivity were strongly associated with breast cancer metastasis in the lungs and extramedullary hematopoiesis in the liver. A greater fraction of platelet aggregates was observed in 4T1-injected mice at the advanced stages of breast cancer. In vitro , platelet activation was elevated after incubation with 4T1 cells, and thrombin-stimulated platelets formed aggregates with 4T1 cells. Neither GPIbα, nor GPIIb/IIIa blocking antibodies, were able to affect platelet-cancer cell aggregation in vitro .The primed circulating platelets became more sensitive to subthreshold stimuli at advanced stages of tumor development, and the formation of platelet-cancer cell aggregates increased with cancer progression. Our findings demonstrate that the age-associated progression of breast cancer cells is connected with increased platelet functioning, and that it can be manifested by the increased number of metastases and extramedullary hematopoiesis in a time-dependent-manner.

Diabetes is associated with an increased risk of cardiovascular disease. This is partially attributed to an altered activation status of blood platelets in this disease. Previously, alterations have been shown in COX-1 and protease activated receptor (PAR)-3 receptor expression in platelets in two animal models of diabetes, there have not been studies which address expression of these proteins in mice with long-term streptozotocin (STZ)-induced diabetes. We have also addressed the effect of diabetes on platelet adhesion under flow conditions. With the use of flow cytometry, we have shown that certain markers of platelet basal activation, such as active form of α II b β 3 and of CD40L were increased in STZ-induced diabetic mice. Platelets from STZ-induced diabetic mice were also more reactive when stimulated with PAR-4 activating peptide as revealed by higher expression of active form of α II b β 3 , membrane-bound on vWillebrand Factor and binding of exogenous fluorescein isothyanate-labelled fibrinogen. Expression of COX-1 and production of thromboxane A 2 in platelets of STZ-induced diabetic mice were higher than in control animals. We observed no effect of diabetes on ability of platelets to form stable adhesions with fibrinogen in flow conditions. We conclude that although certain similarities exist between patterns of activation of platelets in animal models of diabetes, the differences should also be taken into account., (© 2019 The Authors. International Journal of Experimental Pathology © 2019 International Journal of Experimental Pathology.)

Numerous in vitro experiments have confirmed that a dysfunctional endothelium is characterized by, inter alia, a higher affinity for binding of platelets and leukocytes. However, there is still no direct evidence for greater interaction between platelets and intact endothelium in in vivo animal models of diabetes. Therefore, the present study examines the pro-adhesive properties of endothelium change in vivo as an effect of streptozotocin (STZ)-induced diabetes and the role of two key platelet receptors: GPIb-IX-V and GPIIb/IIIa. Mice of C57BL strain with streptozotocin-induced diabetes were used in the study. Flow cytometry was used to assess basal activation and reactivity of platelets. Adhesion of platelets to the vascular wall was visualized with the use of intravital microscopy in mesentery. The contribution of GPIIb/IIIa and GPIb-IX-V was evaluated by the injection of Fab fragments of respective antibodies. The integrity of the endothelium and vWf expression were evaluated histochemically. Basal activation and reactivity of platelets in streptozotocin-diabetic mice were elevated. Blood platelets adhered more often to the vascular wall of diabetic mice than nondiabetic animals: 11.9 (6.4; 32.8) plt/min/mm 2 (median [IQR]) vs 2.7 (1.3; 6.4) plt/min/mm 2 . The injection of anti-GPIbα antibodies decreased the number of adhering platelets from 89.5 (34.0; 113.1) plt/min/mm 2 (median [IQR]) in mice treated with isotype antibodies to 3.1 (1.7; 5.6) plt/min/mm 2 in mice treated with blocking antibodies. The effect of GPIIb/IIIa blockage was not significant. Immunohistochemistry revealed a higher expression of vWF in the endothelium of STZ mice, but no substantial changes in endothelial morphology were detected. To conclude, the study shows that the platelets interact more frequently with the mesenteric vascular bed in mice with 1-month STZ-induced diabetes than in healthy mice. These interactions are mediated via platelet GPIb-IX-V and are driven by increased expression of vWF in endothelial cells.

