Piezo1 helps bile on the pressure

JGP study shows that a mechanosensitive complex containing Piezo1 and Pannexin1 couples osmotic pressure to ATP secretion in bile duct cholangiocytes.

Antiplatelet and Antithrombotic Effects of Epimedium koreanum Nakai

Background and Objective. Epimedium koreanum Nakai is a medicinal plant known for its health beneficial effects on impotence, arrhythmia, oxidation, aging, osteoporosis, and cardiovascular diseases. However, there is no report available that shows its effects on platelet functions. Here, we elucidated antiplatelet and antithrombotic effects of ethyl acetate fraction of E. koreanum. Methodology. We analyzed the antiplatelet properties using standard in vitro and in vivo techniques, such as light transmission aggregometry, scanning electron microscopy, intracellular calcium mobilization measurement, dense granule secretion, and flow cytometry to assess integrin αIIbβ3 activation, clot retraction, and Western blot, on washed platelets. The antithrombotic effects of E. koreanum were assessed by arteriovenous- (AV-) shunt model in rats, and its effects on hemostasis were analyzed by tail bleeding assay in mice. Key Results. E. koreanum inhibited platelet aggregation in agonist-stimulated human and rat washed platelets, and it also reduced calcium mobilization, ATP secretion, and TXB2 formation. Fibrinogen binding, fibronectin adhesion, and clot retraction by attenuated integrin αIIbβ3-mediated inside-out and outside-in signaling were also decreased. Reduced phosphorylation of extracellular signal-regulated kinases (ERK), Akt, PLCγ2, and Src was observed. Moreover, the fraction inhibited thrombosis. HPLC results revealed that the fraction predominantly contained icariin. Conclusion and Implications. E. koreanum inhibited platelet aggregation and thrombus formation by attenuating calcium mobilization, ATP secretion, TXB2 formation, and integrin αIIbβ3 activation. Therefore, it may be considered as a potential candidate to treat and prevent platelet-related cardiovascular disorders.

The features of secretory activity of platelets and fibrinolysis in women with various types of hypertensive disorders in the third trimester of pregnancy

Введение. Гипертензивные расстройства при беременности характеризуются нарушением адаптационных механизмов в системе свертывания крови и фибринолиза, важными участниками которой являются тромбоциты и эндотелий. Выраженность нарушений в тромбоцитарном звене гемостаза и системе фибринолиза при гипертензивных расстройствах при беременности различна. Цель исследования: изучить особенности секреторной активности тромбоцитов и компонентов фибринолитической системы у женщин с гипертензивными расстройствами различного генеза в III триместре беременности. Материалы и методы. Обследовано 238 женщин в III триместре беременности: 70 нормотензивных женщин, 32 — с умеренной преэклампсией (ПЭ), 47 — с тяжелой ПЭ, 58 — с хронической артериальной гипертензией (АГ) и 30 — с ПЭ на фоне хронической АГ. Проведен анализ количества и средней концентрации компонентов тромбоцитов, индуцированной секреции тромбоцитами АТФ, уровня бета-тромбоглобулина и тромбоцитарного фактора 4 в плазме крови. Фибринолитическую систему оценивали путем определения уровня лизиса сгустка, концентраций тканевого активатора плазминогена (t- PA) и ингибитора активатора плазминогена 1-го типа (PAI-1) в плазме крови. Результаты. Полученные данные свидетельствуют о различной степени выраженности нарушений в системе гемостаза при гипертензивных расстройствах различного генеза. При умеренной и тяжелой ПЭ выявлено повышение уровней t- PA и PAI-1, дополнительно при тяжелой ПЭ установлено снижение количества тромбоцитов и секреции активированными тромбоцитами АТФ на фоне повышения их гранулоцитарности. При ПЭ на фоне хронической АГ определено снижение секреции активированными тромбоцитами АТФ на фоне повышения их гранулоцитарности и базальной секреции β-тромбоглобулина и увеличение содержания в плазме крови t- PA и PAI-1. При хронической АГ обнаружено повышение количества тромбоцитов. Заключение. Выявлен- ные изменения позволили сделать вывод о неоднородности изменений в системе гемостаза у беременных с различными формами гипертензивных расстройств в III триместре беременности. Background. Hypertensive disturbances during pregnancy are jointly with the adaptive disorders of both blood coagulation and fibrinolysis, and platelets and endothelium plays important role for that. The disorders severity of platelet hemostasis and fibrinolysis is various in pregnant women with hypertensive disturbances. Objectives: to study the platelets secretory activity and some fibrinolytic components in women with hypertensive disturbances of various genesis in the III trimester of pregnancy. Patients / Methods. We examined 238 women in the III trimester of pregnancy: 70 normotensive women, 32 with moderate preeclampsia (PE), 47 with severe PE, 58 with chronic arterial hypertension (AH), and 30 with PE joint with chronic AH. The lab testing included the number and average concentration of platelet components, platelet-induced secretion of ATP, the β-thromboglobulin and platelet factor 4 in blood plasma. Fibrinolytic system was tested with determining of clot lysis level, tissue plasminogen activator (t- PA) and plasminogen activator inhibitor type 1 (PAI-1) in plasma. Results. Obtained data showed different severity of hemostasis disturbances in hypertensive disturbances of various genesis. In moderate and severe PE increased levels of t- PA and PAI-1 were revealed; in addition, in severe PE, decreased platelets number and ATP secretion by activated platelets was detected with increasing of their granulocyticity. In PE with chronic AH, decreased ATP secretion by activated platelets with increasing of their granulocyticity and basal secretion of beta-thromboglobulin was found, also increased t- PA and PAI-1 plasma content was determined. Increased platelet count was detected in chronic AH. Conclusions. Revealed changes led to the conclusion about the heterogeneity of hemostasis changes in pregnant women with various forms of hypertensive disturbances in the III trimester of pregnancy.

