ABSTRACT
Alibertia edulis leaf extract is commonly used in folk medicine, with rutin caffeic and vanillic acids being its major compounds. The Alibertia edulis leaf extract was investigated for its pharmacological effects via platelet aggregation, calcium mobilization, cyclic nucleotides levels, vasodilator-stimulated phosphoprotein Ser157 and Ser239 and protein kinase Cß2 phosphorylation, thromboxane B2, cyclooxygenases 1 and 2, docking and molecular dynamics. Alibertia edulis leaf extract significantly inhibited (100-1000 µg mL-1) platelet aggregation induced by different agonists. Arachidonic acid increased levels of calcium and thromboxane B2, phosphorylation of vasodilator-stimulated phosphoprotein Ser157 and Ser239, and protein kinase Cß, which were significantly reduced by Alibertia edulis leaf extract, rutin, and caffeic acid as well mixtures of rutin/caffeic acid. Cyclooxygenase 1 activity was inhibited for Alibertia edulis leaf extract, rutin and caffeic acid. These inhibitions were firsrtly explored by specific stabilization of rutin and caffeic acid compared to diclofenac at the catalytic site from docking score and free-energy dissociation profiles. Then, simulations detailed the rutin interactions close to the heme group and Tyr385, responsible for catalyzing the conversion of arachidonic acid to its products. Our results reveal the antiplatelet aggregation properties of Alibertia edulis leaf extract, rutin and caffeic acid providing pharmacological information about its origin from cyclooxygenase 1 inhibition and its downstream pathway.
Subject(s)
Gene Expression Regulation/drug effects , Plant Extracts/pharmacology , Platelet Aggregation/drug effects , Prostaglandin-Endoperoxide Synthases/metabolism , Rubiaceae/chemistry , Thromboxanes/antagonists & inhibitors , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/administration & dosage , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid/pharmacology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/pharmacology , Animals , Arachidonic Acid/administration & dosage , Arachidonic Acid/pharmacology , Calcium/metabolism , Collagen/administration & dosage , Collagen/pharmacology , Cyclooxygenase Inhibitors , Humans , Plant Extracts/chemistry , Plant Leaves/chemistry , Thromboxanes/genetics , Thromboxanes/metabolism , ZebrafishABSTRACT
BACKGROUND: Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. Recent studies have reported the close association between cognitive function in AD and purinergic receptors in the central nervous system. In the current study, we investigated the effect of CD73 inhibitor α, ß-methylene ADP (APCP) on cognitive impairment of AD in mice, and to explore the potential underlying mechanisms. RESULTS: We found that acute administration of Aß142 (i.c.v.) resulted in a significant increase in adenosine release by using microdialysis study. Chronic administration of APCP (10, 30 mg/kg) for 20 d obviously mitigated the spatial working memory impairment of Aß142-treated mice in both Morris water maze (MWM) test and Y-maze test. In addition, the extracellular adenosine production in the hippocampus was inhibited by APCP in Aß-treated mice. Further analyses indicated expression of acetyltransferase (ChAT) in hippocampus of mice of was significantly reduced, while acetylcholinesterase (AChE) expression increased, which compared to model group. We observed that APCP did not significantly alter the NLRP3 inflammasome activity in hippocampus, indicating that anti-central inflammation seems not to be involved in APCP effect. CONCLUSIONS: In conclusion, we report for the first time that inhibition of CD73 by APCP was able to protect against memory loss induced by Aß142 in mice, which may be due to the decrease of CD73-driven adenosine production in hippocampus. Enhancement of central cholinergic function of the central nervous system may also be involved in the effects of APCP.
Subject(s)
Animals , Male , Mice , Adenosine Diphosphate/analogs & derivatives , Neurodegenerative Diseases/prevention & control , Hippocampus , Nucleotidases/antagonists & inhibitors , Acetylcholinesterase , Adenosine Diphosphate/administration & dosage , Alzheimer Disease/prevention & control , Morris Water Maze Test , Mice, Inbred C57BLABSTRACT
Extracellular ATP activates inflammatory responses to tissue injury. It is also implicated in establishing lasting network hyperexcitability in the brain by acting upon independent receptor systems. Whereas the fast-acting P2X channels have well-established roles driving neuroinflammation and increasing hyperexcitability, the slower-acting metabotropic P2Y receptors have received much less attention. Recent studies of P2Y1 receptor function in seizures and epilepsy have produced contradictory results, suggesting that the role of this receptor during seizure pathology may be highly sensitive to context. Here, by using male mice, we demonstrate that the metabotropic P2Y1 receptor mediates either proconvulsive or anticonvulsive responses, dependent on the time point of activation in relation to the induction of status epilepticus. P2Y1 deficiency or a P2Y1 antagonist (MRS2500) administered before a chemoconvulsant, exacerbates epileptiform activity, whereas a P2Y1 agonist (MRS2365) administered at this time point is anticonvulsant. When these drugs are administered after the onset of status epilepticus, however, their effect on seizure severity is reversed, with the antagonist now anticonvulsant and the agonist proconvulsant. This result was consistent across two different mouse models of status epilepticus (intra-amygdala kainic acid and intraperitoneal pilocarpine). Pharmacologic P2Y1 blockade during status epilepticus reduces also associated brain damage, delays the development of epilepsy and, when applied during epilepsy, suppresses spontaneous seizures, in mice. Our data show a context-specific role for P2Y1 during seizure pathology and demonstrate that blocking P2Y1 after status epilepticus and during epilepsy has potent anticonvulsive effects, suggesting that P2Y1 may be a novel candidate for the treatment of drug-refractory status epilepticus and epilepsy.SIGNIFICANCE STATEMENT This is the first study to fully characterize the contribution of a metabotropic purinergic P2Y receptor during acute seizures and epilepsy. The findings suggest that targeting P2Y1 may offer a potential novel treatment strategy for drug-refractory status epilepticus and epilepsy. Our data demonstrate a context-specific role of P2Y1 activation during seizures, switching from a proconvulsive to an anticonvulsive role depending on physiopathological context. Thus, our study provides a possible explanation for seemingly conflicting results obtained between studies of different brain diseases where P2Y1 targeting has been proposed as a potential treatment strategy and highlights that the timing of pharmacological interventions is of critical importance to the understanding of how receptors contribute to the generation of seizures and the development of epilepsy.