Introduction: Recent studies have shown that it may be the concentration of thrombin, which is discriminative in determining of the mechanism of platelet activation via protease activated receptors (PARs). Whether the observed phenomenon of differentiated responses of mouse platelets to various thrombin concentrations in non-diabetic db/+ and diabetic db/db mice depends upon the concerted action of various PARs, remains to be established., Results: We found elevated reactivity of platelets, as well as the enhanced PAR-3 expression in response to both the used concentrations of AYPGKF in db/db mice, as compared to db/+ heterozygotes. At low concentration of thrombin platelets from diabetic mice demonstrated hyperreactivity, reflected by higher expression of PAR-3. For higher thrombin concentration, blood platelets from db/db mice appeared hyporeactive, compared to db/+ animals, while no significant differences in PAR-3 expression were observed between diabetic and non-diabetic mice., Conclusions: The novel and previously unreported finding resulting from our study is that the increased expression of PAR-3 in response to either TRAP for PAR-4 or low thrombin (when PAR-4 is not the efficient thrombin receptor) may be one of the key events contributing to higher reactivity of platelets in db/db mice., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Hop cones (Humulus lupulus L.), very rich source of phenolic compounds, possessing anticancer, antioxidant and anti-inflammatory activities, are considered as beneficial diet ingredients improving human health. In this study, the antiplatelet action of xanthohumol (XN), the principal flavonoid in hop cones, was investigated. XN significantly attenuated ADP-induced blood platelet aggregation (97.2 ± 35.7 AU for 6 μg/ml of XN vs. 120.4 ± 30.1 AU for 0.17% dimethyl sulfoxide (DMSO), p < 0.001) and significantly reduced the expression of fibrinogen receptor (activated form of GPIIbIIIa) on platelets' surface (47.6 ± 15.8 for 1.5 μg/ml XN, 44.6 ± 17.3% for 3 μg/ml XN vs. 54.5 ± 19.2% for control or 43.3 ± 18.4% for 6 μg/ml XN vs. 49.7 ± 19.4% for 0.17% DMSO, p < 0.05 or less). These findings suggest that the phenolic compounds originating from hops (XN) have a novel role as antiplatelet agents and can likely be used as dietary supplements in prophylactic approaches.

Due to its ability to inhibit the blood platelet PGHS-1, acetylsalicylic acid (ASA, Aspirin(®)) is widely used as a preventive agent in atherothrombotic diseases. However, its beneficial effects seem to be lower in diabetic patients, suggesting that protein glycation may impair effective ASA-mediated acetylation process. On the other hand, it is proposed that ASA can prevent some of the late complications of diabetes by lowering the extent of glycation at protein free amino groups. The aim of this work was to evaluate the extents of non-enzymatic N-glycosylation (glycation) and acetylation of blood platelet PGHS-1 (COX-1) and the competition between glycation and acetylation was investigated in order to demonstrate how these two reactions may compete against platelet PGHS-1. When PGHS-1 was incubated with glycating/acetylating agents (glucose, Glu; 1,6-bisphosphofructose, 1,6-BPF; methylglyoxal, MGO, acetylsalicylic acid, ASA), the enzyme was modified in 13.4 ± 1.6, 5.3 ± 0.5, 10.7 ± 1.2 and 6.4 ± 1.1 mol/mol protein, respectively, and its activity was significantly reduced. The prior glycation/carbonylation of PGHS-1 with Glu, 1,6-BPF or MGO decreased the extent of acetylation from 6.4 ± 1.1 down to 2.5 ± 0.2, 3.6 ± 0.3 and 5.2 ± 0.2 mol/mol protein, respectively, but the enzyme still remained susceptible to the subsequent inhibition of its activity with ASA. When PGHS-1 was first acetylated with ASA and then incubated with glycating/carbonylating agents, we observed the following reductions in the enzyme modifications: from 13.4 ± 1.6 to 8.7 ± 0.6 mol/mol protein for Glu, from 5.3 ± 0.5 to 3.9 ± 0.3 mol/mol protein for 1,6-BPF and from 10.7 ± 1.2 to 7.5 ± 0.5 mol/mol protein for MGO, however subsequent glycation/carbonylation did not significantly affect PGHS-1 function. Overall, our outcomes allow to better understand the structural aspects of the chemical competition between glycation and acetylation of PGHS-1., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Direct use of poly(amido)amine (PAMAM) dendrimers as drugs may be limited, due to uncertain (cyto)toxicity. Peripheral blood components, which constitute