Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT): Targeting Pathomechanisms with Bruton Tyrosine Kinase Inhibitors.

A series of cases with rare thromboembolic incidents including cerebral sinus vein thrombosis (some of them fatal) and concomitant thrombocytopenia occurring shortly after vaccination with the coronavirus disease 2019 (COVID-19) vaccine AZD1222 (Vaxzevria) have caused significant concern and led to its temporary suspension in many countries. Immediate laboratory efforts in four of these patients have identified a tentative pathomechanism underlying this syndrome termed initially vaccine-induced prothrombotic immune thrombocytopenia (VIPIT) and renamed recently vaccine-induced immune thrombotic thrombocytopenia (VITT). It encompasses the presence of platelet-activating antibodies to platelet factor-4/heparin complexes, possibly emulated by polyanionic constituents of AZD1222, and thus resembles heparin-induced thrombocytopenia (HIT). Because these immune complexes bind and activate platelets via Fcγ receptor IIA (FcγRIIA), high-dose intravenous immunoglobulin G has been suggested for treatment of VITT in addition to non-heparin anticoagulants. Here we propose inhibitors of Bruton tyrosine kinase (Btk) approved for B cell malignancies (e.g., ibrutinib) as another therapeutic option in VITT, as they are expected to pleiotropically target multiple pathways downstream of FcγRIIA-mediated Btk activation, for example, as demonstrated for the effective inhibition of platelet aggregation, dense granule secretion, P-selectin expression and platelet-neutrophil aggregate formation stimulated by FcγRIIA cross-linking. Moreover, C-type lectin-like receptor CLEC-2- and GPIb-mediated platelet activation, the interactions and activation of monocytes and the release of neutrophil extracellular traps, as encountered in HIT, could be attenuated by Btk inhibitors. As a paradigm for emergency repurposing of approved drugs in COVID-19, off-label use of Btk inhibitors in a low-dose range not affecting haemostatic functions could thus be considered a sufficiently safe option to treat VITT.

Effects of coffee, energy drinks and their components on hemostasis: The hypothetical mechanisms of their action.

Hemostasis is a process which encompasses the clotting, fibrinolysis, blood platelet activation and endothelial cell function. Certain dietary components may modulate some elements of hemostasis, particularly blood platelet function, and modify the progression of cardiovascular diseases. The aim of this review paper is to provide an overview of current knowledge of the role of coffee, energy drinks and their bioactive compounds in such modulation. It describes the effect of coffee, energy drinks and their selected components (e.g. caffeine) on hemostasis, especially blood platelets, and their underlying mechanisms. Like coffee, energy drinks may modify platelet reactivity by changing the activity of signaling enzymes, and by modifying cAMP and reactive oxygen species levels. However, the effects of coffee and energy drinks on platelet activation are dependent on a range of factors, including their bioactive components, platelet activators and the methods used for monitoring platelet activation. While some studies (in vivo models) indicate that energy drinks have pro-aggregatory affects, which may be associated with an elevated risk of thrombosis, others indicate that coffee in fact reduces platelet activation, which may be beneficial for prophylaxis of thrombosis.

Time-Dependent Hypotensive Effect of Aspirin in Mice.

Objective- Evening but not morning administration of low-dose aspirin has been reported to lower blood pressure in hypertensive patients. The present study was designed to determine whether this phenomenon could be replicated in mice, and if so, whether a time-dependent effect of aspirin on blood pressure was because of alteration of circadian clock function. Approach and Results- We recapitulated the protective effect of aspirin (50 µg/d for 7 days) at zeitgeber time 0 (active-to-rest transit), but not at zeitgeber time 12, on a high-salt diet-induced increase of blood pressure. However, the time of aspirin administration did not influence expression of canonical clock genes or their acetylation. We used mouse Bmal1 and Per2-luciferase reporters expressed in U2OS cells to determine the real-time effect of aspirin on circadian function but found that the oscillation of bioluminescence was unaltered. Timing of aspirin administration also failed to alter urinary prostaglandin metabolites or catecholamines, or the acetylation of its COX-1 (cyclooxygenase-1) target in platelets. Conclusions- The time-dependent hypotensive effect of aspirin in humans has been recapitulated in hypertensive mice. However, this does not seem to reflect a direct impact of aspirin on circadian clocks or on acetylation of platelet COX-1.