Subject(s)
Brain/physiopathology , Epilepsy/physiopathology , Receptors, Purinergic P2Y1/physiology , Status Epilepticus/physiopathology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/analogs & derivatives , Animals , Brain/drug effects , Deoxyadenine Nucleotides/administration & dosage , Disease Models, Animal , Electroencephalography , Male , Mice, Inbred C57BL , Mice, Knockout , Purinergic P2Y Receptor Agonists/administration & dosage , Purinergic P2Y Receptor Antagonists/administration & dosage , Receptors, Purinergic P2Y1/geneticsABSTRACT
CD73 inhibitors are considered to be used in the therapies of melanomas, gliomas or breast cancer. However, little is known about their pharmacology and kinetics in mouse experimental models. Thus, this study is aimed to define a metabolic stability and elimination of the adenosine diphosphate (ADP) analog - α,ß-Methylene-ADP also known as AOPCP in BALB/c mice. The process starts with an intravenous injection of AOPCP, next blood and serum samples are collected. Urine samples are possessed by a bladder puncture. Mice aortas are dissected for the e5NT activity evaluation. In order to assess the AOPCP degradation, the incubation of AOPCP in mice blood and plasma is performed. The AOPCP concentration as well as the activity of e5NT were analyzed with the reverse phase-high pressure liquid chromatography (RP-HPLC). The study shows that after 60 minutes of the 20 mg/kg intravenous injection of AOPCP (body weight dose), the concentration of AOPCP in blood diminished rapidly from 38.6 ± 5.0 µM (measured 5 minutes after the injection) to 6.4 ± 1.4 µM. Interestingly, it is also noted that 60 minutes after the incubation of mice blood samples the AOPCP concentration decreases from 50 µM to 30.0 ± 0.3 µM. This study demonstrates a significant and quick decrease of AOPCP concentration in BALB/c mice blood after the intravenous injection and in isolated blood sample incubation. These findings emphasize the quick elimination of AOPCP as well as its instability and suggest that the AOPCP concentration have to be accurately and frequently monitored in all the studies that address its clinical application.
Subject(s)
5'-Nucleotidase/antagonists & inhibitors , Adenosine Diphosphate/analogs & derivatives , Antineoplastic Agents/pharmacokinetics , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/blood , Adenosine Diphosphate/pharmacokinetics , Adenosine Diphosphate/urine , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/blood , Antineoplastic Agents/urine , Dose-Response Relationship, Drug , Injections, Intravenous , Mice, Inbred BALB CABSTRACT
PURPOSE: Altered platelet aggregability has been implicated in the pathogenesis of glaucoma. This study aims to investigate the anti-platelet potential of intraocular pressure lowering drops, with the possibility of establishing it as an additional mechanism of anti-glaucomatous action. MATERIALS AND METHODS: The anti-aggregating effects of a series of anti-glaucomatous eye drops were determined on human platelets in the platelet aggregation model, using four known aggregating factors (platelet activating factor [PAF], adenosine diphosphate [ADP], thrombin receptor-activating peptide [TRAP], and arachidonic acid [AA]). RESULTS: Almost all of the tested samples inhibited platelet aggregation induced by PAF, ADP, TRAP, and AA, except for Alphagan, which did not demonstrate inhibition of ADP- and TRAP-induced aggregation at a wide range of concentrations. Trusopt, Betoptic, and Azarga eye drops were the most potent inhibitors of all four aggregating factors, while Alphagan was the least potent (P<0.05). CONCLUSION: This study shows that anti-glaucomatous eye drops possess anti-platelet effects, and this was shown for the first time by experimenting on human platelets.