the first line of a contact with administered pharmaceuticals, may become vastly affected by PAMAM dendrimers. The aim of this study was to explore how PAMAMs' polycationicity might affect blood platelet activation and reactivity, and thus trigger various haemostatic events. We monitored blood platelet reactivity in rats with experimental diabetes upon a long-term administration of the unmodified PAMAM dendrimers. In parallel, the effects on blood flow in a systemic circulation was recorded intravitally in mice administered with PAMAM G2, G3 or G4. Compounding was the in vitro approach to monitor the impact of PAMAM dendrimers on blood platelet activation and reactivity and on selected haemostatic and protein conformation parameters. We demonstrated the activating effects of polycations on blood platelets. Some diversity of the revealed outcomes considerably depended on the used approach and the particular technique employed to monitor blood platelet function. We discovered undesirable impact of plain PAMAM dendrimers on primary haemostasis and their prothrombotic influence. We emphasize the need of a more profound verifying of all the promising findings collected for PAMAMs with the use of well-designed in vivo preclinical studies., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We explored the hypothesis that zeta potential altered by polycations affects blood platelet activation and reactivity, the phenomena associated with membrane lipid fluidity and platelet mitochondrial bioenergetics. PAMAM dendrimers generation- and dose-dependently enhanced zeta potential of platelets (from -10.7 mV to -4.3 mV). Increased expressions of activation markers, P-selectin and the active complex αIIbβ3, as well as significantly enhanced fibrinogen binding occurred upon the in vitro incubation of blood platelets in the presence of PAMAMs G3 and G4 (resp. 62.1% and 69.4% vs. 1.4% and 2.7% in control for P-selectin, P<0.0001). PAMAM dendrimers increased fluidity of platelet membrane lipid bilayer, while they did not affect platelet mitochondria respiration. Increased platelet activation and their responses to agonists in vitro were statistically associated with the revealed alterations in zeta potential. Our results support the hypothesis that polycation-mediated "neutralized" zeta potential may underlie the activating effects of PAMAMs on blood platelets., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Aims: The high glucose concentration observed in diabetic patients is a recognized factor of mitochondrial damage in various cell types. Its impact on mitochondrial bioenergetics in blood platelets remains largely vague. The aim of the study was to determine how the metabolism of carbohydrates, which has been impaired by streptozotocin-induced diabetes may affect the functioning of platelet mitochondria., Materials and Methods: Diabetes was induced in Sprague Dawley rats by intraperitoneal injection of streptozotocin. Platelet mitochondrial respiratory capacity was monitored as oxygen consumption (high-resolution respirometry). Mitochondrial membrane potential was assessed using a fluorescent probe, JC-1. Activation of circulating platelets was monitored by flow cytometry measuring of the expressions of CD61 and CD62P on a blood platelet surface. To determine mitochondrial protein density in platelets, Western Blot technique was used., Key Findings: The results indicate significantly elevated mitochondria mass, increased mitochondrial membrane potential (ΔΨm) and enhanced respiration in STZ-diabetic animals, although the respiration control ratios appear to remain unchanged. Higher ΔΨm and elevated mitochondrial respiration were closely related to the excessive activation of circulating platelets in diabetic animals., Significance: Long-term diabetes can result in increased mitochondrial mass and may lead to hyperpolarization of blood platelet mitochondrial membrane. These alterations may be a potential underlying cause of abnormal platelet functioning in diabetes mellitus and hence, a potential target for antiplatelet therapies in diabetes., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In diabetes-related states of chronic hyperglycaemia elevated concentrations of glucose may alter the functioning of platelet enzymes involved in arachidonic acid metabolism, including prostaglandin H2 synthase (cyclooxygenase) (PGHS, COX). Therefore, the principal aim of this study was to assess the effects of experimental chronic hyperglycaemia