Neonatal exposure to sevoflurane impairs preference for social novelty in C57BL/6 female mice at early-adulthood.

Social interaction deficit is core symptom of children with autism, owing to interaction of genetic predisposition and environmental toxins. Sevoflurane could induce neurotoxicity in developing brain in rodent models. This study aims to investigate whether sevoflurane anesthesia in neonatal period could impair social behaviors in male and female mice. Twenty-eight male and thirty-one female mice were randomly assigned to receive 3.0% sevoflurane or 60% oxygen on postnatal day 6. They were tested for social interaction behaviors at one- and two-month-old. In addition, the cortex and hippocampus of neonatal mice undergoing sevoflurane anesthesia were harvested for immunoblotting analysis. As a result, both male and female mice undergoing sevoflurane anesthesia showed strong sociability and weak preference for social novelty at juvenile age. In addition, the male mice developed normal preference for social novelty at early-adulthood; However, the female mice remained weak preference for social novelty. Furthurmore, sevoflurane anesthesia could decrease the levels of PSD95 but not Neuroligin-1 in the hippocampus but not cortex of neonatal mice. In conclusion, sevoflurane anesthesia in neonatal period could disturb development of social memory and impair preference for social novelty in female mice at early-adulthood, with the potential mechanism of decreasing PSD95 expression in the hippocampus of C57BL/6 mice.

Targeting Thymidine Phosphorylase With Tipiracil Hydrochloride Attenuates Thrombosis Without Increasing Risk of Bleeding in Mice.

OBJECTIVE: Current antiplatelet medications increase the risk of bleeding, which leads to a clear clinical need in developing novel mechanism-based antiplatelet drugs. TYMP (Thymidine phosphorylase), a cytoplasm protein that is highly expressed in platelets, facilitates multiple agonist-induced platelet activation, and enhances thrombosis. Tipiracil hydrochloride (TPI), a selective TYMP inhibitor, has been approved by the Food and Drug Administration for clinical use. We tested the hypothesis that TPI is a safe antithrombotic medication. Approach and Results: By coexpression of TYMP and Lyn, GST (glutathione S-transferase) tagged Lyn-SH3 domain or Lyn-SH2 domain, we showed the direct evidence that TYMP binds to Lyn through both SH3 and SH2 domains, and TPI diminished the binding. TYMP deficiency significantly inhibits thrombosis in vivo in both sexes. Pretreatment of platelets with TPI rapidly inhibited collagen- and ADP-induced platelet aggregation. Under either normal or hyperlipidemic conditions, treating wild-type mice with TPI via intraperitoneal injection, intravenous injection, or gavage feeding dramatically inhibited thrombosis without inducing significant bleeding. Even at high doses, TPI has a lower bleeding side effect compared with aspirin and clopidogrel. Intravenous delivery of TPI alone or combined with tissue plasminogen activator dramatically inhibited thrombosis. Dual administration of a very low dose of aspirin and TPI, which had no antithrombotic effects when used alone, significantly inhibited thrombosis without disturbing hemostasis. CONCLUSIONS: This study demonstrated that inhibition of TYMP, a cytoplasmic protein, attenuated multiple signaling pathways that mediate platelet activation, aggregation, and thrombosis. TPI can be used as a novel antithrombotic medication without the increase in risk of bleeding.

Towards the Therapeutic Use of Thrombospondin 1/CD47 Targeting TAX2 Peptide as an Antithrombotic Agent.

OBJECTIVE: TSP-1 (thrombospondin 1) is one of the most expressed proteins in platelet α-granules and plays an important role in the regulation of hemostasis and thrombosis. Interaction of released TSP-1 with CD47 membrane receptor has been shown to regulate major events leading to thrombus formation, such as, platelet adhesion to vascular endothelium, nitric oxide/cGMP (cyclic guanosine monophosphate) signaling, platelet activation as well as aggregation. Therefore, targeting TSP-1:CD47 axis may represent a promising antithrombotic strategy. Approach and Results: A CD47-derived cyclic peptide was engineered, namely TAX2, that targets TSP-1 and selectively prevents TSP-1:CD47 interaction. Here, we demonstrate for the first time that TAX2 peptide strongly decreases platelet aggregation and interaction with collagen under arterial shear conditions. TAX2 also delays time for complete thrombotic occlusion in 2 mouse models of arterial thrombosis following chemical injury, while Thbs1-/- mice recapitulate TAX2 effects. Importantly, TAX2 administration is not associated with increased bleeding risk or modification of hematologic parameters. CONCLUSIONS: Overall, this study sheds light on the major contribution of TSP-1:CD47 interaction in platelet activation and thrombus formation while putting forward TAX2 as an innovative antithrombotic agent with high added-value.