Subject(s)
Adenosine Diphosphate/pharmacology , Arachidonic Acid/pharmacology , Glaucoma/drug therapy , Mediator Complex/pharmacology , Ophthalmic Solutions/pharmacology , Platelet Activating Factor/pharmacology , Adenosine Diphosphate/administration & dosage , Arachidonic Acid/administration & dosage , Blood Platelets/drug effects , Humans , Mediator Complex/administration & dosage , Ophthalmic Solutions/administration & dosage , Platelet Activating Factor/administration & dosage , Platelet Aggregation/drug effectsABSTRACT
BACKGROUND: Although scientific evidence is limited, clopidogrel is frequently used as prophylaxis for arterial thromboembolism in cats with hypertrophic cardiomyopathy (HCM). OBJECTIVES: Evaluating effects of clopidogrel therapy in asymptomatic cats with HCM on (1) conventional whole blood aggregation (WBA), (2) alternative platelet aggregation assessed with tubes of the Plateletworks® assay and (3) standard coagulation parameters. ANIMALS AND METHODS: Prospective, randomized, double-blind, placebo-controlled pilot study. Fourteen asymptomatic HCM cats were randomly allocated to receive placebo (n = 5) or clopidogrel (18.75 mg/cat q24h, n = 9) as part of a larger study. Aggregation responses (to 20 µM adenosine diphosphate (ADP) and 10 µg/ml collagen) in WBA and the Plateletworks® assay and standard coagulation parameters were evaluated at baseline and after seven days of therapy. RESULTS: Clopidogrel therapy significantly reduced aggregation responses to ADP and collagen in the Plateletworks® agonists tubes (ADP and collagen: P < 0.001), but did not significantly reduce aggregation responses to ADP and collagen in the WBA technique (ADP: P = 0.07, collagen: P = 0.30). Clopidogrel therapy did not show a significant effect on prothrombin time, activated partial thromboplastin time, antithrombin, D-dimers and fibrinogen concentrations. CONCLUSION AND CLINICAL IMPORTANCE: Clopidogrel therapy at a dose of 18.75 mg/cat q24h for seven days causes a significant decrease in in vitro platelet aggregation evaluated with the Plateletworks® assay, without affecting standard coagulation parameters in cats with asymptomatic HCM.
Subject(s)
Blood Coagulation/drug effects , Cardiomyopathy, Hypertrophic/veterinary , Cat Diseases/drug therapy , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Ticlopidine/analogs & derivatives , Adenosine Diphosphate/administration & dosage , Animals , Asymptomatic Diseases/therapy , Cardiomyopathy, Hypertrophic/drug therapy , Cardiomyopathy, Hypertrophic/etiology , Cat Diseases/etiology , Cats , Clopidogrel , Double-Blind Method , Female , Male , Pilot Projects , Platelet Aggregation Inhibitors/adverse effects , Platelet Aggregation Inhibitors/therapeutic use , Prospective Studies , Ticlopidine/adverse effects , Ticlopidine/pharmacology , Ticlopidine/therapeutic useABSTRACT
It is known that adenosine 5'-triphosphate (ATP) is released along with the neurotransmitter acetylcholine (ACh) from motor nerve terminals. At mammalian neuromuscular junctions (NMJs), we have previously demonstrated that ATP is able to decrease ACh secretion by activation of P2Y receptors coupled to pertussis toxin-sensitive Gi/o protein. In this group, the receptor subtypes activated by adenine nucleotides are P2Y12 and P2Y13. Here, we investigated, by means of pharmacological and immunohistochemical assays, the P2Y receptor subtype that mediates the modulation of spontaneous and evoked ACh release in mouse phrenic nerve-diaphragm preparations. First, we confirmed that the preferential agonist for P2Y12-13 receptors, 2-methylthioadenosine 5'-diphosphate trisodium salt hydrate (2-MeSADP), reduced MEPP frequency without affecting MEPP amplitude as well as the amplitude and quantal content of end-plate potentials (EPPs). The effect on spontaneous secretion disappeared after the application of the selective P2Y12-13 antagonists AR-C69931MX or 2-methylthioadenosine 5'-monophosphate triethylammonium salt hydrate (2-MeSAMP). 2-MeSADP was more potent than ADP and ATP in reducing MEPP frequency. Then we demonstrated that the selective P2Y13 antagonist MRS-2211 completely prevented the inhibitory effect of 2-MeSADP on MEPP frequency and EPP amplitude, whereas the P2Y12 antagonist MRS-2395 failed to do this. The preferential agonist for P2Y13 receptors inosine 5'-diphosphate sodium salt (IDP) reduced spontaneous and evoked ACh secretion and MRS-2211 abolished IDP-mediated modulation. Immunohistochemical studies confirmed the presence of P2Y13 but not P2Y12 receptors at the end-plate region. Disappearance of P2Y13 receptors after denervation suggests the presynaptic localization of the receptors. We conclude that, at motor nerve terminals, the Gi/o protein-coupled P2Y receptors implicated in presynaptic inhibition of spontaneous and evoked ACh release are of the subtype P2Y13. This study provides new insights into the types of purinergic receptors that contribute to the fine-tuning of cholinergic transmission at mammalian neuromuscular junction.