Modulatory effect of eugenol on arginase, nucleotidase, and adenosine deaminase activities of platelets in a carrageenan-induced arthritis rat model: A possible anti-arthritic mechanism of eugenol.

This study investigated the effect of eugenol on arginase, nucleotidase and adenosine deaminase activities in platelets of carrageenan-induced arthritic rat model to explain a possible anti-arthritic mechanism of eugenol. Fifty adult female rats (140-250 g) were divided into ten (10) groups (n = 5). Group I received oral administration of corn oil, group II received 2.50 mg/kg of eugenol, group III and IV rats received oral administration of 5.0 and 10.0 mg/kg of eugenol respectively, group V received 0.20 mg/kg of dexamethasone orally, group VI rats was injected with 1% carrageenan (arthritic rats) and received saline solution orally (arthritic control rat group), group VII, VIII and IX: arthritic rats received 2.50, 5.0 or 10 mg/kg of eugenol orally respectively, group X: arthritic rats was administered with 0.20 mg/kg of dexamethasone orally. The animals were treated for 21 days, thereafter, tibiofemoral histological examination, thiobabituric acid reactive substances level, arginase, nucleoside triphosphate diphosphohydrolase, 5´-nucleotidase and adenosine deaminase activities were assessed. Tibiofemoral histological examination result showed that infiltration of inflammatory cells was significantly decreased with an increase in eugenol dose. Activities of arginase, adenosine triphosphate and adenosine monophosphate hydrolyses were significantly decreased while adenosine diphosphate hydrolysis and adenosine deaminase activities were significantly increased in arthritic rat groups administered with different doses of eugenol. Therefore, eugenol might be a natural complement and alternative promising anti-arthritic agent. These possible anti-arthritic mechanisms may be partly through the modulation of arginase and adenosine nucleotides hydrolyzing enzyme activities as well as the antioxidative action of eugenol.

Imaging analyses of coagulation-dependent initiation of fibrinolysis on activated platelets and its modification by thrombin-activatable fibrinolysis inhibitor.

Using intravital confocal microscopy, we observed previously that the process of platelet phosphatidylserine (PS) exposure, fibrin formation and lysine binding site-dependent plasminogen (plg) accumulation took place only in the centre of thrombi, not at their periphery. These findings prompted us to analyse the spatiotemporal regulatory mechanisms underlying coagulation and fibrinolysis. We analysed the fibrin network formation and the subsequent lysis in an in vitro experiment using diluted platelet-rich plasma supplemented with fluorescently labelled coagulation and fibrinolytic factors, using confocal laser scanning microscopy. The structure of the fibrin network formed by supplemented tissue factor was uneven and denser at the sites of coagulation initiation regions (CIRs) on PS-exposed platelets. When tissue-type plasminogen activator (tPA; 7.5 nM) was supplemented, labelled plg (50 nM) as well as tPA accumulated at CIRs, from where fibrinolysis started and gradually expanded to the peripheries. The lysis time at CIRs and their peripheries (50 µm from the CIR) were 27.9 ± 6.6 and 44.4 ± 9.7 minutes (mean ± SD, n=50 from five independent experiments) after the addition of tissue factor, respectively. Recombinant human soluble thrombomodulin (TMα; 2.0 nM) attenuated the CIR-dependent plg accumulation and strongly delayed fibrinolysis at CIRs. A carboxypeptidase inhibitor dose-dependently enhanced the CIR-dependent fibrinolysis initiation, and at 20 µM it completely abrogated the TMα-induced delay of fibrinolysis. Our findings are the first to directly present crosstalk between coagulation and fibrinolysis, which takes place on activated platelets' surface and is further controlled by thrombin-activatable fibrinolysis inhibitor (TAFI).

