cangrelor and Disease-Models--Animal

Sepsis is a common complication of severe wound injury and infection, with a very high mortality rate. The P2Y12 receptor inhibitor, cangrelor, is an antagonist anti-platelet drug.. In our study, we investigated the protective mechanisms of cangrelor in CLP-induced pulmonary injury in sepsis, using C57BL/6 mouse models.. TdT-mediated dUTP Nick-End Labeling (TUNEL) and Masson staining showed that apoptosis and fibrosis in lungs were alleviated by cangrelor treatment. Cangrelor significantly promoted surface expression of CD40L on platelets and inhibited CLP-induced neutrophils in Bronchoalveolar lavage fluid (BALF) (p < 0.001). We also found that cangrelor decreased the inflammatory response in the CLP mouse model and inhibited the expression of inflammatory cytokines, IL-1β (p  < 0.01), IL-6 (p < 0.05), and TNF-α (p < 0.001). Western blotting and RT-PCR showed that cangrelor inhibited the increased levels of G-protein-coupled receptor 17 (GPR17) induced by CLP (p < 0.001).. Our study indicated that cangrelor repressed the levels of GPR17, followed by a decrease in the inflammatory response and a rise of neutrophils in BALF, potentially reversing CLP-mediated pulmonary injury during sepsis.

Topics: Acute Lung Injury; Adenosine Monophosphate; Animals; Cecum; Disease Models, Animal; Ligation; Mice; Mice, Inbred C57BL; Punctures; Purinergic P2Y Receptor Antagonists; Sepsis

Pulmonary fibrosis is a progressive chronic inflammatory lung disease whose pathogenesis is complicated. Platelets and neutrophils play important roles in the progression of pulmonary inflammation. We have reported that cangrelor, a non-sepesific GPR17 antagonist, alleviates pulmonary fibrosis partly by inhibiting macrophage inflammation in mice. Cangrelor is also a well-known anti-platelet agent. To test whether cangrelor mitigated pulmonary fibrosis partly through the inhibition of platelets, bleomycin (BLM) was used to induce pulmonary fibrosis in C57BL/6 J mice. We found that cangrelor (10 mg/kg) not only significantly decreased BLM-induced release of inflammatory cytokines (PF4, CD40 L and MPO), but also decreased the increment of platelets, neutrophils and platelet-neutrophil aggregates in the fibrotic lung and in the peripheral blood of BLM-treated mice. In addition, cangrelor decreased the number of CD40 and MPO double positive neutrophils and the expression level of CD40 in BLM-treated mouse lungs. Based on these results we conclude that cangrelor alleviates BLM-induced lung inflammation and pulmonary fibrosis in mice, partly through inhibition of platelet activation, therefore reducing the infiltration of neutrophils due to the adhesion of platelets and neutrophils mediated by CD40 - CD40 L interaction. Cangrelor could be a potential therapeutic medicine for pulmonary fibrosis.

Topics: Adenosine Monophosphate; Animals; Bleomycin; CD40 Antigens; Cytokines; Disease Models, Animal; Inflammation Mediators; Lung; Male; Mice; Mice, Inbred C57BL; Neutrophil Infiltration; Neutrophils; Platelet Activation; Platelet Aggregation Inhibitors; Pulmonary Fibrosis

Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic

Topics: Adenosine Monophosphate; Animals; Bacterial Toxins; Blood Platelets; Deoxyadenine Nucleotides; Disease Models, Animal; Escherichia coli Proteins; Hemolysin Proteins; Humans; Male; Mice, Inbred BALB C; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Sepsis; Treatment Outcome; Urinary Tract Infections; Uropathogenic Escherichia coli

Platelets from patients with diabetes mellitus are hyperactive. Hyperactivated platelets may contribute to cardiovascular complications and inadequate responses to antiplatelet agents in the setting of diabetes mellitus. However, the underlying mechanism of hyperactivated platelets is not completely understood.. We measured P2Y. Platelet P2Y