Safety and Functional Pharmacokinetic Profile of APAC, a Novel Intravascular Antiplatelet and Anticoagulant.

ABSTRACT: Vascular intervention-induced platelet and coagulation activation is often managed with a combination of antiplatelets and anticoagulants, with evident benefits, but with a risk of systemic bleeding. Antiplatelet and anticoagulant (APAC) is a dual antiplatelet and anticoagulant heparin bioconjugate, which targets vascular injury sites to act as a local antithrombotic. We assessed the nonclinical safety and exposure of intravenously infused APAC in rats and cynomolgus monkeys by using single-day and 14-day repeat dose toxicology and pharmacodynamic markers. Activated partial thromboplastin time (APTT) was used as a functional surrogate of anticoagulant exposure of APAC. Routine clinical in-life observations were followed by clinical pathology and necropsy. The no-observed-adverse-effect level (NOAEL) in rats for the single APAC dose was 20 mg/kg and for the repeated administration was 10 mg/kg/d. Monkeys tolerated a single APAC dose of 10 mg/kg, although the red blood cell count reduced 16%-19% correlating with tissue hemorrhage at vein puncture and affected muscle sites during handling of the animals. However, after 2-week recovery, all clinical signs were normal. The single dose NOAEL exceeded 3 mg/kg. The repeat administration of 3-6 mg/kg/d of APAC was tolerated, but some clinical signs were observed. The NOAEL for repeated dosing was 0.5 mg/kg/d. APAC prolonged APTT dose-dependently in both species, returning to baseline after 1.5 (<10 mg/kg) or essentially by 6 hours also under repetitive dosing. The toxicology profile supports the safety of an intravenous APAC dose of 0.5 mg/kg/d for possible clinical applications. APTT is an acceptable indicator of the immediate systemic anticoagulation effect of APAC.

Cistanches alleviates sevoflurane-induced cognitive dysfunction by regulating PPAR-γ-dependent antioxidant and anti-inflammatory in rats.

This study aimed to investigate the protective effects and underlying mechanisms of cistanche on sevoflurane-induced aged cognitive dysfunction rat model. Aged (24 months) male SD rats were randomly assigned to four groups: control group, sevoflurane group, control + cistanche and sevoflurane + cistanche group. Subsequently, inflammatory cytokine levels were measured by ELISA, and the cognitive dysfunction of rats was evaluated by water maze test, open-field test and the fear conditioning test. Three days following anaesthesia, the rats were killed and hippocampus was harvested for the analysis of relative biomolecules. The oxidative stress level was indicated as nitrite and MDA concentration, along with the SOD and CAT activity. Finally, PPAR-γ antagonist was used to explore the mechanism of cistanche in vivo. The results showed that after inhaling the sevoflurane, 24- but not 3-month-old male SD rats developed obvious cognitive impairments in the behaviour test 3 days after anaesthesia. Intraperitoneal injection of cistanche at the dose of 50 mg/kg for 3 consecutive days before anaesthesia alleviated the sevoflurane-induced elevation of neuroinflammation levels and significantly attenuated the hippocampus-dependent memory impairments in 24-month-old rats. Cistanche also reduced the oxidative stress by decreasing nitrite and MDA while increasing the SOD and CAT activity. Moreover, such treatment also inhibited the activation of microglia. In addition, we demonstrated that PPAR-γ inhibition conversely alleviated cistanche-induced protective effect. Taken together, we demonstrated that cistanche can exert antioxidant, anti-inflammatory, anti-apoptosis and anti-activation of microglia effects on the development of sevoflurane-induced cognitive dysfunction by activating PPAR-γ signalling.