Effects of chlorogenic acid, caffeine and coffee on components of the purinergic system of streptozotocin-induced diabetic rats.

We evaluated the effect of chlorogenic acid (CGA), caffeine (CA) and coffee (CF) on components of the purinergic system from the cerebral cortex and platelets of streptozotocin-induced diabetic rats. Animals were divided into eight groups: control animals treated with (I) water (WT), (II) CGA (5 mg/kg), (III) CA (15 mg/kg) and (IV) CF (0.5 g/kg), and diabetic animals treated with (V) WT, (VI) CGA (5 mg/kg), (VII) CA (15 mg/kg) and (VIII) CF (0.5 g/kg). Our results showed an increase (173%) in adenosine monophosphate (AMP) hydrolysis in the cerebral cortex of diabetic rats. In addition, CF treatment increased adenosine diphosphate (ADP) and AMP hydrolysis in group VIII synaptosomes. Platelets showed an increase in ectonucleotidase activity in group V, and all treatments reduced the increase in adenosine triphosphate and ADP hydrolysis. Furthermore, there was an increase in platelet aggregation of 72% in the diabetic rats, and CGA and CF treatment reduced platelet aggregation by nearly 60% when compared to diabetic rats. In this context, we can suggest that CGA and CF treatment should be considered a therapeutic and scientific target to be investigated in diseases associated with hyperglycemia.

Effect of dietary supplementation of Padauk (Pterocarpus soyauxii) leaf on high fat diet/streptozotocin induced diabetes in rats' brain and platelets.

BACKGROUND: This study investigated the effects of Padauk leaf on brain malondialdehyde (MDA) content, acetylcholinesterase (AChE) activities, ectonucleotidases and adenosine deaminase (ADA) activities in the platelet of high fat diet and streptozotocin (STZ)-induced diabetic rats. METHODS: The animals were divided into six groups (n=7): normal control rats; diabetic rats+high fat diet (HFD); diabetic rats+HFD+Metformin; diabetic rats+HFD+acarbose; diabetic rats+HFD+10% Padauk leaf; normal rats+basal diet+10% Padauk leaf. After 30days of experiment comprising of acclimatization, dietary manipulation, pre-treatment with STZ and supplementation with Padauk leaf, the animals were sacrificed and the rats' brain and blood were collected for subsequent analysis. RESULTS: The results demonstrated that the elevated MDA content and AChE activity in the diabetic rats were significantly reduced when compared with the control rats. Furthermore, the increased NTPDases, 5'-nucleotidase and ADA activities in the diabetic rats were significantly reduced when compared with the control rats. CONCLUSION: This study demonstrated that Padauk leaf exhibited modulatory effects on purinergic and cholinergic enzymes involved in the prevention of platelet abnormality and consequent vascular complications in diabetic state.

Red Wine Inhibits Aggregation and Increases ATP-diphosphohydrolase (CD39) Activity of Rat Platelets in Vitro.

Moderate consumption of red wine has been shown to exert a peculiar cardioprotective effect compared with other alcoholic beverages; inhibition of platelet aggregation seems to be one of the mechanisms underlying this beneficial effect. CD39/ATP-diphosphohydrolase is an integral membrane glycoprotein metabolizing ATP and ADP to AMP; in concert with CD73/ecto-5'-nucleotidase, it contributes to extracellular adenosine accumulation. CD39 is considered a key modulator of thrombus formation; it inhibits platelet aggregation by promoting ADP hydrolysis. There is evidence that red wine consumption increases CD39 activity in platelets from streptozotocin-induced diabetic rats. Here we show that two kinds of Aglianico red wines inhibit aggregation and increase ATP--and ADPase activity in rat platelets.