Topics: Adenosine Monophosphate; Animals; Blood Platelets; Cell Adhesion Molecules; Cell Line; Chlorides; Cyclic AMP; Diabetes Mellitus, Experimental; Diabetes Mellitus, Type 2; Disease Models, Animal; Drug Inverse Agonism; Ferric Compounds; Fibrinolytic Agents; Humans; Male; Microfilament Proteins; NF-kappa B; Phosphoproteins; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Reactive Oxygen Species; Receptors, Purinergic P2Y12; Thrombosis

Unlike currently approved adenosine diphosphate receptor antagonists, the new diadenosine tetraphosphate derivative GLS-409 targets not only P2Y12 but also the second human platelet adenosine diphosphate receptor P2Y1 and may, therefore, be a promising antiplatelet drug candidate. The current study is the first to investigate the in vivo antithrombotic effects of GLS-409.. We studied (1) the in vivo effects of GLS-409 on agonist-stimulated platelet aggregation in anesthetized rats, (2) the antithrombotic activity of GLS-409 and the associated effect on the bleeding time in a canine model of platelet-mediated coronary artery thrombosis, and (3) the inhibition of agonist-stimulated platelet aggregation by GLS-409 versus selective P2Y1 and P2Y12 inhibition in vitro in samples from healthy human subjects before and 2 hours after aspirin intake. In vivo treatment with GLS-409 significantly inhibited adenosine diphosphate- and collagen-stimulated platelet aggregation in rats. Further, GLS-409 attenuated cyclic flow variation, that is, platelet-mediated thrombosis, in vivo in our canine model of unstable angina. The improvement in coronary patency was accompanied by a nonsignificant 30% increase in bleeding time. Of note, GLS-409 exerted its effects without affecting rat and canine hemodynamics. Finally, in vitro treatment with GLS-409 showed effects similar to that of cangrelor and the combination of cangrelor with the selective P2Y1 inhibitor MRS 2179 on agonist-stimulated platelet aggregation in human platelet-rich plasma and whole blood before and 2 hours after aspirin intake.. Synergistic inhibition of both P2Y1 and P2Y12 adenosine diphosphate receptors by GLS-409 immediately attenuates platelet-mediated thrombosis and effectively blocks agonist-stimulated platelet aggregation irrespective of concomitant aspirin therapy.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Adult; Animals; Aspirin; Blood Coagulation; Blood Platelets; Coronary Thrombosis; Dinucleoside Phosphates; Disease Models, Animal; Dogs; Dose-Response Relationship, Drug; Female; Fibrinolytic Agents; Humans; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Platelet Function Tests; Purinergic P2Y Receptor Antagonists; Rats, Sprague-Dawley; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Time Factors; Young Adult

Recent reports indicate that elevating DNA glycosylase/AP lyase repair enzyme activity offers marked cytoprotection in cultured cells and a variety of injury models. In this study, we measured the effect of EndoIII, a fusion protein construct that traffics Endonuclease III, a DNA glycosylase/AP lyase, to the mitochondria, on infarct size in a rat model of myocardial ischemia/reperfusion. Open-chest, anesthetized rats were subjected to 30 min of occlusion of a coronary artery followed by 2 h of reperfusion. An intravenous bolus of EndoIII, 8 mg/kg, just prior to reperfusion reduced infarct size from 43.8 ± 1.4% of the risk zone in control animals to 24.0 ± 1.3% with no detectable hemodynamic effect. Neither EndoIII's vehicle nor an enzymatically inactive EndoIII mutant (K120Q) offered any protection. The magnitude of EndoIII's protection was comparable to that seen with the platelet aggregation inhibitor cangrelor (25.0 ± 1.8% infarction of risk zone). Because loading with a P2Y12 receptor blocker to inhibit platelets is currently the standard of care for treatment of acute myocardial infarction, we tested whether EndoIII could further reduce infarct size in rats treated with a maximally protective dose of cangrelor. The combination reduced infarct size to 15.1 ± 0.9% which was significantly smaller than that seen with either cangrelor or EndoIII alone. Protection from cangrelor but not EndoIII was abrogated by pharmacologic blockade of phosphatidylinositol-3 kinase or adenosine receptors indicating differing cellular mechanisms. We hypothesized that EndoIII protected the heart from spreading necrosis by preventing the release of proinflammatory fragments of mitochondrial DNA (mtDNA) into the heart tissue. In support of this hypothesis, an intravenous bolus at reperfusion of deoxyribonuclease I (DNase I) which should degrade any DNA fragments escaping into the extracellular space was as protective as EndoIII. Furthermore, the combination of EndoIII and DNase I produced additive protection. While EndoIII would maintain mitochondrial integrity in many of the ischemic cardiomyocytes, DNase I would further prevent mtDNA released from those cells that EndoIII could not save from propagating further necrosis. Thus, our mtDNA hypothesis would predict additive protection. Finally to demonstrate the toxicity of mtDNA, isolated hearts were subjected to 15 min of global ischemia. Infarct size doubled when the coronary vasculature was filled with mtDNA fragments durin

