Collaborative science in action: A 20 year perspective from the Health and Environmental Sciences Institute (HESI) Cardiac Safety Committee.

The Health and Environmental Sciences Institute (HESI) is a nonprofit organization dedicated to resolving global health challenges through collaborative scientific efforts across academia, regulatory authorities and the private sector. Collaborative science across non-clinical disciplines offers an important keystone to accelerate the development of safer and more effective medicines. HESI works to address complex challenges by leveraging diverse subject-matter expertise across sectors offering access to resources, data and shared knowledge. In 2008, the HESI Cardiac Safety Committee (CSC) was established to improve public health by reducing unanticipated cardiovascular (CV)-related adverse effects from pharmaceuticals or chemicals. The committee continues to significantly impact the field of CV safety by bringing together experts from across sectors to address challenges of detecting and predicting adverse cardiac outcomes. Committee members have collaborated on the organization, management and publication of prospective studies, retrospective analyses, workshops, and symposia resulting in 38 peer reviewed manuscripts. Without this collaboration these manuscripts would not have been published. Through their work, the CSC is actively addressing challenges and opportunities in detecting potential cardiac failure modes using in vivo, in vitro and in silico models, with the aim of facilitating drug development and improving study design. By examining past successes and future prospects of the CSC, this manuscript sheds light on how the consortium's multifaceted approach not only addresses current challenges in detecting potential cardiac failure modes but also paves the way for enhanced drug development and study design methodologies. Further, exploring future opportunities and challenges will focus on improving the translational predictability of nonclinical evaluations and reducing reliance on animal research in CV safety assessments.

Discovery of TRPA1 Antagonist GDC-6599: Derisking Preclinical Toxicity and Aldehyde Oxidase Metabolism with a Potential First-in-Class Therapy for Respiratory Disease.

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 metabolite─generated by aldehyde oxidase (AO)─possessing 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.

A prospective cohort study to identify clinical diagnostic and prognostic markers of primary immune thrombocytopenia in dogs.

BACKGROUND: Primary immune thrombocytopenia (pITP) in dogs presents a diagnostic challenge, and clinical markers of severity are lacking. OBJECTIVES: Identify clinicopathologic features that differentiate pITP from secondary ITP (sITP) and markers related to bleeding severity, transfusion, and survival of dogs with pITP. ANIMALS: Ninety-eight thrombocytopenic dogs (58 pITP and 40 sITP). METHODS: Client-owned dogs with platelet counts <50 000/µL were enrolled in a prospective, multi-institution cohort study. History and treatment information, through a maximum of 7 days, was recorded on standard data forms. Bleeding severity was scored daily using a bleeding assessment tool (DOGiBAT). At-admission blood samples were collected for CBC, biochemistry, C-reactive protein concentration, and coagulation panels, and to measure platelet surface-associated immunoglobulin G (PSAIg) and expression of platelet membrane proteins and phospholipids. Dogs with evidence of coincident disease were classified as sITP. RESULTS: No definitive pITP diagnostic test was found. However, pITP cases were characterized by lower platelet counts, D dimer concentrations, and platelet membrane protein expression than sITP cases. Differentiation between pITP and sITP was further enhanced using logistic regression modeling combining patient sex, coagulation profile, platelet count, D dimer, and PSAIg. A second model of pITP severity indicated that low hematocrit and high BUN concentration were associated with non-survival. Low hematocrit at admission, but not platelet count or DOGiBAT score, was associated with transfusion. CONCLUSIONS AND CLINICAL IMPORTANCE: Pending validation studies, models constructed from at-admission clinicopathologic findings may improve differentiation of pITP from sITP and identify the most severe pITP cases at the time of presentation.

Increased thrombin activatable fibrinolysis inhibitor activity is associated with hypofibrinolysis in dogs with sepsis.

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.

Anticoagulant effects of rivaroxaban, prednisone, alone and in combination, in healthy dogs.

BACKGROUND: The potential effects of glucocorticoid administration on rivaroxaban's anticoagulant bioactivity in dogs, and an appropriate rivaroxaban dosage regimen for dogs receiving glucocorticoid therapy are unknown. HYPOTHESIS/OBJECTIVES: The objective was to determine whether glucocorticoid administration influences the anticoagulant effects of rivaroxaban in healthy dogs. We hypothesized that administration of rivaroxaban and prednisone would reduce the anticoagulant intensity compared with rivaroxaban alone. ANIMALS: Nine healthy dogs. METHODS: Randomized, cross-over study. Dogs were administered prednisone (2 mg/kg, PO, q24h), rivaroxaban (1.5 mg/kg, PO, q24h), or prednisone and rivaroxaban, and the coagulation status was evaluated using prothrombin time (PT), and rivaroxaban-calibrated anti-Xa activity (RIVA, results were log10 transformed for analysis), before drug administration and on days 2, 4, and 8. Linear mixed models and correlation were used to evaluate associations in variables (P < .05 was considered significant). RESULTS: There were no differences in RIVA results for the rivaroxaban and prednisone/rivaroxaban groups on day 8 (P = .599, median 87 [range 45-156] to 167 [56-333], respectively, median difference 90 ng/mL [95% CI:87.3-161.8]) There was a strong correlation between RIVA and PT results when days 2, 4, and 8 were combined (r = .846, P < .001), and increased during drug administration, day 2 (r = .810, P < .001), day 4 (r = .863, P < .001), and day 8 (r = .885, P < .001). CONCLUSIONS AND CLINICAL IMPORTANCE: Clotting times in the PT correlate with rivaroxaban levels and may prove useful for drug monitoring. Prednisone administration had no apparent influence on the anticoagulant effects of rivaroxaban in healthy dogs, suggesting that combined therapy will not require dosage adjustments.