Subject(s)
Acetylcholine/metabolism , Miniature Postsynaptic Potentials , Neuromuscular Junction/metabolism , Receptors, Purinergic P2/physiology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/analogs & derivatives , Adenosine Monophosphate/administration & dosage , Adenosine Monophosphate/analogs & derivatives , Animals , Female , Male , Mice , Neuromuscular Junction/drug effects , Purinergic P2 Receptor Agonists/administration & dosage , Purinergic P2Y Receptor Antagonists/administration & dosage , Receptors, Purinergic P2Y12/physiology , Thionucleotides/administration & dosageABSTRACT
OBJECTIVES: Fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes can accumulate via dodecapeptide HHLGGAKQAGDV interactions at bleeding sites where they release adenosine 5'-diphosphate that is rapidly metabolized to adenosine, which has tissue-protective effects. We investigated the efficacy of fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes to treat blast lung injury, with a focus on adenosine signaling. DESIGN: Controlled animal study. SETTING: University research laboratory. SUBJECTS: Adult male C57BL/6 mice. INTERVENTIONS: Mice were pretreated with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes, dodecapeptide HHLGGAKQAGDV-(phosphate-buffered saline)-liposomes, adenosine 5' diphosphateliposomes, or phosphate-buffered saline-liposomes. Five minutes after treatment the mice received a single laser-induced shock wave (1.8 J/cm) that caused lethal blast lung injury, and their survival times and lung injuries were then assessed. We also evaluated the therapeutic effect of posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes or H12-(phosphate-buffered saline)-liposomes 1 minute after laser-induced shock wave exposure. To examine the effect of adenosine signaling, adenosine A2A receptor (ZM241385) or adenosine A2B receptor (PSB 1115) antagonists were administered to the mice 1 hour before the pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes that was followed by laser-induced shock wave exposure. MEASUREMENTS AND MAIN RESULTS: Pre- and posttreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes significantly increased mouse survival [fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes: 58% survival vs H12-(phosphate-buffered saline)-liposomes: 8%; p < 0.05 (posttreatment)] and mitigated pulmonary tissue damage/hemorrhage and neutrophil accumulation after laser-induced shock wave exposure. fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes accumulated at pulmonary vessel injury sites after laser-induced shock wave exposure with both pre- and posttreatment. Furthermore, pretreatment with fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes reduced albumin and macrophage inflammatory protein-2 levels in bronchoalveolar lavage fluid. Although fibrinogen γ-chain (dodecapeptide HHLGGAKQAGDV)-coated adenosine 5'-diphosphate-encapsulated liposomes pretreatment did not affect blood coagulation activity in the injured mice, its beneficial effect on blast lung injury was significantly abrogated by A2A or A2B adenosine receptor antagonists (A2A antagonist: 17% survival; A2B antagonist: 33% vs dimethyl sulfoxide control: 80%; p < 0.05, respectively). CONCLUSIONS: Fibrinogen γ-chain (dodecapeptide HHLGGAKQA GDV)-coated adenosine 5'-diphosphate-encapsulated liposomes may be effective against blast lung injury by promoting tissue-protective adenosine signaling and could represent a novel controlled-release drug delivery system.
Subject(s)
Adenosine Diphosphate/administration & dosage , Blast Injuries/therapy , Fibrinogen/administration & dosage , Lung Injury/therapy , Platelet Aggregation Inhibitors/administration & dosage , Adenosine/physiology , Animals , Blast Injuries/etiology , Blast Injuries/pathology , Disease Models, Animal , Liposomes , Lung Injury/etiology , Lung Injury/pathology , Male , Mice , Mice, Inbred C57BL , Oligopeptides/administration & dosage , Signal TransductionABSTRACT
BACKGROUND: High-residual-on-treatment platelet reactivity still represents a challenging issue, potentially vanishing the benefits of dual antiplatelet treatment in patients with coronary artery disease. However, very few is known on the determinants of suboptimal response to antiplatelet agents. Recent interests have emerged on the potential prothrombotic effect of parathyroid hormone (PTH). Therefore, the aim of the present study was to assess the impact of parathyroid hormone (PTH) on platelet reactivity in patients receiving DAPT after an acute coronary syndrome or PCI. METHODS: Patients treated with DAPT (ASA and clopidogrel or ticagrelor) were scheduled for platelet function assessment at 30- to 90-days postdischarge. By whole blood impedance aggregometry, HRPR was considered for ASPI test >862 AU*min (for ASA) and ADP test values ≥417 AU*min (for ADP antagonists). RESULTS: We included 362 patients on DAPT, 125 (34.5%) receiving clopidogrel, and 237 (65.5%) on ticagrelor. Patients were divided according to PTH quartiles values (<45.8; 45.8-60.3; 60.4-88; ≥88.1 pg/mL). Higher PTH was associated with older age (P = 0.001); renal failure (P < 0.001), higher HDL cholesterol (P = 0.006) and creatinine (P < 0.001) and lower 25-OH cholecalciferol (P < 0.001). Suboptimal response to ASA was infrequent (2.8%), and not influenced by the levels of PTH (P = 0.57). ADP-mediated platelet aggregation was significantly increased in patients with higher PTH (P = 0.03), with an absolute increase in the prevalence of HRPR to ADP antagonists for higher PTH (24.7% vs. 40%, P = 0.007 for 4th vs. 1-3rd quartiles, adjusted OR[95%CI] = 2.04[1.14-3.64], P = 0.02). By the use of the ROC curve, we identified PTH levels above 96.7 pg/mL as the best predictor of HRPR with ADP antagonists (adjusted OR[95%CI] = 2.52[1.31-4.87], P = 0.006). Higher rate of HRPR was confirmed for PTH >96.7 pg/mL among the subgroup of patients on clopidogrel (51.5 vs. 85.7%, P = 0.001; adjusted OR[95%CI] = 12.5[2.6-60.9], P = 0.002), but not among ticagrelor-treated patients (11.3 vs. 16.7%, P = 0.31; adjusted OR[95%CI] = 1.55[0.56-4.6], P = 0.42). CONCLUSION: In patients receiving dual antiplatelet therapy for coronary artery disease, higher PTH levels are associated with an increased ADP-mediated platelet reactivity and suboptimal response to clopidogrel, especially for values above 96.7 pg/mL, while not influencing the effectiveness of ASA and ticagrelor.