Panax quinquefolium saponin combined with dual antiplatelet drugs inhibits platelet adhesion to injured HUVECs via PI3K/AKT and COX pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Panax quinquefolium saponin (PQS) is the active component extracted from traditional Chinese medicine Panax quinquefolius L. and has been widely used as a supplement to dual antiplatelet drugs (DA) for treatment of coronary artery disease (CAD) for two decades; however, the efficacy of PQS combined with DA against platelet adhesion to endothelial cells (ECs), an essential step in thrombosis, remains unclear. AIM OF THE STUDY: To compare PQS combined with DA and DA alone in inhibiting platelet adhesion to injured human umbilical vein endothelial cells (HUVECs) and to explore the possible mechanisms focusing on PI3K/AKT, COX-2/6-keto-PGF1α, and COX-1/TXB2 pathways. METHODS: HUVECs injured by oxidized low-density lipoprotein (ox-LDL) were randomly allocated into control, model, DA, PQS+DA (P+DA), LY294002 (a PI3K inhibitor)+DA (L+DA), and LY294002+PQS+DA (LP+DA) groups. HUVEC apoptosis, platelet adhesion to injured HUVECs, and platelet CD62p expression were assayed by fluorescence activated cell sorting (FACS). The concentrations of 6-keto-PGF1α and TXB2 in the supernatant were measured by radioimmunoassay. Protein expression of phosphorylated-PI3K, PI3K, phosphorylated-AKT, AKT, COX-1, and COX-2 in both platelets and HUVECs was evaluated by western blot. RESULTS: Compared to DA alone, PQS combined with DA reduced platelet adhesion to HUVECs and HUVEC apoptosis more potently, increased the concentration of supernatant 6-keto-PGF1α and up-regulated phospho-AKT protein in HUVECs. LY294002 mitigated the effects of PQS on HUVEC apoptosis and platelet adhesion. CONCLUSIONS: These findings show that PQS as a powerful supplement to DA, attenuated HUVEC apoptosis and improved the DA-mediated reduction of platelet adhesion to injured HUVECs and the underlying mechanisms may be associated with PI3K/AKT and COX pathways in HUVECs and platelets. PQS might provide a new complementary approach to improve the prognosis of thrombotic diseases in future.

Dietary Supplementation of Ginger and Turmeric Rhizomes Modulates Platelets Ectonucleotidase and Adenosine Deaminase Activities in Normotensive and Hypertensive Rats.

Hypertension is associated with platelet alterations that could contribute to the development of cardiovascular complications. Several studies have reported antiplatelet aggregation properties of ginger (Zingiber officinale) and turmeric (Curcuma longa) with limited scientific basis. Hence, this study assessed the effect of dietary supplementation of these rhizomes on platelet ectonucleotidase and adenosine deaminase (ADA) activities in Nω-nitro-l-arginine methyl ester hydrochloride (l-NAME) induced hypertensive rats. Animals were divided into seven groups (n = 10): normotensive control rats; induced (l-NAME hypertensive) rats; hypertensive rats treated with atenolol (10 mg/kg/day); normotensive and hypertensive rats treated with 4% supplementation of turmeric or ginger, respectively. After 14 days of pre-treatment, the animals were induced with hypertension by oral administration of l-NAME (40 mg/kg/day). The results revealed a significant (p < 0.05) increase in platelet ADA activity and ATP hydrolysis with a concomitant decrease in ADP and AMP hydrolysis of l-NAME hypertensive rats when compared with the control. However, dietary supplementation with turmeric or ginger efficiently prevented these alterations by modulating the hydrolysis of ATP, ADP and AMP with a concomitant decrease in ADA activity. Thus, these activities could suggest some possible mechanism of the rhizomes against hypertension-derived complications associated to platelet hyperactivity. Copyright © 2016 John Wiley & Sons, Ltd.

Evaluating the inhibitory potential of Withania somnifera on platelet aggregation and inflammation enzymes: An in vitro and in silico study.