A Pharmacodynamic Study of CN-218, a Novel Antiplatelet and Antithrombotic Agent Primarily Targeting the P2Y12 Receptor.

PURPOSE: Drugs inhibiting the platelet P2Y12 receptor, such as clopidogrel and prasugrel, are potent antithrombotic agents and are widely used in cardiovascular disease. However, the adverse effects of these drugs have limited their clinical use. For example, clopidogrel resistance occurs in approximately one third of patients, while prasugrel increases the risk of major bleeding. Therefore, new generations of such drugs are of clinical interest. METHODS: In this study, the pharmacodynamics of a new P2Y12 antagonist, CN-218, was compared with that of clopidogrel and prasugrel in rats and mice. The differences between CN-218 and clopidogrel include deuteration of the 7-position methyl carboxylate and the introduction of cinnamate in the 2-position of thiophene. RESULTS: CN-218 had an antiaggregatory efficacy that was at least five times more potent than that of clopidogrel but not as potent as that of prasugrel. It had a significant impact on activated partial thromboplastin time (APTT), whereby the APTT of CN-218-treated rats was approximately 9 s longer than that of the vehicle- or clopidogrel-treated group, while it had no impact on prothrombin time (PT) in rats. CN-218 had a similar potent antithrombotic effect to that of prasugrel and clopidogrel and also reduced the risk of bleeding compared to prasugrel. CONCLUSION: CN-218 may be a promising antithrombotic agent, with potent antiplatelet and significant anticoagulant activity, as well as a lower risk of bleeding compared to clopidogrel and prasugrel.

The activation of high mobility group Box1 and toll-like receptor 4 is involved in clopidogrel-induced gastric injury through p38 MAPK.

Clopidogrel-induced gastric injury is an important clinical problem. However, the exact mechanism was still unclarified. This study aimed to investigate the role of high mobility group box protein 1 (HMGB1) and the toll-like receptor 4 (TLR4) pathway in normal human gastric epithelial (GES-1) cells under clopidogrel exposure. Morphological alterations of the gastric epithelial cells were observed under a microscope. A laser scanning confocal microscope was used to determine the distribution of HMGB1 and TLR4 in clopidogrel-induced injury. MTT was used to compare the viability of GES-1 cell among the pretreated Cli-095 (TLR4 inhibitor) group, pretreated ethyl pyruvate (HMGB1 inhibitor) group, clopidogrel group, and control group. Moreover, expression of the HMGB1, TLR4, tight junction (TJ) proteins occludin and the phosphorylation of the mitogen-activated protein kinases (MAPK) p38 were examined by western blot. We found the expression of HMGB1 and TLR4 in the cytoplasm increased after clopidogrel administration. Besides, inhibited TLR4, which is a receptor of HMGB1, prevented the injury and occludin reducing caused by clopidogrel challenge. Furthermore, blocking HMGB1 or TLR4 activity by ethyl pyruvate (HMGB1 inhibitor) or cli-095(TLR4 inhibitor) can partially diminish the activation of p38MAPK. Gastric mucosal damages observed by clopidogrel challenge are associated with HMGB1, TLR4, through p38MAPK signal pathway.

Trowaglerix Venom Polypeptides As a Novel Antithrombotic Agent by Targeting Immunoglobulin-Like Domains of Glycoprotein VI in Platelet.