Subject(s)
Acute Coronary Syndrome/therapy , Adenosine/analogs & derivatives , Aspirin/therapeutic use , Coronary Artery Disease/therapy , Homeodomain Proteins/blood , Platelet Aggregation Inhibitors/therapeutic use , Platelet Aggregation/drug effects , Ticlopidine/analogs & derivatives , Transcription Factors/blood , Acute Coronary Syndrome/blood , Adenosine/adverse effects , Adenosine/therapeutic use , Adenosine Diphosphate/administration & dosage , Aged , Aspirin/adverse effects , Biomarkers/blood , Clopidogrel , Coronary Artery Disease/blood , Databases, Factual , Drug Resistance , Drug Therapy, Combination , Female , Humans , Logistic Models , Male , Middle Aged , Multivariate Analysis , Odds Ratio , Percutaneous Coronary Intervention , Platelet Aggregation Inhibitors/adverse effects , Platelet Function Tests , Risk Factors , Ticagrelor , Ticlopidine/adverse effects , Ticlopidine/therapeutic use , Time Factors , Treatment Outcome , Up-RegulationABSTRACT
The study was carried out to evaluate variability of morpho-genetic potential of plasma rich in thrombocytes. The analysis of ADP-induced aggregation of thrombocytes separated from blood samples of 32 male volunteers aged 18-42 (22±3) was applied. The bimodal character of reaction of thrombocytes to ADP with peaks of aggregation in range of 30-50% and 60-80%. Besides individual differences in range of aggregation differences in time of reaching peak of aggregation and entering plateau were established. In some of volunteers the reversible character of aggregation curve reflecting absence of phase of thrombogenesis stabilization was established. The characteristics of aggregation curve, associated with activation of signal system, launching of degranulation of thrombocytes and exposition of GPIIaIIIb can reflect velocity and effectiveness of secretion of granules of thrombocytes at application of standard doses of stimulants of aggregation. These specifics substantiate necessity of preliminary evaluation of aggregation response of thrombocytes for prognosis of effectiveness of degranulation and release out of granules of thrombocytes biologically active molecules determining morpho-genetic potential of plasma rich with thrombocytes.
Subject(s)
Blood Platelets/pathology , Platelet Aggregation/physiology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/metabolism , Adolescent , Adult , Blood Platelets/drug effects , Humans , Male , Platelet Aggregation/drug effects , Reference Standards , Young AdultABSTRACT
BACKGROUND: The marine-derived kinase inhibitor fascaplysin down-regulates the PI3K pathway in cancer cells. Since this pathway also plays an essential role in platelet signaling, we herein investigated the effect of fascaplysin on thrombosis. METHODS: Fascaplysin effects on platelet activation, platelet aggregation and platelet-leukocyte aggregates (PLA) formation were analyzed by flow cytometry. Mouse dorsal skinfold chambers were used to determine in vivo the effect of fascaplysin on photochemically induced thrombus formation and tail-vein bleeding time. RESULTS: Pre-treatment of platelets with fascaplysin reduced the activation of glycoprotein (GP)IIb/IIIa after protease-activated receptor-1-activating peptide (PAR-1-AP), adenosine diphosphate (ADP) and phorbol-12-myristate-13-acetate (PMA) stimulation, but did not markedly affect the expression of P-selectin. This was associated with a decreased platelet aggregation. Fascaplysin also decreased PLA formation after PMA but not PAR-1-AP and ADP stimulation. This may be explained by an increased expression of CD11b on leukocytes in PAR-1-AP- and ADP-treated whole blood. In the dorsal skinfold chamber model of photochemically induced thrombus formation, fascaplysin-treated mice revealed a significantly extended complete vessel occlusion time when compared to controls. Furthermore, fascaplysin increased the tail-vein bleeding time. CONCLUSION: Fascaplysin exerts anti-thrombotic activity, which represents a novel mode of action in the pleiotropic activity spectrum of this compound.
Subject(s)
Indoles/pharmacology , Platelet Activation/drug effects , Platelet Aggregation/drug effects , Thrombosis/drug therapy , Adenosine Diphosphate/administration & dosage , Animals , Disease Models, Animal , Flow Cytometry , Leukocytes/metabolism , Mice , Mice, Inbred BALB C , Oligopeptides/administration & dosage , Platelet Glycoprotein GPIIb-IIIa Complex/metabolism , Tetradecanoylphorbol Acetate/administration & dosage , Thrombosis/pathologyABSTRACT
A preclinical study of dodecapeptide ((400)HHLGGAKQAGDV(411)) (H12)-(adenosine diphosphate, ADP)-liposomes for use as a synthetic platelet (PLT) substitute under conditions of red blood cell (RBC) transfusion-induced dilutional thrombocytopenia is limited to pharmacological effect. In this study, the pharmacokinetics of H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were evaluated. As evidenced by the use of (14) C, (3) H double-radiolabeled H12-(ADP)-liposomes in which the encapsulated ADP and liposomal membrane were labeled with (14) C and (3) H, respectively, the H12-(ADP)-liposomes remained intact in the blood circulation for up to 3 h after injection, and were mainly distributed to the liver and spleen. The encapsulated ADP was mainly eliminated in the urine, whereas the outer membrane was mainly eliminated in the feces. These successive pharmacokinetic properties of the H12-(ADP)-liposomes in RBC transfusion-induced dilutional thrombocytopenic rats were similar to those in healthy rats, except for the shorter retention time in the circulation. When H12-(ADP)-liposomes were repeatedly injected into RBC transfusion-induced dilutional thrombocytopenic rats at intervals of 5 days at a dose of 10 mg lipids/kg, the second dose of injected H12-(ADP)-liposomes were rapidly cleared from the circulation, namely, via the accelerated blood clearance phenomenon. These novel pharmacokinetic findings provide useful information for the further development of H12-(ADP)-liposomes as a PLT substitute.