Context Withania somnifera (L.) Dunal is traditionally used for treating various ailments, but lacks scientific evaluation. Objective This study evaluates Withania somnifera (WS) for its effect on platelet activity and inflammatory enzymes. Materials and methods Aqueous and ethanolic (1:1) leaf extracts were subjected to in vitro indirect haemolytic activity using Naja naja venom, human platelet aggregation was quantified for lipid peroxidation using arachidonic acid (AA) as agonist and 5-lipoxygenase (5-LOX) levels were determined using standard spectrometric assays. Further, molecular docking was performed by the ligand fit method using molegro software package (Molegro ApS, Aarhus, Denmark). Results The study found that aqueous and ethanol extracts have very negligible effect (15%) with an IC50 value of 13.8 mg/mL on PLA2 from Naja naja venom. Further, extracts of WS also had very little effect (18%) with an IC50 value of 16.6 mg/mL on malondialdehyde (MDA) formation. However, a 65% inhibition of 5-LOX with an IC50 value of 0.92 mg/mL was observed in 1:1 ethanol extracts. The same was evident from SAR model with the active ingredient withaferin A binding predominantly on Phe 77, Tyr 98, Arg 99, Asp 164, Leu 168, Ser 382, Arg 395, Tyr 396 and Tyr 614 with an atomic contact energy value of -128.96 compared to standard phenidone (-103.61). Thus, the current study validates the application of WS for inflammatory diseases. Conclusion This study reveals the inhibitory potential of W. somnifera on inflammatory enzymes and platelet aggregation. Thus, WS can serve as a newer, safer and affordable medicine for inflammatory diseases.

[Effect of different fractions of Taohong Siwu decoction on ADP-induced platelet aggregation and thrombin activity].

To evaluate the effect of different fractions of Taohong Siwu decoction on ADP-induced platelet aggregation and thrombin activity, and to exploit the bioactive constituents, ADP-induced platelet aggregation rate in rabbits was determined by using the method of turbidity method. A bioassay called thrombin time was developed for determining anti-thrombin activities. UHPLC-Q-TOF-MS method was used to qualitatively analyze the chemical constituents of different parts. Alcohol precipitation deposition fraction, alcohol precipitation supernatant fraction and 20% to 30% alcohol elution fraction could significantly inhibit ADP-induced platelet aggregation. Alcohol precipitation supernatant fraction, water insoluble fraction and 40% to 70% alcohol elution fraction could significantly inhibit thrombin activity. The main components of alcohol precipitation deposition fraction, alcohol precipitation supernatant fraction and 20% to 40% alcohol elution fraction were analyzed and identified as aromatic acids, glycosides and phthalides. The bioactive constituents of Taohong Siwu decoction for inhibiting ADP-induced platelet aggregation and thrombin activity include aromatic acids, glycosides and phthalides. This experiment provides scientific basis to further explore the bioactive constituents and mechanism of Taohong Siwu decoction in treating blood stasis syndrome.

Platelet microparticles are internalized in neutrophils via the concerted activity of 12-lipoxygenase and secreted phospholipase A2-IIA.

Platelets are anucleated blood elements highly potent at generating extracellular vesicles (EVs) called microparticles (MPs). Whereas EVs are accepted as an important means of intercellular communication, the mechanisms underlying platelet MP internalization in recipient cells are poorly understood. Our lipidomic analyses identified 12(S)-hydroxyeicosatetranoic acid [12(S)-HETE] as the predominant eicosanoid generated by MPs. Mechanistically, 12(S)-HETE is produced through the concerted activity of secreted phospholipase A2 IIA (sPLA2-IIA), present in inflammatory fluids, and platelet-type 12-lipoxygenase (12-LO), expressed by platelet MPs. Platelet MPs convey an elaborate set of transcription factors and nucleic acids, and contain mitochondria. We observed that MPs and their cargo are internalized by activated neutrophils in the endomembrane system via 12(S)-HETE. Platelet MPs are found inside neutrophils isolated from the joints of arthritic patients, and are found in neutrophils only in the presence of sPLA2-IIA and 12-LO in an in vivo model of autoimmune inflammatory arthritis. Using a combination of genetically modified mice, we show that the coordinated action of sPLA2-IIA and 12-LO promotes inflammatory arthritis. These findings identify 12(S)-HETE as a trigger of platelet MP internalization by neutrophils, a mechanism highly relevant to inflammatory processes. Because sPLA2-IIA is induced during inflammation, and 12-LO expression is restricted mainly to platelets, these observations demonstrate that platelet MPs promote their internalization in recipient cells through highly regulated mechanisms.