Topics: Adenosine Monophosphate; Animals; Deoxyribonuclease I; Disease Models, Animal; Endodeoxyribonucleases; Hemodynamics; Male; Mitochondria; Myocardial Infarction; Myocardial Reperfusion Injury; Purinergic P2Y Receptor Antagonists; Rats; Rats, Sprague-Dawley; Recombinant Fusion Proteins

In our previous in vitro study, we reported a constitutively active chimeric P2Y(12) (cP2Y(12)) and found that AR-C78511 is a potent inverse agonist at this receptor. The role of cP2Y(12) in platelet activation and thrombosis is not clear.. To investigate the physiologic implications of cP2Y(12) for platelet activation and thrombus formation, and to evaluate the antiplatelet activity of AR-C78511 as an inverse agonist.. We generated transgenic mice conditionally and platelet-specifically expressing cP2Y(12). High-level expression of cP2Y(12) in platelets increased platelet reactivity, as shown by increased platelet aggregation in response to multiple platelet agonists. Moreover, transgenic mice showed a shortened bleeding time, and more rapid and stable thrombus formation in mesenteric artery injured with FeCl(3). The constitutive activity of cP2Y(12) in platelets was confirmed by decreased platelet cAMP levels and constitutive Akt phosphorylation in the absence of agonists. AR-C78511 reversed the cAMP decrease in transgenic mouse platelets, and exhibited a superior antiplatelet effect to that of AR-C69931MX in transgenic mice.. These findings further emphasize the importance of P2Y(12) in platelet activation, hemostasis, and thrombosis, as well as the prothrombotic role of the constitutive activity of P2Y(12). Our data also validate the in vivo inverse agonist activity of AR-C78511, and confirm its superior antiplatelet activity over neutral antagonists.

Topics: Adenosine Monophosphate; Animals; Bleeding Time; Blood Platelets; Chlorides; Cyclic AMP; Disease Models, Animal; Drug Inverse Agonism; Ferric Compounds; Fibrinolytic Agents; Genotype; Mice; Mice, Transgenic; Phenotype; Phosphorylation; Platelet Activation; Platelet Aggregation; Platelet Aggregation Inhibitors; Proto-Oncogene Proteins c-akt; Purinergic P2Y Receptor Antagonists; Receptors, Purinergic P2Y12; Thrombosis