OBJECTIVE: Currently prescribed antiplatelet drugs have 1 common side effect-an increased risk of hemorrhage and thrombocytopenia. On the contrary, bleeding defects associated with glycoprotein VI (GPVI) expression deficiency are usually slightly prolonged bleeding times. However, GPVI antagonists are lacking in clinic. APPROACH AND RESULTS: Using reverse-phase high-performance liquid chromatography and sequencing, we revealed the partial sequence of trowaglerix α subunit, a potent specific GPVI-targeting snaclec (snake venom C-type lectin protein). Hexapeptide (Troα6 [trowaglerix a chain hexapeptide, CKWMNV]) and decapeptide (Troα10) derived from trowaglerix specifically inhibited collagen-induced platelet aggregation through blocking platelet GPVI receptor. Computational peptide design helped to design a series of Troα6/Troα10 peptides. Protein docking studies on these decapeptides and GPVI suggest that Troα10 was bound at the lower surface of D1 domain and outer surface of D2 domain, which was at the different place of the collagen-binding site and the scFv (single-chain variable fragment) D2-binding site. The newly discovered site was confirmed by inhibitory effects of polyclonal antibodies on collagen-induced platelet aggregation. This indicates that D2 domain of GPVI is a novel and important binding epitope on GPVI-mediated platelet aggregation. Troα6/Troα10 displayed prominent inhibitory effect of thrombus formation in fluorescein sodium-induced platelet thrombus formation of mesenteric venules and ferric chloride-induced carotid artery injury thrombosis model without prolonging the in vivo bleeding time. CONCLUSIONS: We develop a novel antithrombotic peptides derived from trowaglerix that acts through GPVI antagonism with greater safety-no severe bleeding. The binding epitope of polypeptides on GPVI is novel and important. These hexa/decapeptides have therapeutic potential for developing ideal small-mass GPVI antagonists for arterial thrombogenic diseases.

Evaluation of recombinant factor VIIa, tranexamic acid and desmopressin to reduce prasugrel-related bleeding: A randomised, placebo-controlled study in a rabbit model.

BACKGROUND: Prasugrel is a thienopyridine that inhibits platelet aggregation more rapidly and effectively than clopidogrel, with an increased bleeding risk. OBJECTIVE: The current study aimed to evaluate the efficacy of three nonspecific haemostatic drugs - recombinant activated factor VII (rFVIIa), tranexamic acid and desmopressin (DDAVP) - to limit blood loss after administration of prasugrel in a rabbit model of bleeding while also evaluating any prothrombotic effects. DESIGN: Randomised, placebo-controlled study. SETTING: Faculty of Medicine, University of Geneva, Switzerland, in 2013. ANIMALS: Anaesthetised and artificially ventilated rabbits (n=56). INTERVENTIONS: Animals were randomly allocated to one of five groups: control (placebo-placebo), prasugrel-placebo, rFVIIa (prasugrel-rFVIIa 150 µg kg), tranexamic acid (prasugrel-tranexamic acid 20 mg kg) or DDAVP (prasugrel-DDAVP 1 µg kg). Two hours after an oral prasugrel loading dose (4 mg kg), a stenosis and an injury were inflicted on the carotid artery to induce cyclic flow reductions (CFRs) due to thrombosis. Haemostatic drugs were administered during the ensuing observation period. MAIN OUTCOME MEASURES: Standardised hepatosplenic sections were performed to evaluate the primary endpoint of blood loss, monitored for 15 min. Ear-immersion bleeding time and incidence of CFRs were secondary endpoints. RESULTS: Prasugrel decreased ADP-induced platelet aggregation (light transmission method) from 66 ±â€Š4% (mean ±â€ŠSD) to 41 ±â€Š7% (P < 0.001) and doubled blood loss: 10.7 g (10.1 to12.7) [median (interquartile range)] vs. 20.0 g (17.0 to 24.4), P = 0.003 in the control and prasugrel-placebo groups, respectively. rFVIIa, tranexamic acid and DDAVP reduced neither hepatosplenic blood loss [19.7 g (14.0 to 27.6), 25.2 g (22.6 to 28.7) and 22.9 g (16.8 to 28.8), respectively] nor bleeding time compared with placebo. Regarding safety, rVIIa induced three or more CFRs in 5/12 rabbits, vs. 0/12 in the prasugrel-placebo group (P = 0.037), whereas tranexamic acid and DDAVP did not increase them. CONCLUSION: The three studied haemostatic drugs rFVIIa, tranexamic acid and DDAVP failed to reduce prasugrel-related bleeding in this model. rFVIIa-treated rabbits were more prone to arterial thrombotic events. TRIAL REGISTRATION: NA.

A novel STAT3 inhibitor negatively modulates platelet activation and aggregation.