Pharmacokinetic, partial pharmacodynamic and initial safety analysis of (-)-epicatechin in healthy volunteers.

(-)-Epicatechin ((-)-EPI), a naturally occurring flavanol, has emerged as a likely candidate for cocoa-based product reported reductions in cardiometabolic risk. The present study aimed to determine the safety, tolerability, pharmacokinetics and pharmacodynamics of purified (-)-EPI administered to healthy volunteers. In this phase I, open-label, two-part single- and multiple-dose study, subjects received either a single dose (n = 9) of 50, 100 or 200 mg or multiple doses (n = 8) of 50 mg daily (q.d.) or twice daily (b.i.d) for 5 days. Blood was collected at 0, 0.5, 1, 2, 4 and 6 h after (-)-EPI administration in the single and multiple dose groups (blood collection repeated in day 5). Samples were analyzed by HPLC-HR-ESI-MS for EPI and metabolite quantification. In the q.d. and b.i.d. groups, blood samples were analyzed for NO surrogates and follistatin levels as well as, platelet mitochondrial complexes I, V and citrate synthase activity levels. (-)-EPI was well tolerated and readily absorbed with further phase 2 metabolism. On day 5, in the q.d. and b.i.d. groups, there were significant increases in plasma nitrite of 30% and 17%, respectively. In the q.d. group on day 5 vs. day 1, platelet mitochondrial complexes I, IV and citrate synthase activities demonstrated a significant increase of ∼92, 62 and 8%, respectively. Average day 5 follistatin AUC levels were ∼2.5 fold higher vs. day 1 AUC levels in the b.i.d. group. (-)-EPI was safe to use, with no observed adverse effects, and our findings suggest that increases in NO metabolites, mitochondrial enzyme function and plasma follistatin levels may underlie some of the beneficial effects of cocoa products or (-)-EPI as reported in other studies.

Effect of Scutia buxifolia Reissek in nucleotidase activities and inhibition of platelet aggregation.

This study aimed to determine the effect of lyophilized aqueous extracts of Scutia buxifolia on NTPDase and 5'-nucleotidase activity on platelets and lymphocytes as well as the profile of the platelet aggregation. In vitro tests were used to investigate the effect of the aqueous crude extract obtained from S. buxifolia leaves (SbL) and stem bark (SbS) on enzymatic activities and platelet aggregation. The platelets and lymphocytes were exposed to lyophilized aqueous extracts of S. buxifolia at concentrations of 1-200 µg/mL in the presence of ATP, ADP, AMP as substrates, during the enzymatic assay, as well as the platelet aggregation. The results showed that SbS and SbL potently inhibited NTPDase and 5'-nucleotidase in platelets and lymphocytes. ADP-induced aggregation was inhibited by the SbS (50, 100, and 200 µg/mL) and SbL (200 µg/mL). In addition, these results suggest that S. buxifolia have compounds, such as gallic acid, chlorogenic acid, caffeic acid, quercetin, rutin, and kaempferol, which cause a decrease the NTPDase and 5'-nucleotidase activity, resulting in alterations in adenine nucleotide levels and protection against ADP-induced platelet aggregation.