Mitoquinone (MitoQ) Inhibits Platelet Activation Steps by Reducing ROS Levels

Platelet activation plays a key role in cardiovascular diseases. The generation of mitochondrial reactive oxygen species (ROS) has been described as a critical step required for platelet activation. For this reason, it is necessary to find new molecules with antiplatelet activity and identify their mechanisms of action. Mitoquinone (MitoQ) is a mitochondria-targeted antioxidant that reduces mitochondrial overproduction of ROS. In this work, the antiplatelet effect of MitoQ through platelet adhesion and spreading, secretion, and aggregation was evaluated. Thus MitoQ, in a non-toxic effect, decreased platelet adhesion and spreading on collagen surface, and expression of P-selectin and CD63, and inhibited platelet aggregation induced by collagen, convulxin, thrombin receptor activator peptide-6 (TRAP-6), and phorbol 12-myristate 13-acetate (PMA). As an antiplatelet mechanism, we showed that MitoQ produced mitochondrial depolarization and decreased ATP secretion. Additionally, in platelets stimulated with antimycin A and collagen MitoQ significantly decreased ROS production. Our findings showed, for the first time, an antiplatelet effect of MitoQ that is probably associated with its mitochondrial antioxidant effect.

CHANGES IN THE INDICATORS OF THROMBOELASTOGRAPHY AND PLATELET FUNCTION IN PREGNANT WOMEN WITH VARIOUS FORMS OF HYPERTENSIVE DISORDERS IN THE THIRD TRIMESTER OF PREGNANCY

The aim of this research is the study of haemostasis of pregnant women suffering from various forms of hypertensive disorders in their III trimester of pregnancy. 165 women at 26-41 weeks of pregnancy were examined: 22 women had moderate preeclampsia, 31 had severe preeclampsia, 45 women suffered from chronic hypertension, 20 women have developed preeclampsia on the background of chronic hypertension and 47 women had no hypertensive disorders (control group). The hemostasis system has been assessed using the results of the following investigations: thromboelastography, induced platelet aggregation with ADP and adrenaline at a dosage of 1.25 and 2.5 μg/ml respectively and collagen at a dosage of 20 mg/ml, platelet ATP secretion and the average concentration of platelet components. Thromboelastography has been performed using TEG® 5000 thromboelastograph (Haemoscope Corporation, USA). The study of platelet aggregation and platelet ATP secretion has been performed at automatic aggregometer CHRONO-LOG® Model 700 (USA). The mean platelet component concentration has been measured using SIEMENS ADVIA 2120i automated hematology analyzer (Siemens Healthcare Diagnostics Inc., USA). Thromboelastogram analysis showed a decrease in the plasma hemostasis activity in all groups of women with hypertensive disorders. The functional activity of platelets of women with moderate preeclampsia and chronic arterial hypertension did not change in comparison with to the control group. The disorder of dense platelet granules degranulation and decrease in their aggregation ability have been detected in a cohort with severe preeclampsia. The decrease in adrenaline induced platelet aggregation has been noted in the group of women suffering from preeclampsia on the background of chronic arterial hypertension. Thromboelastography analysis (R, K, angle α, TMA, Cl, LY30) may be useful for the differential diagnosis of severe preeclampsia and chronic arterial hypertension. The results of the study led to the conclusion that it is advisable to use low doses of ADP and adrenaline as inducers of platelet aggregation, considering their granulocyticity and the ability to secrete ATP.