The signal transducer and activator of transcription 3 (STAT3) plays a critical role in platelet functions. This study sought to understand the effects of the STAT3 inhibitor SC99 on platelet activation and aggregation. Immunoblotting assays were applied to measure the effects of SC99 on the STAT3 signaling pathway. A ChronoLog aggregometer was used to evaluate platelet aggregation. A flow cytometer was used to evaluate P-selectin expression in the presence of SC99. AlamarBlue and Annexin-V staining were used to evaluate platelet viability and apoptosis, respectively. A fluorescence microscope was applied to analyze platelet spreading. SC99 inhibited the phosphorylation of JAK2 and STAT3 in human platelets but had no effects on the phosphorylation of AKT, p65 or Src, all of which are involved in platelet activation. Further studies revealed that SC99 inhibited human platelet aggregation induced by collagen and thrombin in a dose-dependent manner. SC99 inhibited thrombin-induced P-selectin expression and fibrinogen binding to single platelets. Moreover, SC99 inhibited platelet spreading on fibrinogen and clot retraction mediated by outside-in signaling. SC99 inhibited platelet aggregation in mice but it did not significantly prolong the bleeding time. Taken together, the present study revealed that SC99 inhibited platelet activation and aggregation as a STAT3 inhibitor. This agent can be developed as a promising treatment for thrombotic disorders.

Chemical composition, vasorelaxant, antioxidant and antiplatelet effects of essential oil of Artemisia campestris L. from Oriental Morocco.

BACKGROUND: Artemisia campestris L. (Asteraceae) is a medicinal herb traditionally used to treat hypertension and many other diseases. Hence, this study is aimed to analyze the essential oil of A. campestris L (AcEO) and to investigate the antiplatelet, antioxidant effects and the mechanisms of its vasorelaxant effect. METHODS: The chemical composition of AcEO was elucidated using GC/MS analysis. Then, the antioxidant effect was tested on DPPH radical scavenging and on the prevention of ß-carotene bleaching. The antiplatelet effect was performed on the presence of the platelet agonists: thrombin and ADP. The mechanism of action of the vasorelaxant effect was studied by using the cellular blockers specified to explore the involvement of NO/GC pathway and in the presence of calcium channels blockers and potassium channels blockers. RESULTS: AcEO is predominated by the volatiles: spathulenol, ß-eudesmol and p-cymene. The maximal antioxidant effect was obtained with the dose 2 mg/ml of AcEO. The dose 1 mg/ml of AcEO showed a maximum antiplatelet effect of, respectively 49.73% ±9.54 and 48.20% ±8.49 on thrombin and ADP. The vasorelaxation seems not to be mediated via NOS/GC pathway neither via the potassium channels. However, pretreatment with calcium channels blockers attenuated this effect, suggesting that the vasorelaxation is mediated via inhibition of L-type Ca2+ channels and the activation of SERCA pumps of reticulum plasma. CONCLUSION: This study confirms the antioxidant, antiplatelet and vasorelaxant effects of A.campestris L essential oil. However, the antihypertensive use of this oil should be further confirmed by the chemical fractionation and subsequent bio-guided assays.

The role of the Wnt/ß-catenin-Annexin A1 pathway in the process of sevoflurane-induced cognitive dysfunction.

Postoperative cognitive decline (POCD) is a common geriatric complication, and sevoflurane is a widely accepted inducer of POCD. Although the aetiology of POCD is not clear, a breach in the blood-brain barrier (BBB) is involved in early POCD. Annexin A1 has shown protective effects on BBB function. The objective of this study was to investigate both the effects of sevoflurane on the components of the BBB and the underlying mechanism. In vivo treatment with 3.6% sevoflurane for 6 h disrupted BBB components led to fibrinogen invasion and down-regulation of Annexin A1 expression at 24 h after inhalation. The administration of human recombinant Annexin A1 (hr Annexin A1) attenuated the disruption of BBB components, thereby reducing fibrinogen invasion. In addition, the administration of hr Annexin A1 improved cognitive function after the inhalation of 3.6% sevoflurane for 6 h. Moreover, in cultured endothelial cells, 3.6% sevoflurane for 6 h increased GSK-3ß and decreased ß-catenin levels at 24 h after inhalation. The activation/inhibition of the Wnt/ß-catenin signalling pathway attenuated/worsened the sevoflurane-induced decrease in Annexin A1. Our findings indicate that in endothelial cells, treatment with 3.6% sevoflurane for 6 h inhibits the Wnt/ß-catenin signalling pathway, thereby increasing GSK-3ß and decreasing ß-catenin. By inhibiting this pathway, the gas anaesthetic sevoflurane down-regulated Annexin A1, which consequently breached the BBB and induced POCD. We propose the following cascade for sevoflurane-induced cognitive dysfunction: in microvascular endothelial cells, treatment with 3.6% sevoflurane for 6 h inhibits the Wnt/ß-catenin signalling pathway, increasing GSK-3ß and decreasing ß-catenin, which down-regulates the expression of Annexin A1. This cascade leads to a breach in the blood-brain barrier, a process which is involved in the occurrence of early postoperative cognitive decline. Cover Image for this issue: doi: 10.1111/jnc.13314.