Tanshinone IIA, a major component of Salvia milthorriza Bunge, inhibits platelet activation via Erk-2 signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: The roots of Salvia milthorriza Bunge (Lamiaceae) known as "Danshen", are used in Traditional Chinese Medicine as a remedy for activating blood and eliminating stasis. TIIA, a diterpenoid of Salvia milthorriza, is one of active components in Danshen that exhibits a significant improvement of the blood flow in the coronary circulatory system and a reduction of myocardial infarction. However, its effect on platelet and underlying mechanism remains largely unknown. On this basis, this compound could be a promising agent to improve blood viscosity and microcirculation and to prevent CVD. MATERIALS AND METHODS: In order to investigate the effects of TIIA on platelet functionality and its interaction with various platelet activation pathways, rat PRP were incubated with TIIA for 1 min at 37°C prior the addition of the stimuli (ADP or collagen). Aggregation was monitored in a light transmission aggregometer measuring changes in turbidity with continuous observation up to 10 min after the addition of the stimuli. MAPK signaling pathway and tubulin acetylation were analyzed by a Western blot technique. The effect of the TIIA was also studied in vivo on bleeding time in mice. RESULTS: TIIA selectively inhibited rat platelet aggregation induced by reversible ADP stimuli (3 µM) in a concentration-dependent manner (0.5-50 µM). Nevertheless, TIIA was less active against the irreversible stimuli induced by ADP (10 µM) and collagen (10 µg/mL). Moreover, experiments performed on platelet lysates collected at different time-point after the addition of the stimuli shown that TIIA modulated tubulin acetylation and inhibited Erk-2 phosphorylation. Concomitantly, TIIA administrated i.p. at 10 mg/kg significantly amplified the mice bleeding time with an increase of 58% compared to its control (2.06±0.29 min vs 1.30±0.07). ASA was used as reference drug for in vitro and in vivo experiments. CONCLUSIONS: This study clarifies the intracellular signaling pathway involved in antiplatelet action of TIIA and also gives preliminary evidences for its anticoagulant activity. On this basis, this compound could be a promising agent to improve blood viscosity and microcirculation and to prevent CVD.

Polyunsaturated fatty acids balance affects platelet NOX2 activity in patients with liver cirrhosis.

BACKGROUND: NADPH-oxidase-2 up-regulation has been suggested in liver damage perpetuation via an oxidative stress-mediated mechanism. n-6/n-3 polyunsaturated fatty acids ratio derangement has been reported in liver disease. AIM: To explore polyunsaturated fatty acids balance and its interplay with platelet oxidative stress in liver cirrhosis. METHODS: A cross-sectional study in 51 cirrhotic patients and sex- and age-matched controls was performed. Serum polyunsaturated fatty acids and oxidative stress markers (urinary isoprostanes and serum soluble NADPH-oxidase-2-derived peptide) were measured. The effect on platelet oxidative stress of n-6/n-3 polyunsaturated fatty acids ratio in vitro and in vivo (1-week supplementation with 3g/daily n-3-polyunsaturated fatty acids) was tested. RESULTS: Compared to controls, cirrhotic patients had significantly higher n-6/n-3 polyunsaturated fatty acids ratio. n-6/n-3 polyunsaturated fatty acids ratio correlated significantly with disease severity and oxidative stress markers. In vitro experiments showed that in Child-Pugh C patients' platelets incubation with low n-6/n-3 polyunsaturated fatty acids ratio resulted in dose-dependent decrease of radical oxigen species (-39%), isoprostanes (-25%) and NADPH-oxidase-2 regulation (-51%). n-3 polyunsaturated fatty acids supplemented patients showed significant oxidative stress indexes reduction. CONCLUSIONS: In cirrhosis, n-6/n-3 polyunsaturated fatty acids imbalance up-regulates platelet NADPH-oxidase-2 with ensuing oxidative stress. Further study to evaluate if n-3 supplementation may reduce disease progression is warranted.

Fatty acids and TxA(2) generation, in the absence of platelet-COX-1 activity.