on platelet PGHS-1 (COX-1) expression and activity. Blood platelet activation and reactivity were assessed in Sprague-Dawley rats with the 5-month streptozotocin (STZ) diabetes. The PGHS-1 abundance in platelets was evaluated with flow cytometry and Western blotting, while its activity monitored using a high resolution respirometry and the peroxidase fluorescent assay. The production of prostaglandin E2 (PGE2) and thromboxane B2 (TXB2) in platelets were assayed immunoenzymatically. Circulating platelets from diabetic were characterised by increased size, elevated 'priming' and altered reactivity, compared to non-diabetic animals. Both Western blot analysis and flow cytometry revealed significantly elevated expressions of platelet PGHS-1 in STZ-diabetic rats (p < 0.05). We also observed significantly elevated platelet PGHS-1-related arachidonic acid metabolism in diabetic vs. non-diabetic animals, with the use of polarographic (p < 0.05) and total activity assay (p < 0.001). Such increases were accompanied by the elevated production of PGE2 (p < 0.001) and TXB2 (p < 0.05) in diabetic animals. The increased PGHS-1-dependent oxygen consumption and the total activity of PGHS-1 in diabetic animals remained very significant (p < 0.001) also upon adjusting for blood platelet PGHS-1 abundance. Therefore, our results further contribute to the explanation of the increased metabolism of arachidonic acid observed in diabetes.

Currently, there are several animal models of diabetes mellitus and hypertension, but relatively little is known about blood platelet function in these models. The aim of this work was to characterise and compare platelet reactivity and activation in db/db mice (mouse model of diabetes) and mice receiving L-NAME (model of chronic inhibition of NO synthesis), using various platelet function assays. We found higher platelet activation (circulating resting platelets) in db/db mice than in db/+heterozygotes, as evidenced by elevated expressions of CD62P and CD40L and a lower expression of CD42b. The expression of COX-1 was significantly increased, and the phosphorylation of vasodilator stimulated phosphoprotein (VASP) Ser(157) significantly reduced in platelets from db/db mice. Similarly, we observed platelet hyperreactivity in db/db mice following the in vitro responses to 20μg/ml collagen (reflected by increased expressions of CD62P and CD40L, and reduced CD42b), 20μM ADP (reduced CD42b) and lower concentrations of thrombin (0.025 U/ml) (increased CD62P, JON/A, bound vWF, and bound fibrinogen). Otherwise, platelet hyporeactivity was revealed for higher thrombin (0.25 U/ml) (reduced CD62P and bound vWF), while hyperreactivity occurred for CD40L and bound Fg in db/db mice compared to non-diabetic control, db/+. Plasma levels of sCD40L, but not of sCD62P, were increased in db/db mice; also plasma TXB2 concentrations were over 3.5-fold higher in this group than in the heterozygous db/+mice (P<0.01). In contrast, in the mice administered with L-NAME, no statistical differences in expressions of platelet activation markers were found between mice supplemented with L-NAME and controls. Likewise, the TXB2 level did not differ between L-NAME mice and controls, but L-NAME mice had significantly higher plasma levels of sCD62P and sCD40L than controls. In conclusion, these two studied models differ in the overall picture of blood platelet activation and reactivity, as they demonstrated opposite time sequence patterns of platelet activation in circulating blood. More generally, our study provides another argument for the opinion that multiparametric analysis of platelet function offers a much better tool for investigation and minimizes the likelihood of artefacts., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The effects of blood platelet inhibitors are often not quite equivalent under in vivo and in vitro conditions. Amongst various models of human pathology using laboratory animals, mice offer several benefits that make them convenient tools for studying the putative therapeutic value of various compounds. However, despite its advantages, the mouse model has methodological limitations concerning the small amount of blood available and technical difficulties with its collection. Among the variety of available methods used to study blood platelet activation and/or reactivity, flow cytometry seems an attractive technique that largely minimizes the constraints of using small rodents and enables outcomes of laboratory