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Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metaboliteâgenerated by aldehyde oxidase (AO)âpossessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.
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Doenças Respiratórias , Canais de Potencial de Receptor Transitório , Humanos , Canais de Potencial de Receptor Transitório/metabolismo , Canal de Cátion TRPA1 , Aldeído Oxidase/metabolismo , Oxirredutases/metabolismo , Proteínas do Citoesqueleto/metabolismoRESUMO
Introduction: Disorders of coagulation are well-recognized in dogs with sepsis, but data regarding fibrinolysis disorders are limited. We aimed to characterize fibrinolysis in dogs with sepsis compared to healthy controls. We hypothesized that dogs with sepsis would be hypofibrinolytic, and that hypofibrinolysis would be associated with non-survival. Methods: This was a prospective observational cohort study. We enrolled 20 client-owned dogs with sepsis admitted to the Cornell University Hospital for Animals and 20 healthy pet dogs. Coagulation and fibrinolytic pathway proteins including antiplasmin activity (AP), antithrombin activity (AT), thrombin activatable fibrinolysis inhibitor activity (TAFI), D-dimer concentration, fibrinogen concentration, and plasminogen activity were measured and compared between groups. Overall coagulation potential, overall fibrinolysis potential, and overall hemostatic potential were calculated from the curve of fibrin clot formation and lysis over time. Results: Compared to healthy controls, dogs with sepsis had lower AT (P = 0.009), higher AP (P = 0.002), higher TAFI (P = 0.0385), and higher concentrations of fibrinogen (P < 0.0001) and D-dimer (P = 0.0001). Dogs with sepsis also had greater overall coagulation potential (P = 0.003), overall hemostatic potential (P = 0.0015), and lower overall fibrinolysis potential (P = 0.0004). The extent of fibrinolysis was significantly negatively correlated with TAFI. No significant differences were observed between survivors and non-survivors. Discussion: Dogs with sepsis were hypercoagulable and hypofibrinolytic compared to healthy dogs, suggesting potential utility of thromboprophylaxis in this patient population. The association between high TAFI and low overall fibrinolysis potential might provide a potential mechanism for this hypofibrinolysis.
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OBJECTIVE: Rivaroxaban is a new anticoagulant option for dogs, yet its reported oral bioavailability is as low as 60%. The objective of this study was to examine the influence of feeding and gastroprotectant medications on the bioactivity (anti-Xa activity) of rivaroxaban in healthy dogs. DESIGN: Prospective experimental study. SETTING: University research laboratory. ANIMALS: Five healthy neutered male purpose-bred Beagles. INTERVENTIONS: Dogs were administered a median dose of 1.8 mg/kg rivaroxaban (range, 1.6-1.8 mg/kg) orally once daily for 2 consecutive days with either (1) no food, (2) food, (3) sucralfate 30 minutes before rivaroxaban, or (4) omeprazole at the same time as rivaroxaban. Blood was collected from preplaced jugular catheters immediately before and at 6 time points after rivaroxaban administration (2, 4, 8, 24, 36, and 48 hours). A rivaroxaban calibrated anti-Xa activity assay (RIVA) was used to monitor anticoagulant effect. MEASUREMENTS AND MAIN RESULTS: Rivaroxaban administration resulted in significant increases in RIVA (P = 0.02), with peak activities occurring 2 to 4 hours after dosingduring each study arm. No feeding was associated with significantly higher RIVA at the 36-hour time point compared to all other treatment arms (P < 0.0001), and feeding resulted in high RIVA at the 48-hour time point compared with sucralfate administration (P = 0.003). No significant changes in RIVA were otherwise identified with respect to feeding or gastroprotectant administration (P = 0.2). CONCLUSIONS AND CLINICAL IMPORTANCE: Although administration without food demonstrated an apparent increase in RIVA 36 hours after drug administration, clinically relevant differences among treatment groups were not identified in combined analyses of time points. Based on these results, dogs treated with rivaroxaban do not require special modification of feeding practices or gastroprotectant drug administration.