BACKGROUND AND AIMS: Omega-3 fatty acids suppress Thromboxane A(2) (TxA(2)) generation via mechanisms independent to that of aspirin therapy. We sought to evaluate whether baseline omega-3 fatty acid levels influence arachidonic acid proven platelet-cyclooxygenase-1 (COX-1) independent TxA(2) generation (TxA(2) generation despite adequate aspirin use). METHODS AND RESULTS: Subjects with acute myocardial infarction, stable CVD or at high risk for CVD, on adequate aspirin therapy were included in this study. Adequate aspirin action was defined as complete inhibition of platelet-COX-1 activity as assessed by <10% change in light transmission aggregometry to ≥1 mmol/L arachidonic acid. TxA(2) production was measured via liquid chromatography-tandem mass spectrometry for the stable TxA(2) metabolite 11-dehydro-thromboxane B2 (UTxB2) in urine. The relationship between baseline fatty acids, demographics and UTxB(2) were evaluated. Baseline omega-3 fatty acid levels were not associated with UTxB(2) concentration. However, smoking was associated with UTxB(2) in this study. CONCLUSION: Baseline omega-3 fatty acid levels do not influence TxA(2) generation in patients with or at high risk for CVD receiving adequate aspirin therapy. The association of smoking and TxA(2) generation, in the absence of platelet COX-1 activity, among aspirin treated patients warrants further study.

Antioxidant and antiplatelet effects of atorvastatin by Nox2 inhibition.

In recent years, it became evident that reactive oxygen species (ROS) are implicated in the thrombotic process. Statins are lipid-lowering agents able to lower serum cholesterol levels and retard atherosclerotic complications and their clinical sequelae. There is evidence that, among statins, atorvastatin may exert antiplatelet effects by interfering with redox signaling. Recent studies demonstrated that atorvastatin possesses antiplatelet activity via inhibition of platelet formation of NADPH oxidase-derived ROS. This effect results in down-regulation of isoprostanes, which are pro-aggregating molecules, and up-regulation of nitric oxide, which is a platelet inhibitor; such changes occurred immediately after atorvastatin administration and were independent from lipid-lowering property. Experimental and clinical studies documented that statins possess antithrombotic effects, which may account for the reduction of thrombotic-related vascular outcomes. This has been evidenced in different cardiovascular clinical settings such as percutaneous coronary intervention (PCI), myocardial infarction (MI), and venous thrombosis. Future studies should be addressed to analyze if the antiplatelet effect of atorvastatin may preferentially occur at high dosage. Interestingly, the antiplatelet effects of statins could be useful in clinical settings where the clinical efficacy of aspirin and other antiplatelet drugs is still uncertain.

Moderate red wine and grape juice consumption modulates the hydrolysis of the adenine nucleotides and decreases platelet aggregation in streptozotocin-induced diabetic rats.

This study investigated the ex vivo effects of the moderate red wine (RW) and grape juice (GJ) consumption, and the in vitro effects of the resveratrol, caffeic acid, gallic acid, quercetin, and rutin on NTPDase (nucleoside triphosphate diphosphohydrolase), ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP), 5'-nucleotidase, and adenosine deaminase (ADA) activities in platelets and platelet aggregation from streptozotocin-induced diabetic rats. The animals were divided into six groups (n = 10): control/saline, control/GJ, control/RW, diabetic/saline, diabetic/GJ, and diabetic/RW. RW and GJ were administered for 45 days; after this period, the blood was collected for experimental determinations. Results showed that NTPDase, E-NPP, 5'-nucleotidase, and ADA activities as well as platelet aggregation were increased in the diabetic/saline group compared to the control/saline group. Treatment with RW and GJ increased ectonucleotidases activities and prevented the increase in the ADA activity in the diabetic/GJ and diabetic/RW groups. Platelet aggregation was also decreased by the treatment with RW and GJ in the diabetic/GJ and diabetic/RW groups. In the in vitro tests, resveratrol, caffeic acid, and gallic acid increased ATP, ADP, and AMP hydrolysis, while quercetin and rutin decreased the hydrolysis of these nucleotides in platelets of diabetic rats. The ADA activity and platelet aggregation were reduced in platelets of diabetic rats in the presence of all polyphenols tested in vitro. These findings suggest that RW, GJ, and all polyphenols tested were able to modulate the ectoenzymes activities. Moreover, a decrease in the platelet aggregation was observed and it could contribute to the prevention of platelet abnormality, and consequently vascular complications in diabetic state.