Characterization of a novel function-blocking antibody targeted against the platelet P2Y1 receptor.

OBJECTIVE: Platelet hyperactivity is associated with vascular disease and contributes to the genesis of thrombotic disorders. ADP plays an important role in platelet activation and activates platelets through 2 G-protein-coupled receptors, the Gq-coupled P2Y1 receptor (P2Y1R), and the Gi-coupled P2Y12 receptor. Although the involvement of the P2Y1R in thrombogenesis is well established, there are no antagonists that are currently available for clinical use. APPROACH AND RESULTS: Our goal is to determine whether a novel antibody targeting the ligand-binding domain, ie, second extracellular loop (EL2) of the P2Y1R (EL2Ab) could inhibit platelet function and protect against thrombogenesis. Our results revealed that the EL2Ab does indeed inhibit ADP-induced platelet aggregation, in a dose-dependent manner. Furthermore, EL2Ab was found to inhibit integrin GPIIb-IIIa activation, dense and α granule secretion, and phosphatidylserine exposure. These inhibitory effects translated into protection against thrombus formation, as evident by a prolonged time for occlusion in a FeCl3-induced thrombosis model, but this was accompanied by a prolonged tail bleeding time. We also observed a dose-dependent displacement of the radiolabeled P2Y1R antagonist [(3)H]MRS2500 from its ligand-binding site by EL2Ab. CONCLUSIONS: Collectively, our findings demonstrate that EL2Ab binds to and exhibits P2Y1R-dependent function-blocking activity in the context of platelets. These results add further evidence for a role of the P2Y1R in thrombosis and validate the concept that targeting it is a relevant alternative or complement to current antiplatelet strategies.

Antiplatelet pyrazolopyridines derivatives: pharmacological, biochemical and toxicological characterization.

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N'-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3 h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE2 and TXB2 through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.

The effectiveness of platelet supplementation for the reversal of ticagrelor-induced inhibition of platelet aggregation: An in-vitro study.

BACKGROUND: Management of ticagrelor-induced bleeding is challenging, as no antidote is currently available. Platelet transfusion, usually proposed to reverse antiplatelet drugs, has been suggested to be ineffective but few data are available. OBJECTIVE: To assess the efficacy of platelet supplementation to restore platelet aggregation inhibited by ticagrelor. DESIGN: In vitro study. SETTING: Blood samples were obtained from the French Blood Bank Institute. PARTICIPANTS: Healthy blood donors. INTERVENTIONS: Whole blood from healthy donors was spiked with ticagrelor or aspirin (used as a positive control). MAIN OUTCOME MEASURES: Platelet aggregation was investigated with impedance aggregometry on whole blood [expressed in ohms (V)] and light transmission aggregometry (expressed in %) on platelet-rich plasma using ADP or arachidonic acid as agonists for ticagrelor or aspirin, respectively. Platelet supplementation was defined as the addition of washed platelet suspension increasing at least 60% of whole blood platelet count. RESULTS: Ticagrelor (3.25 mM) inhibited ADP-induced platelet aggregation compared with control either in whole blood (2 vs. 13 V, P < 0.05) or in platelet-rich plasma (15 vs. 75% P < 0.05). Aspirin (25 mM) inhibited arachidonic acid-induced aggregation (1 vs. 7.5 V, P < 0.05 in whole blood and 5 vs. 77.5%, P = 0.01 in platelet-rich plasma). Platelet supplementation completely restored arachidonic acid-induced platelet aggregation in whole blood (10 vs. 1 V, P = 0.008) and platelet-rich plasma (73 vs. 5%, P < 0.01) in aspirin-treated samples, whereas it failed to correct ADP-induced aggregation (2 vs. 2 V in whole blood and 13.5 vs. 15% in platelet-rich plasma, P > 0.05) in ticagrelor-treated samples. We also report a case of a ticagrelor-treated patient in whom platelet transfusion failed to restore ADP-induced platelet aggregation. CONCLUSION: Platelet supplementation restored platelet aggregation in aspirin-spiked but not in ticagrelor-spiked samples. These results do not support the use of platelet transfusion to reverse the effects of ticagrelor.