The addition of phosphodiesterase (PDE) inhibitors has been reported to potentiate the antithrombotic effects of P2Y₁₂ antagonists without increasing bleeding risk. In this study, we report that a potent antiplatelet agent, 2-ethylthio-6-phenethylaminoadenosine (BF061), inhibits platelet activation and thrombosis via P2Y₁₂ antagonism and PDE inhibition. We explored the antiplatelet mechanism of BF061 by measuring cAMP, cGMP levels, PDE activity, and the interaction between ADP and P2Y₁₂ using atomic force microscopy. The antithrombotic effect of BF061 was evaluated in mice using intravital microscopy in FeCl₃₋induced mesenteric and laser-induced cremasteric arterial thrombosis models. BF061 robustly inhibited platelet aggregation and ATP release induced by multiple platelet agonists via P2Y₁₂ antagonism and PDE inhibition. Interestingly, despite being structurally similar to BF061, P2Y₁₂ receptor antagonist AR-C69931MX had no effect on human platelet PDE. In FeCl3-induced mesenteric arterial thrombosis model, BF061 effectively prevented thrombus formation similarly to clopidogrel; it also reduced thrombus volume in laser-injured cremaster arteriole model. In contrast, BF061 induced dramatically less bleeding at an antithrombotic dose compared to clopidogrel. In summary, we developed a novel antiplatelet and antithrombotic agent targeting both P2Y₁₂ and PDE. Given the prevalence of combined antiplatelet therapy in clinical practice, an antiplatelet agent bearing dual activities may have therapeutic advantage as a potential antithrombotic drug.

Topics: Adenosine; Adenosine Monophosphate; Animals; Disease Models, Animal; Drug Synergism; Enzyme Inhibitors; Fibrinolytic Agents; Humans; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Phosphoric Diester Hydrolases; Platelet Activation; Platelet Aggregation Inhibitors; Purinergic P2Y Receptor Antagonists; Thrombosis

The mechanisms underlying neuropathic pain are poorly understood. However, several studies have implied a role for reactive microglia located in the dorsal horn in neuropathic pain. To clarify the roles of activated microglia in neuropathic pain, we investigated the interactions among microglia and other neural components in the dorsal horn using electron microscopy. Microglia were more abundantly localized in layers II-III of the dorsal horn than in other areas, and some of them adhered to and engulfed both injured and uninjured myelinated axons. This microglial engulfment was rarely observed in the normal dorsal horn, and the number of microglia attached to myelinated axons was markedly increased on postoperative day 7 on the operated side. However, after blocking the P2Y12 ATP receptor in microglia by intrathecal administration of its antagonist, AR-C69931MX, the increase in the number of microglia attached to myelinated axons, as well as the development of tactile allodynia, were markedly suppressed, although the number of activated microglia did not change remarkably. These results indicate that engulfment of myelinated axons by activated microglia via P2Y12 signaling in the dorsal horn may be a critical event in the pathogenesis of neuropathic pain.

Topics: Adenosine Monophosphate; Animals; Calcium-Binding Proteins; Cell Proliferation; Disease Models, Animal; Functional Laterality; Gene Expression Regulation; Hyperalgesia; Male; Microfilament Proteins; Microglia; Microscopy, Immunoelectron; Nerve Fibers, Myelinated; Neuralgia; Posterior Horn Cells; Purinergic P2Y Receptor Antagonists; Rats; Rats, Wistar; Receptors, Purinergic P2Y12; Signal Transduction; Spinal Cord; Spinal Nerves

To determine the antithrombotic effects of SCH 602539, an analog of the selective protease-activated receptor (PAR)-1 antagonist vorapaxar (formerly SCH 530348) currently in advanced clinical development, and the P2Y(12) ADP receptor antagonist cangrelor, alone and in combination.. Multiple platelet activation pathways contribute to thrombosis. The effects of SCH 602539 and cangrelor alone and in combination on cyclic flow reductions were evaluated in a Folts model of thrombosis in cynomolgus monkeys. The effects of these treatments on ex vivo platelet aggregation and coagulation parameters were also monitored. Dose-dependent inhibition of cyclic flow reductions was observed after treatment with SCH 602539 alone and cangrelor alone (P<0.05 versus vehicle for the 2 highest concentrations of each agent). The combination of SCH 602539 and cangrelor was associated with synergistic antithrombotic effects (P<0.05 versus vehicle for all combinations tested). The 2 highest doses of SCH 602539 inhibited platelet aggregation in response to PAR-1-selective high-affinity thrombin receptor agonist peptide by greater than 80% but did not affect platelet aggregation induced by other agonists; also, they did not affect any coagulation parameters.. The combined inhibition of the PAR-1 and the P2Y(12) ADP platelet activation pathways had synergistic antithrombotic and antiplatelet effects. The addition of a PAR-1 antagonist to a P2Y(12) ADP receptor antagonist may provide incremental clinical benefits in patients with atherothrombotic disease, both in short- and long-term settings. These hypotheses need to be tested clinically.