research to be transferred successfully to clinical practice. In this study we aimed at a critical evaluation of the optimal discriminative flow cytometric protocol, useful for reliable studies of the effect of cangrelor, a P2Y12 receptor antagonist, on mouse platelets under in vitro and in vivo conditions., Methods: Blood samples were drawn from two-month-old female BALB/c mice. Protocols differing in methods of anesthesia, blood withdrawal, anticoagulation, gating antibodies, blood preparation and fixation were tested to optimize the one best suited to discrimination between resting and activated platelets. The antiplatelet capabilities of cangrelor were tested in vitro (140 μM in whole blood) and in vivo (7.8 mg/kg b.w. administered once, directly into the bloodstream through the vena cava of the anesthetized animal, 15 min prior to blood withdrawal). Expressions of P-selectin, activated α(IIb)β3 complex and GPIba were monitored using two-color flow cytometry., Results: "Washed blood" anticoagulated with low molecular weight heparin demonstrated the best discrimination between circulating (resting) platelets and upon their in vitro response to thrombin, collagen or ADP in freshly-stained unfixed cell suspensions. Cangrelor inhibited the expression of the active form of the integrin a(IIb)β3 to approximately the same extent under in vitro and in vivo conditions (84.5 ± 7.7% vs. 75.4 ± 19.5% for the in vitro and in vivo approaches, respectively, n.s.)., Conclusions: The agreement between the in vivo and in vitro approaches with respect to cangrelor-inhibited hallmarks of blood platelet activation and reactivity supports our proposal that flow cytometry is useful and reliable for determining the effects of antiplatelet agents on the activation of circulating platelets in the mouse model, as well as the in vitro response of platelets to agonists.

O-phospho-L-threonine is a convenient sample to setup a (1)H-(31)P-(13)C double cross-polarization (DCP) Hartmann-Hahn match. The (1)H-(31)P-(13)C technique is extremely sensitive to the rate of the sample spinning. Both zero-quantum (ZQ) and double-quantum (DQ) cross-polarization operate at an average spinning rate (6-7 kHz). At higher spinning rates (10 kHz), the DQCP mechanism dominates and leads to a reduction of signal intensity, in particular for lower (31)P RF field strength. The application of two shape pulses during the second cross-polarization greatly improves the signal to noise ratio allowing the recording of better quality spectra. (31)P-(13)C spectrally induced filtering in combination with cross-polarization (SPECIFIC-CP) experiments can be carried out under ZQCP and DQCP conditions if careful attention is paid to the choice of RF field amplitudes and carriers Ω. Application of 1D and 2D (1)H-(31)P-(13)C experiments is demonstrated on model samples; disodium ATP hydrate and O-phospho-L-tyrosine., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Two analogous Schiff bases, (S,E)-2-((1-hydroxy-3-methyl-1,1-diphenylbutan-2-ylimino)methyl)phenol (1) and (S,Z)-2-hydroxy-6-((1-hydroxy-3-methyl-1,1-diphenylbutan-2-ylamino)methylene)cyclohexa-2,4-dienone (2), exist in the solid state as phenol-imine and keto-amine tautomers, respectively. Their crystal structures were solved using the X-ray diffraction method. Sample 1 forms orthorhombic crystals of space group P2(1)2(1)2(1), while 2 forms monoclinic crystals of space group P2(1). In each sample, one molecule is in the asymmetric unit of the crystal structure. One-dimensional and two-dimensional solid state NMR techniques were used for structure assignment and for inspection of the (13)C and (15)N δ(ii) of the chemical shift tensor (CST) values. NMR study indicates that the span (Ω = δ(11)-δ(33)) and the skew (κ = 3(δ(22)-δ(iso)/Ω) are extremely sensitive to change in the tautomeric form of the Schiff bases. Theoretical calculations of NMR shielding parameters for 1 and 2 and a model compound with reduced aliphatic residue were performed using the GIAO method with B3LYP functional and 6-311++g(d,p) basis sets. From comparative analysis of the experimental and theoretical parameters, it was concluded that the position of hydrogen in the intramolecular bridge has tremendous influence on (13)C and (15)N CST parameters. Inspection of Ω and κ parameters allowed for the establishment of the nature of the hydrogen bonding and the assignment of the equilibrium proton position in the intramolecular bridges in the solid state.