The mechanism and risk factors of clopidogrel-induced liver injury.

CONTEXT AND OBJECTIVE: Clopidogrel (CLP) is a prodrug which is widely used as a platelet aggregation inhibitor. Hepatotoxicity is rare but a potentially serious adverse reaction that is associated with CLP. Thiophene in CLP (the thienopyridine derivative) is a group that is easily oxidated by cytochrome P450 enzymes (CYP450s) to generate reactive metabolites (RMs), it may be implicated in the mechanism of CLP-induced hepatotoxicity. CYP2C19 and CYP2B6 are important CYP450s involved in the metabolism and activation of CLP, and the aim of this study is to investigate whether the metabolites of CYP2C19 and CYP2B6 are associated with the CLP-induced liver injury. METHOD: Primary rat hepatocytes are applied to evaluate the hepatotoxicity of CLP. Glutathione-depleted mouse model is used to evaluate whether this toxicity of CLP is metabolized by CYP450s. We also used HepG2 cells co-incubated with recombinant CYP2B6 and CYP2C19 enzymes to further assess whether the metabolites of CYP2C19 and CYP2B6 are associated with the CLP-induced hepatocellular toxicity. RESULT: CLP in high dose (100 µM and 300 µM) showed cytotoxicity in primary rat hepatocytes assay. Administration of CLP with l-buthionine-S, R-sulfoxinine (BSO) for seven days enhanced the liver injury of CLP. The level of ALT, AST and TBIL in plasma increased significantly, and the histopathological results showed the obvious liver injury; Pretreatment of 1-aminobenzotriazole, a nonspecific inhibitor of CYP450s, suppressed CLP-induced hepatotoxicity; CLP showed a dose-dependent toxicity in HepG2/CYP2C19 enzyme and HepG2/CYP2B6 enzyme models. CONCLUSION: High activities of CYP2C19 and CYP2B6 are the risk factors for hepatocellular toxicity of CLP.

Aspirin and Clopidogrel Inhibit Aneurysm Healing after HydroCoil Implantation in External Carotid Artery Aneurysm Model.

PURPOSE: To understand whether the use of antiplatelet agents leads to less intra-aneurismal tissue formation following coil implantation in a rat end-pouch external carotid artery (ECA) aneurysm model. METHODS: End-pouch ECA aneurysms were created in adult rats and were then embedded with either platinum or HydroCoils. Rats were treated either with aspirin, clopidogrel, aspirin + clopidogrel, or saline for 2 weeks after coil implantation. At 2 weeks after coil implantation, rats were sacrificed and the aneurysm pouch was removed for histological and immunohistochemical analysis. A blinded single observer calculated the percentage of the organized area and the residual length of elastic lamina within the aneurysm. Student's t-test was used to compare data from image analysis between the different groups. RESULTS: Within the platinum group, the organized tissue area was not affected by antiplatelet administration (aspirin versus saline, P = .83; clopidogrel versus saline, P = .46; aspirin + clopidogrel versus saline, P = .54). For the HydroCoil group, the organized tissue area was significantly reduced (aspirin versus saline, P = .02; clopidogrel versus saline, P = .04; aspirin + clopidogrel versus saline, P = .02) in rats treated with antiplatelet agents; however, no difference (aspirin versus clopidogrel, P = .8; aspirin versus aspirin + clopidogrel, P = .3; clopidogrel versus aspirin + clopidogrel, P = .5) was found among type or combination of antiplatelets administered. HydroCoil-treated aneurysms had a similar number of macrophages compared to the platinum group (P = .3819); however, the HydroCoil group had significant suppression of macrophages in the groups treated with combined antiplatelets (P = .02). CONCLUSION: Following HydroCoil implantation, the area of organized tissue is diminished significantly in a rat end-pouch ECA aneurysm model treated with antiplatelets.