Subject(s)
Adenosine Diphosphate/pharmacokinetics , Blood Platelets/cytology , Blood Substitutes/pharmacokinetics , Erythrocyte Transfusion/adverse effects , Oligopeptides/pharmacokinetics , Thrombocytopenia/etiology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/analogs & derivatives , Amino Acid Sequence , Animals , Blood Substitutes/administration & dosage , Blood Substitutes/chemistry , Disease Models, Animal , Liposomes , Male , Oligopeptides/administration & dosage , Oligopeptides/chemistry , Rats , Rats, Sprague-Dawley , Thrombocytopenia/drug therapyABSTRACT
Adenosine diphosphate (ADP)-encapsulated liposomes coated with a fibrinogen γ-chain dodecapeptide [H12 (dodecapeptide ((400) HHLGGAKQAGDV(411) ))-(ADP)-liposome] is a synthetic platelet substitute, in which the surface is covered with polyethylene glycol (PEG). It has been reported that repeated injections of PEGylated liposomes induce an accelerated blood clearance (ABC) phenomenon, which involves a loss in the long-circulation half-life of the material when administered repeatedly to the same animals. The objective of this study was to determine whether the ABC phenomenon was induced by repeated injections of H12-(ADP)-liposome in healthy and anticancer drug-induced thrombocytopenia model rats. The findings show that the ABC phenomenon was induced by healthy rats that were repeatedly injected with H12-(ADP)-liposomes at the interval of 5 days at a dose of 10 mg lipids/kg. The ABC phenomenon involves the production of anti-H12-(ADP)-liposome immunoglobulin M (IgM) and complement activation. On the other hand, when thrombocytopenia model rats were repeatedly injected with H12-(ADP)-liposomes under the same conditions, no ABC phenomenon, nor was any suppression of anti-H12-(ADP)-liposome IgM-mediated complement activation observed. We thus conclude that the repeated injection of H12-(ADP)-liposome treatment in rat model with anticancer drug-induced thrombocytopenia did not induce the ABC phenomenon.
Subject(s)
Adenosine Diphosphate/administration & dosage , Antineoplastic Agents/adverse effects , Blood Platelets/drug effects , Blood Substitutes/administration & dosage , Fibrinogen/administration & dosage , Liposomes/administration & dosage , Animals , Blood Platelets/metabolism , Half-Life , Immunoglobulin M/metabolism , Injections/methods , Male , Polyethylene Glycols/administration & dosage , Rats , Rats, Sprague-Dawley , Thrombocytopenia/chemically inducedABSTRACT
BACKGROUND: Epidemiological studies have shown the prevention of cardiovascular diseases through the regular consumption of vegetables. Eruca sativa Mill., commonly known as rocket, is a leafy vegetable that has anti-inflammatory activity. However, its antiplatelet and antithrombotic activities have not been described. METHODS: Eruca sativa Mill. aqueous extract (0.1 to 1 mg/mL), was evaluated on human platelets: (i) P-selectin expression by flow cytometry; (ii) platelet aggregation induced by ADP, collagen and arachidonic acid; (iii) IL-1ß, TGF-ß1, CCL5 and thromboxane B2 release; and (iv) activation of NF-κB and PKA by western blot. Furthermore, (v) antithrombotic activity (200 mg/kg) and (vi) bleeding time in murine models were evaluated. RESULTS: Eruca sativa Mill. aqueous extract (0.1 to 1 mg/mL) inhibited P-selectin expression and platelet aggregation induced by ADP. The release of platelet inflammatory mediators (IL-1ß, TGF-ß1, CCL5 and thromboxane B2) induced by ADP was inhibited by Eruca sativa Mill. aqueous extract. Furthermore, Eruca sativa Mill. aqueous extract inhibited NF-κB activation. Finally, in murine models, Eruca sativa Mill. aqueous extract showed significant antithrombotic activity and a slight effect on bleeding time. CONCLUSION: Eruca sativa Mill. presents antiplatelet and antithrombotic activity.