Topics: Adenosine Monophosphate; Animals; Disease Models, Animal; Drug Therapy, Combination; Fibrinolytic Agents; Macaca fascicularis; Platelet Aggregation Inhibitors; Purinergic P2Y Receptor Antagonists; Receptor, PAR-1; Thrombosis

Photochemically induced thrombosis (a thrombin-dependent process) was measured in rats treated with moderate doses of anticoagulants, but which appeared to be unchanged. We considered the possibility that platelet-inhibiting agents, which also indirectly inhibit coagulation, would act as more potent antithrombotic agents. Inhibitors used as such were prostaglandin E1 (PGE1), which elevates cyclic AMP levels, and the P2Y12 ADP-receptor antagonist, AR-C69931MX. Effects of these agents were investigated in an ex vivo model system, in which whole blood under coagulant conditions was perfused over fibrinogen at defined wall shear rate. Perfusion of blood (rat or human) in the presence of tissue factor resulted in deposition of activated platelets and subsequent aggregate formation, along with exposure of procoagulant phosphatidylserine (PS) on the platelet surface and formation of fibrin fibers. In the presence of PGE1 aggregation was completely inhibited, but platelet adhesion and PS exposure were only party reduced, while fibrin formation was hardly affected. Treatment with AR-C69931MX caused similar, but less complete effects. These results indicate that in tissue factor-triggered blood under conditions of flow: (i) the platelet procoagulant response is independent of aggregate formation; (ii) the platelet-inhibiting effect of PGE1 and AR-C69931MX is sufficient to suppress aggregation, but not platelet adhesion and coagulation. These platelet inhibitors thus maintain their aggregation-inhibiting effect at sites of thrombin formation.

Topics: Adenosine Monophosphate; Alprostadil; Animals; Blood Coagulation; Disease Models, Animal; Drug Interactions; Fibrin; Fibrinogen; Fibrinolytic Agents; Male; Perfusion; Phosphatidylserines; Platelet Activation; Platelet Aggregation; Rats; Rats, Wistar; Thrombin; Thromboplastin

Reperfusion therapy for myocardial infarction is limited by a significant reocclusion rate and less optimal myocardial tissue perfusion due to excessive platelet accumulation and recruitment at the sites of vascular injury. We assessed the influence of a selective P2Y(12)-receptor antagonist (AR-C69931MX), in conjunction with thrombolytic therapy, on the prevention of platelet aggregation and thrombus formation.. A canine coronary electrolytic injury thrombosis model was used. Tissue-type plasminogen activator (t-PA; 1 mg/kg in phase I, 0.5 mg/kg in phase II in the AR-C69931MX group, and 1 mg/kg in the placebo group in phase I and II) was administered 30 minutes after thrombus formation; either saline or AR-C69931MX (4 micro g x kg(-1) x min(-1)) was given to all animals intravenously 10 minutes before t-PA administration for a total of 2 hours. All animals received heparin (80 U/kg) as an intravenous bolus followed by a continuous infusion of 17 U x kg(-1) x h(-1). Myocardial tissue perfusion was evaluated by use of the colored microsphere technique and real-time myocardial contrast echocardiography. The incidences of reocclusion and cyclic flow variation were significantly decreased in the AR-C69931MX group (P<0.05). Myocardial tissue flow with AR-C69931MX treatment improved significantly at 20 and 120 minutes after reflow, whereas tissue flow with placebo remained at a level similar to that during occlusion (P<0.05).. The adjunctive administration of AR-C69931MX blocked ADP-mediated platelet aggregation and recruitment and prevented platelet-mediated thrombosis, resulting in prolongation of reperfusion time and a decrease in reocclusion and cyclic flow variations. Importantly, myocardial tissue perfusion was significantly improved in the P2Y(12) antagonist group.