Comparison of cytotoxic and anti-platelet activities of polyphenolic extracts from Arnica montana flowers and Juglans regia husks.

Polyphenolic compounds of plant origin are well known to be beneficial to human health: they exert protective effects on haemostasis and have a particular influence on blood platelets. However, the anti-platelet properties of polyphenolic compounds observed so far have not been weighed against their potential cytotoxic action against platelets. The aim of this study was to demonstrate that anti-platelet and cytotoxic effects on blood platelets may interfere and therefore, may often lead to confusion when evaluating the properties of plant extracts or other agents towards blood platelets. The anti-platelet and cytotoxic in vitro effects of plant extracts obtained from the husks of walnuts (J. regia) and flowers of arnica (A. montana) on platelet reactivity and viability were examined. Platelet function was assessed using standard methods (flow cytometry: P-selectin expression, activation of GPIIbIIIa complex, vasodilator-stimulated phosphoprotein, VASP index; turbidimetric and impedance aggregometry) and newly set assays (flow cytometric monitoring of platelet cytotoxicity). The results reveal that none of the studied plant extracts demonstrated cytotoxicity towards blood platelets. The phenolic acid-rich extract of A. montana (7.5 and 15 µg/ml) significantly reduced the ADP-induced aggregation in both whole blood and PRP, and decreased the platelet reactivity index (PRI; VASP phosphorylation) in whole blood, while showing excellent antioxidant capacity. The extract of J. regia husks significantly reduced ADP-induced platelet aggregation in whole blood when applied at 7.5 µg/ml, and only slightly decreased the PRI at 15 µg/ml. Both examined extracts suppressed platelet hyper-reactivity, and such influence did not interfere with cytotoxic effects of the extracts. Thus, its high polyphenol content, excellent antioxidant capacity and distinct anti-platelet properties, in combination with its lack of toxicity, make the extract of A. montana flowers a possible candidate as an anti-platelet agent or a compounding diet supplement.

Bioactivation potential of thiophene-containing drugs.

Thiophene is a five-membered, sulfur-containing heteroaromatic ring commonly used as a building block in drugs. It is considered to be a structural alert, as its metabolism can lead to the formation of reactive metabolites. Thiophene S-oxides and thiophene epoxides are highly reactive electrophilic thiophene metabolites whose formation is cytochrome P450-dependent. These reactive thiophene-based metabolites are quite often responsible for drug-induced hepatotoxicity. Tienilic acid is an example of a thiophene-based drug that was withdrawn from the market after only a few months of use, due to severe cases of immune hepatitis. However, inclusion of the thiophene moiety in drugs does not necessarily result in toxic effects. The presence of other, less toxic metabolic pathways, as well as an effective detoxification system in our body, protects us from the bioactivation potential of the thiophene ring. Thus, the presence of a structural alert itself is insufficient to predict a compound's toxicity. The question therefore arises as to which factors significantly influence the toxicity of thiophene-containing drugs. There is no easy way to answer this question. However, the findings presented here indicate that, for a number of reasons, daily dose and alternative metabolic pathways are important factors when predicting toxicity and will therefore be discussed together with examples.

Gastric-sparing nitric oxide-releasable 'true' prodrugs of aspirin and naproxen.

Nitric oxide-releasing non-steroidal anti-inflammatory drugs (NO-NSAIDs) are gaining attention as potentially gastric-sparing NSAIDs. Herein, we report a novel class of '1-(nitrooxy)ethyl ester' group-containing NSAIDS as efficient NO releasing 'true' prodrugs of aspirin and naproxen. While an aspirin prodrug exhibited comparable oral bioavailability and antiplatelet activity (i.e., TXB2 inhibition) to those of aspirin, a naproxen prodrug exhibited better bioavailability than naproxen. These promising NO-NSAIDs protected experimental rats from gastric damage. We therefore believe that these promising NO-NSAIDs could represent a new class of potentially 'Safe NSAIDs' for the treatment of arthritic pain, inflammation and cardiovascular disorders in the case of NO-aspirin.