Extracellular ATP released from Candida albicans activates non-peptidergic neurons to augment host defense

Intestinal microbes release ATP to modulate local immune responses. Herein we demonstrates that Candida albicans, an opportunistic commensal fungus, also modulates immune responses via secretion of ATP. We found that ATP secretion from C. albicans varied between standard laboratory strains. A survey of eighty-nine clinical isolates revealed heterogeneity in ATP secretion, independent of growth kinetics and intracellular ATP levels. Isolates from blood released less ATP than commensals, suggesting that ATP secretion assists with commensalism. To confirm this, cohorts of mice were infected with strains matched for origin, and intracellular ATP concentration, but high or low extracellular ATP. In all cases fungal burden was inversely correlated with ATP secretion. Mice lacking P2RX7, the key ATP receptor expressed by immune cells in the skin, showed no alteration in fungal burden. Rather, treatments with a P2RX2/3 antagonist result in increased fungal burden. P2RX2/3 is expressed by non-peptidergic neurons that terminate in the epidermis. Cultured sensory neurons flux Ca2+ when exposed to supernatant from heat-killed C. albicans (HKCA), and these non-peptidergic fibers are the dominant subset that respond to HKCA. Ca2+ flux, but not CGRP-release, can be abrogated by pretreatment of HKCA supernatant with apyrase. To determine whether non-peptidergic neurons participate in host defense, we generated MRGPRD-DTR mice. Infection in these mice resulted in increased CFU only for those C. albicans strains with high ATP secretion. Taken together, our findings indicate that C. albicans releases ATP, which is recognized by non-peptidergic nerves in the skin resulting in augmented anti-Candida immune responses.Author SummaryBacterial release of ATP has been shown to modulate immune responses. Candida albicans displays heterogeneity in ATP release among laboratory strains and commensal clinical isolates release more ATP than invasive isolates. C. albicans strains with high ATP secretion show lower fungal burden following epicutaneous infection. Mice lacking P2RX7, the key ATP receptor expressed by immune cells, showed no alteration in fungal burden. In contrast, treatment with P2RX2/3 antagonists resulted in increased fungal burden. P2RX3 is expressed by a subset of non-peptidergic neurons that terminate in the epidermis. These non-peptidergic fibers are the predominant responders when cultured sensory neurons are exposed to heat-killed C. albicans in vitro. Mice lacking non-peptidergic neurons have increased infection when exposed to high but not low ATP-secreting isolates of C. albicans. Taken together, our findings indicate that C. albicans releases ATP which is recognized by non-peptidergic nerves in the skin resulting in augmented anti-Candida immune responses.Bullet pointsATP released from heat killed C. albicans activates non-peptidergic sensory neuronsLive C. albicans clinical isolates release variable amounts of ATPElevated levels of ATP released by C. albicans correlates with reduced infectivity in vivoMRGPRD-expressing cutaneous neurons are required for defense against ATP-secreting C. albicans

Wide field of view quantitative imaging of cellular ATP release

Although several mechanical stressors promote ATP secretion from eukaryotic cells, few mechanosensitive pathways for ATP release have been precisely characterized and none have been clearly identified. To facilitate progress, we report here a wide field of view (∼20 × 20 mm sample area) imaging technique paired with a quantitative image analysis to accurately map the dynamics of ATP release from a cell population. The approach has been tested on A549 cells stretched at high initial strain rate (2–5 s−1) or swelled by hypotonic shock. The amount of ATP secreted in response to a series of five graded stretch pulses (5–37% linear deformation, 1-s duration at 25°C) changed nonmonotonically with respect to strain amplitude and was inhomogeneous across the cell monolayer. In a typical experiment, extracellular ATP density averaged 250 fmol/mm2, but the area of detectable signal covered only ∼40% of the cells. In some areas, ATP accumulation peaked around 900 fmol/mm2, which corresponded to an estimated concentration of 4.5 µM. The total amount of ATP released from the combined stretch pulses reached 384 ± 224 pmol/million cells ( n = 4). Compared with stretch, hypotonic shock (50%, 30°C) elicited a more homogeneous ATP secretion from the entire cell population but at a lower yield totaling 28 ± 12 pmol/million cells ( n = 4). The quantitative extracellular ATP mapping of several thousand cells at once, with this wide field of view imaging system, will help identify ATP release pathways by providing unique insights on the dynamics and inhomogeneities of the cellular ATP secretion that are otherwise difficult to assess within the smaller field of view of a microscope.