Subject(s)
Brassicaceae/chemistry , Fibrinolytic Agents/pharmacology , NF-kappa B/metabolism , Plant Extracts/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Adenosine Diphosphate/administration & dosage , Adenosine Diphosphate/adverse effects , Animals , Blood Platelets/drug effects , Blood Platelets/metabolism , Chemokine CCL5/metabolism , Disease Models, Animal , Humans , Interleukin-1beta/metabolism , Mice , Mice, Inbred C57BL , P-Selectin/genetics , P-Selectin/metabolism , Platelet Aggregation/drug effects , Thrombosis/drug therapy , Thromboxane B2/metabolism , Transforming Growth Factor beta1/metabolismABSTRACT
Bleeding risk with antiplatelet therapy is an increasing clinical challenge. However, the inter-individual variation in this risk is poorly understood. We assessed whether the level of plasma creatine kinase, the enzyme that utilizes ADP and phosphocreatine to rapidly regenerate ATP, may modulate bleeding risk through a dose-dependent inhibition of ADP-induced platelet activation. Exogenous creatine kinase (500 to 4000 IU/L, phosphocreatine 5 mM) added to human plasma induced a dose-dependent reduction to complete inhibition of ADP-induced platelet aggregation. Accordingly, endogenous plasma creatine kinase, studied in 9 healthy men (mean age 27.9 y, SE 3.3; creatine kinase 115 to 859 IU/L, median 358), was associated with reduced ADP-induced platelet aggregation (Spearman's rank correlation coefficient, -0.6; p < 0.05). After exercise, at an endogenous creatine kinase level of 4664, ADP-induced platelet aggregation was undetectable, normalizing after rest, with a concomitant reduction of creatine kinase to normal values. Thus, creatine kinase reduces ADP-induced platelet activation. This may promote bleeding, in particular when patients use platelet P2Y12 ADP receptor inhibitors.
Subject(s)
Adenosine Diphosphate/administration & dosage , Creatine Kinase/blood , Hemorrhage/blood , Platelet Aggregation/drug effects , Adult , Blood Platelets/drug effects , Hemorrhage/pathology , Humans , Male , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2Y12/drug effectsABSTRACT
AIMS: The aim of this study was to elucidate myocardial interstitial serotonin (5-HT) kinetics in the heart, including 5-HT reuptake and enzymatic degradation to 5-hydroxyindole acetic acid (5-HIAA) via monoamine oxidase (MAO). MAIN METHODS: Using microdialysis technique in anesthetized rats, we simultaneously monitored myocardial interstitial levels of 5-HT and its major metabolite, 5-HIAA, in the left ventricle and examined the effects of local administration of a MAO inhibitor, pargyline, or a 5-HT uptake inhibitor, fluoxetine. KEY FINDINGS: Pargyline increased dialysate 5-HT concentration from 1.8±0.3 at baseline to 3.9±0.5nM but decreased dialysate 5-HIAA concentration from 20.7±1.0 at baseline to 15.8±1.4nM at 60-80min of administration. Fluoxetine increased dialysate 5-HT concentration from 1.9±0.4 at baseline to 6.5±0.9nM at 60-80min of administration, but did not change dialysate 5-HIAA concentration. Local administration of ADP (100mM) increased dialysate 5-HT and 5-HIAA concentrations. Pargyline did not affect ADP-induced increase in dialysate 5-HT concentration but suppressed ADP-induced increase in dialysate 5-HIAA concentration during 60min of ADP administration. Fluoxetine increased dialysate 5-HT concentration at 40-60min of ADP administration, but did not affect ADP-induced increase in dialysate 5-HIAA concentration. SIGNIFICANCE: Simultaneous monitoring of myocardial interstitial 5-HT and 5-HIAA levels provides valuable information on 5-HT kinetics including reuptake and enzymatic degradation by MAO, which play a role in the regulation of myocardial interstitial 5-HT levels at baseline and when 5-HT levels are elevated.
Subject(s)
Hydroxyindoleacetic Acid/metabolism , Microdialysis/methods , Monoamine Oxidase/metabolism , Myocardium/metabolism , Serotonin/metabolism , Adenosine Diphosphate/administration & dosage , Animals , Fluoxetine/administration & dosage , Fluoxetine/pharmacology , Heart Ventricles/metabolism , Male , Monoamine Oxidase/drug effects , Monoamine Oxidase Inhibitors/pharmacology , Pargyline/administration & dosage , Pargyline/pharmacology , Rats , Rats, Wistar , Selective Serotonin Reuptake Inhibitors/administration & dosage , Selective Serotonin Reuptake Inhibitors/pharmacology , Time FactorsABSTRACT
Several prospective studies have shown that high on-clopidogrel platelet reactivity (HPR) in patients undergoing percutaneous coronary intervention (PCI) is a risk factor for ischemic events. All studies were insufficiently powered to detect differences in stroke between patients with HPR and those without. Therefore, we performed a systematic review and meta-analysis of available publications aimed at determining whether patients undergoing PCI with HPR are also at increased risk of stroke. We searched for prospective studies enrolling patients undergoing PCI and treated with aspirin and clopidogrel that reported on clinical relevance of HPR to adenosine diphosphate. Study end point was the rate of stroke. We also investigated whether there was an interaction on the relative risk of stroke between HPR, clinical presentation, duration of follow-up, or laboratory methods. Fourteen studies including 11,959 patients were deemed eligible. On pooled analysis, the risk of stroke was higher in patients with HPR compared with patients with no HPR (1.2% vs 0.7%, relative risk on fixed effect 1.84, 95% confidence interval 1.21 to 2.80). There was no heterogeneity among the studies (I(2) = 0%, p = 0.5). Clinical presentation (p = 0.39 for interaction), duration of follow-up (p = 0.87 for interaction), and laboratory method for detection of HPR (p = 0.99 for interaction) did not affect the relative increase in the risk of stroke in patients with HPR compared with patients with no HPR. In conclusion, in patients with coronary artery disease undergoing PCI, the presence of HPR to adenosine diphosphate is a risk factor for stroke.