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Antiulcerosos/farmacologia , Anticoagulantes/farmacocinética , Inibidores do Fator Xa/farmacocinética , Refeições , Rivaroxabana/farmacocinética , Administração Oral , Animais , Antiulcerosos/administração & dosagem , Anticoagulantes/administração & dosagem , Testes de Coagulação Sanguínea/veterinária , Cães , Inibidores do Fator Xa/administração & dosagem , Masculino , Estudos Prospectivos , Valores de Referência , Rivaroxabana/administração & dosagemRESUMO
OBJECTIVE: To evaluate a panel of coagulation assays for their potential utility in rivaroxaban monitoring as alternatives to the rivaroxaban-specific anti-Xa activity (RIVA). DESIGN: Prospective experimental study. SETTING: University research laboratory. ANIMALS: Five healthy neutered male Beagles. INTERVENTIONS: Dogs were administered a median dose of 1.8 mg/kg rivaroxaban (range, 1.6-1.8 mg/kg) orally once daily for 2 consecutive days as part of a pharmacodynamic study. Blood was collected from a preplaced jugular catheter at time points relative to their rivaroxaban administration (0, 2, 4, 8, 24, 36, and 48 h) for measurement of RIVA, prothrombin time (PT), activated partial thromboplastin time, RapidTEG, and thrombin generation variables. MEASUREMENTS AND MAIN RESULTS: One hundred forty data points were available for analysis. There was poor correlation between RIVA and RapidTEG variables: R time (R) (min) (r = 0.554, P < 0.0001), K time (K) (min) (r = -0.204, P = 0.016), alpha angle (degrees) (r = 0.152, P = 0.073), Maximum amplitude (MA) (mm) (r = 0.106, P = 0.215), and G value (G) (dynes/s) (r = 0.108, P = 0.205). A good correlation was noted between thrombin generation variables and RIVA: lag time (min) (r = 0.827, P < 0.0001), peak (nM) (r = -0.752, P < 0.0001), and endogenous thrombin potential (nM·min) (r = -0.762, P < 0.0001). There was an excellent correlation between PT and RIVA (r = 0.915, P < 0.0001) and a good correlation between activated partial thromboplastin time and RIVA (r = 0.772, P < 0 .0001). CONCLUSIONS: Of all the coagulation tests investigated, the PT correlated best with RIVA. There is potential for PT being a convenient second-line monitoring option in dogs receiving rivaroxaban, but further work is necessary to validate other PT assays. Thromboelastography performed with strong activators correlated poorly with anti-Xa activity.
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Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Inibidores do Fator Xa/farmacologia , Rivaroxabana/farmacologia , Administração Oral , Animais , Anticoagulantes/administração & dosagem , Testes de Coagulação Sanguínea/veterinária , Cães , Inibidores do Fator Xa/administração & dosagem , Masculino , Tempo de Tromboplastina Parcial/veterinária , Testes Imediatos , Estudos Prospectivos , Tempo de Protrombina/veterinária , Valores de Referência , Rivaroxabana/administração & dosagem , Tromboelastografia/veterináriaRESUMO
Factor XII (FXII) is a coagulation protein that initiates surface-activation of the coagulation cascade in vitro. The protein's in vivo role, however, remains poorly defined. Factor XII deficiency, or Hageman trait, is a rare hereditary disorder that is not associated with bleeding, and wide variations in FXII activity (FXII:C) exist among healthy people. While FXII-deficient knockout mice appear to be resistant to arterial thrombosis, human F12 polymorphisms that influence FXII:C have not been associated with thrombotic risk in population surveys. Factor XII deficiency is a naturally occurring hereditary trait in domestic cats. We undertook phenotypic and genotypic analyses of FXII-deficient cats for comparative studies with the human disease counterpart. A retrospective review of feline submissions to our laboratory revealed that FXII deficiency is common in domestic cats, and also present in many different breeds. The trait has a geographic bias toward the Midwestern United States. Clinical history, coagulation assays, and samples for F12 sequencing were obtained from 26 FXII deficient cats. None of the cats had experienced abnormal bleeding and their residual FXII:C was related to F12 mutation number and mutation-type. We found 2 high frequency F12 mutations: an exon 13 missense mutation (c.1631Gâ¯>â¯C) and an exon 11 deletion mutation (c.1321delC), and additional sequence variants throughout the gene. Factor XII deficiency in pet cat populations provides an animal model system to help clarify the biologic actions and clinical relevance of FXII protein.