Topics: Adenosine Monophosphate; Angioplasty, Balloon, Coronary; Animals; Bleeding Time; Blood Coagulation; Coronary Circulation; Coronary Restenosis; Coronary Stenosis; Disease Models, Animal; Dogs; Drug Combinations; Echocardiography; Female; Heparin; Infusions, Intravenous; Male; Membrane Proteins; Myocardial Infarction; Myocardial Reperfusion; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y12; Thrombosis; Tissue Plasminogen Activator

In vivo, activated platelets contribute to the initiation of thrombin generation through the exposure of phosphatidylserine to form a procoagulant catalytic surface and through platelet-leukocyte interactions, which lead to the exposure of leukocyte tissue factor (TF). On the basis of observations that the platelet P2Y1 and P2Y12 receptors both contribute to thrombosis and thrombin formation in an in vivo model of TF-induced thromboembolism, we further characterized the role of these receptors in thrombin generation.. By using the selective P2 antagonists MRS2179 and AR-C69931MX, the P2Y12 receptor was found to be involved in thrombin-induced exposure of PS on isolated platelets and consequently in TF-induced thrombin formation in platelet-rich plasma. By contrast, the P2Y1 receptor was not involved in phosphatidylserine exposure nor in thrombin generation in platelet-rich plasma. In addition, both receptors were found to contribute to the interactions between platelets and leukocytes mediated by platelet P-selectin exposure, which result in TF exposure at the surface of leukocytes.. Overall, these results point to a differential involvement of the 2 platelet ADP receptors in the generation of thrombin and provide further evidence for the relevance of molecules targeting these receptors as antithrombotic agents.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Animals; Blood Platelets; Disease Models, Animal; Humans; Leukocytes; Male; Membrane Proteins; Mice; Mice, Inbred C57BL; P-Selectin; Peptide Fragments; Phosphatidylserines; Plasma; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2; Receptors, Purinergic P2Y1; Receptors, Purinergic P2Y12; Thrombin; Thrombosis

P2Y1, P2X1, and P2T receptors mediate ADP-induced platelet aggregation. The antithrombotic effects of AR-C69931MX (N6-[2-methylthio)ethyl]-2-[3,3,3-trifluoropropylthio]-5'-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid), a selective P2T platelet receptor antagonist, was assessed in a canine model of arterial thrombosis. Placebo or AR-C69931MX (4.0 microg/kg/min for 6 h) pretreatment was administered as an intravenous infusion beginning 15 min before inducing vessel wall injury. A 300-microA anodal current was applied to the intima of the carotid artery for 180 min or discontinued 30 min after cessation of blood flow due to thrombus formation. Each of five control animals developed occlusive thrombi within 3 h after induction of vessel wall injury. In contrast, carotid artery blood flow in five of six AR-C69931MX-treated animals was maintained for the duration of the protocol. Ex vivo platelet aggregation in response to adenosine diphosphate was inhibited at the first measurement time point of 75 min after the start of drug infusion and remained inhibited during drug administration. Bleeding time values were increased in the drug-treated group. Values for both the ex vivo platelet aggregation and the bleeding times returned to control values shortly after discontinuation of AR-C69931MX. The results indicate that AR-C69931MX antagonizes the ex vivo and in vivo aggregatory actions of ADP, and displays a rapid onset and offset of action with the ability to prevent occlusive arterial thrombus formation. AR-C69931MX may be suitable for the management of patients who require short-term modulation of platelet function.

Topics: Adenosine Diphosphate; Adenosine Monophosphate; Animals; Arterial Occlusive Diseases; Bleeding Time; Blood Flow Velocity; Blood Platelets; Carotid Arteries; Carotid Artery Thrombosis; Disease Models, Animal; Dogs; Female; Hemodynamics; Injections, Intravenous; Male; Membrane Proteins; Platelet Aggregation; Platelet Aggregation Inhibitors; Purinergic P2 Receptor Antagonists; Receptors, Purinergic P2Y12; Tunica Intima