Subject(s)
Adenosine Diphosphate/administration & dosage , Blood Platelets/drug effects , Myocardial Ischemia/therapy , Percutaneous Coronary Intervention , Platelet Aggregation/drug effects , Stroke , Ticlopidine/analogs & derivatives , Clopidogrel , Dose-Response Relationship, Drug , Global Health , Humans , Incidence , Myocardial Ischemia/blood , Platelet Aggregation Inhibitors/administration & dosage , Platelet Function Tests , Postoperative Period , Risk Factors , Stroke/blood , Stroke/epidemiology , Stroke/etiology , Ticlopidine/administration & dosageABSTRACT
Chitosan nanoparticles (NPs) decorated with adenosine diphosphate (ADP) (ANPs) or fibrinogen (FNPs) were used to fabricate hemostatic NPs that can shorten blood clotting time and prevent severe local hemorrhage. The structure and mechanical properties of the blood clot induced with ANP (clot/ANP) or FNP (clot/FNP) were also investigated. The NPs, ANPs, and FNPs, which had particle sizes of 245.1 ± 14.0, 251.0 ± 9.8, and 326.5 ± 14.5 nm and zeta potentials of 24.1 ± 0.5, 20.6 ± 1.9, and 15.3 ± 1.5 mV (n=4), respectively, were fabricated by ionic gelation and then decorated with ADP and fibrinogen. The zeta potentials and Fourier transform infrared (FTIR) spectroscopy of the NPs confirmed that their surfaces were successfully coated with ADP and fibrinogen. The scanning electron microscope (SEM) micrographs of the structure of the clot induced with "undecorated" chitosan NPs (clot/NP), clot/ANP, and clot/FNP (at 0.05 wt%) were different, after citrated bloods had been recalcified by a calcium chloride solution containing NPs, ANPs, or FNPs. This indicated that many NPs adhered on the membrane surfaces of red blood cells, that ANPs induced many platelet aggregates, and that FNPs were incorporated into the fibrin network in the clots. Measurements of the blood clotting times (Tc) of blood clot/NPs, clot/ANPs, and clot/FNPs, based on 90% of ultimate frequency shifts measured on a quartz crystal microbalance (QCM), were significantly (P<0.05) (n=4) shorter than that of a clot induced by a phosphate-buffered solution (PBS) (clot/PBS) (63.6% ± 3.1%, 48.3% ± 6.2%, and 63.2% ± 4.7%, respectively). The ΔF2 values in the spectra of frequency shifts associated with the propagation of fibrin networks in the clot/ANPs and clot/FNPs were significantly lower than those of clot/PBS. Interestingly, texture profile analysis of the compressional properties showed significantly lower hardness and compressibility in clot/NPs and clot/ANPs (P<0.05 or better) (n=4) compared with clot/PBS and clot/FNPs. Accordingly, among the hemostatic NPs, ANP substantially reduced blood clotting times, ΔF2 values, and compression flow properties of the clot. Hence, ANPs have potential applications for preventing severe local hemorrhage.
Subject(s)
Adenosine Diphosphate/administration & dosage , Blood Coagulation/drug effects , Blood Coagulation/physiology , Chitosan/administration & dosage , Nanocapsules/administration & dosage , Adenosine Diphosphate/chemistry , Blood Coagulation Tests/methods , Chitosan/chemistry , Compressive Strength/drug effects , Compressive Strength/physiology , Drug Combinations , Hardness/drug effects , Hardness/physiology , Hemostatics/administration & dosage , Hemostatics/chemical synthesis , Humans , Nanocapsules/chemistryABSTRACT
In contrast to short thrombelastography (s-TEG) which utilises adenosine diphosphate (ADP) alone, the VerifyNow P2Y12 assay (VN-P2Y12) additionally uses prostaglandin E1 (PGE1) as agonist to assess response to P2Y12 inhibitors. Based upon previous observations, we hypothesised that VN-P2Y12 overestimates the therapeutic effects of clopidogrel. Simultaneous assay with s-TEG and VN-P2Y12 was performed in 43 healthy volunteers and 170 patients either on or off clopidogrel. Furthermore, in 27 patients on clopidogrel 75 mg we compared the effects of adding 22 nM PGE1 to ADP on platelet aggregation in s-TEG to ADP alone. A higher proportion of individuals had a result indicating high platelet reactivity (HPR) with s-TEG than VN-P2Y12 in (i) 43 clopidogrel naïve volunteers (95.3% vs 81.4%, p = NS); (ii) 28 volunteers loaded with clopidogrel 600 mg (39.3% vs 10.7 %, p = < 0.01); (iii) 123 clopidogrel naïve patients (93.5% vs 78%, p = < 0.0001); (iv) 47 patients on clopidogrel 75 mg (42.6% vs 4.3%, p = < 0.0001). In 59 patients loaded with clopidogrel 600 mg/900 mg, a greater proportion had a "therapeutic response" with VN-P2Y12 compared to s-TEG, regardless of the threshold for defining HPR with VN-PY12 (P2Y12 reaction units ≥ 230 or 208). Furthermore, adding PGE1 to ADP in s-TEG potentiated the anti-aggregatory effects of clopidogrel compared with ADP alone. In conclusion, VN-P2Y12 overestimates the functional effects of clopidogrel in some individuals, possibly because it utilises PGE1 in addition to ADP. This could have implications for the ability of VN-P2Y12 to stratify patients as "responders" or "non-responders" to clopidogrel.