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Gatos/genética , Deficiência do Fator XII/genética , Fator XII/genética , Animais , Éxons/genética , Fator XII/fisiologia , Deficiência do Fator XII/veterinária , Mutação , Polimorfismo Genético/genética , Estudos Retrospectivos , Deleção de Sequência , Estados UnidosRESUMO
Cold stress syndrome (CSS) in the Florida manatee Trichechus manatus latirostris has been defined as morbidity and mortality resulting from prolonged exposure to water temperatures <20°C. The pathophysiology is described as multifactorial, involving nutritional, immunological and metabolic disturbances; however, the exact mechanisms are unknown. We hypothesized that thromboembolic complications contribute to the pathophysiology of CSS in addition to the previously described factors. During the winter of 2014-2015, 10 Florida manatees with clinical signs of CSS were presented to Lowry Park Zoo, Tampa, FL, USA. Thromboelastography (TEG) and coagulation panels were performed at admission. In addition, coagulation panel data from 23 retrospective CSS cases were included in the analyses. There were numerous differences between mean values of TEG and coagulation parameters for healthy manatees and those for CSS cases. Among TEG parameters, reaction time (R), clot formation time (K) and percentage of clot lysed after 30 min (LY30) values were significantly different (p < 0.05) between the 2 groups. CSS cases also had significantly higher mean D-dimer concentration and coagulation factor XI activity, prolonged mean activated partial thromboplastin time (aPTT) and significantly decreased mean antithrombin activity. These combined abnormalities include clinicopathologic criteria of disseminated intravascular coagulation, indicating an increased risk of thromboembolic disease associated with manatee CSS.
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Transtornos da Coagulação Sanguínea/veterinária , Temperatura Baixa , Estresse Fisiológico , Trichechus manatus/sangue , Animais , Transtornos da Coagulação Sanguínea/etiologia , Estudos RetrospectivosRESUMO
BACKGROUND: Thrombin generation assays (TGA) have potential applications as measures of hemostatic balance in animal models. However, variations in plasma processing greatly influence human TGA, and may also impact on the translational value of TGA in animal studies. OBJECTIVES: The purpose of the study was to compare the performance characteristics of Sprague-Dawley rat plasma prepared by single vs double centrifugation protocols in TGA and fibrinolysis assays. METHODS: Platelet-poor plasma (PPP) from adult rats (n = 20 males; 20 females) was prepared by centrifugation at 1200g × 12 min, or 2 sequential centrifugations of 2500g × 15 min. Plasma aliquots were assayed fresh and after freeze-thaw in a commercial fluorogenic TGA (Technothrombin TGA, Technoclone) using 2 different trigger reagents containing approximately 7 pM human tissue factor. In addition to TGA variables (lag time, peak thrombin, endogenous thrombin potential), we compared clotting time test and fibrinogen concentration, residual platelet and platelet-derived microparticle (PMP) counts measured by flow cytometry, and variables of fibrin clot formation and lysis measured in turbidimetric assays. RESULTS: Single-centrifugation PPP demonstrated significantly greater thrombin-generating potential regardless of trigger reagent, yielded higher residual platelet and procoagulant PMP counts, and more stable fibrin clot profiles. The influence of a freeze-thaw cycle on TGA varied depending on trigger reagent, and male sex was associated with an overall "procoagulant" phenotype. CONCLUSIONS: Preanalytic processing and sex have significant effects on many functional measures of hemostasis in rats. A standardized double centrifugation protocol to prepare PPP is recommended for future studies.