To Clot or Not to Clot: Deepening Our Understanding of Alterations in the Hemostatic System.

The session on the hemostatic system focused on new developments in coagulation and platelet biology as well as how therapeutic agents may affect hemostasis. The classic cascade model of coagulation was compared with the more recent models of cell-based and vascular-based coagulation, which may provide better insight on how the coagulation cascade works in vivo. A review of platelet biology highlighted that, as platelets age, desialylated platelets form and are recognized by Ashwell-Morell receptor (AMR), leading to hepatic uptake and subsequent increase in thrombopoietin (TPO) production. Administration of therapeutics that induce thrombocytopenia was also discussed, including Mylotarg, which is an antibody-drug conjugate that was shown to decrease human megakaryocyte development but had no effect on platelet aggregation. An acetyl co-A carboxylase inhibitor was shown to cause thrombocytopenia by inhibiting de novo lipogenesis, which is critical for the formation of the megakaryocyte demarcation membrane system responsible for platelet production. It was also illustrated how preclinical translation models have been very helpful in the development of adeno-associated virus (AAV) hemophilia B gene therapy and what old and new preclinical tools we have that can predict the risk of a prothrombotic state in people.

Complex Feline Disease Mapping Using a Dense Genotyping Array.

The current feline genotyping array of 63 k single nucleotide polymorphisms has proven its utility for mapping within breeds, and its use has led to the identification of variants associated with Mendelian traits in purebred cats. However, compared to single gene disorders, association studies of complex diseases, especially with the inclusion of random bred cats with relatively low linkage disequilibrium, require a denser genotyping array and an increased sample size to provide statistically significant associations. Here, we undertook a multi-breed study of 1,122 cats, most of which were admitted and phenotyped for nine common complex feline diseases at the Cornell University Hospital for Animals. Using a proprietary 340 k single nucleotide polymorphism mapping array, we identified significant genome-wide associations with hyperthyroidism, diabetes mellitus, and eosinophilic keratoconjunctivitis. These results provide genomic locations for variant discovery and candidate gene screening for these important complex feline diseases, which are relevant not only to feline health, but also to the development of disease models for comparative studies.

Coagulation testing in green iguanas (Iguana iguana) with development of prothrombin time assays using reptile and avian thromboplastin.

BACKGROUND: Captive reptiles often present with clinical signs suggestive of a clotting disorder or severe illness that can induce or exacerbate a coagulopathy. However, coagulopathies in reptiles are difficult to characterize due to lack of species-appropriate reagents to perform coagulation tests. The objective of this study was to develop screening tests to evaluate the extrinsic and common pathways of coagulation in green iguanas (Iguana iguana). KEY FINDINGS: Reptile and avian thromboplastin, extracted from reptile and avian brains, respectively, were used to initiate coagulation in prothrombin time (PT) assays and commercially available reagents were used to determine Russell's viper venom time, thrombin time, and fibrinogen using the Clauss method. Coagulation assays were performed on citrate-anticoagulated plasma from 18 healthy green iguanas. Results were summarized as median (minimum-maximum): PT (reptile thromboplastin), 34.8 seconds (27.1-42.1 s), PT (avian thromboplastin), 78.5 seconds (51.6-114.23 s), Russell's viper venom time, 56.15 seconds (18.4-79.7 s), thrombin time, 10 seconds (7.0-36.5 s), and fibrinogen, 258 mg/dl (89-563.0) (2.58 [0.89-5.63 g/L]). SIGNIFICANCE: Commercial reagents can be used to evaluate the common pathway and fibrinogen; however, avian- or reptile-sourced thromboplastin is preferred for a reliable coagulation trigger to perform the PT assay and evaluate the extrinsic pathway.

Prevalence of anticoagulant rodenticide exposure in red-tailed hawks (Buteo jamaicensis) and utility of clotting time assays to detect coagulopathy.

Anticoagulant rodenticides (ARs) continue to be used across the United States as a method for controlling pest rodent species. As a consequence, wild birds of prey are exposed to these toxicants by eating poisoned prey items. ARs prevent the hepatic recycling of vitamin K and thereby impede the post-translational processing of coagulation factors II, VII, IX, and X that are required for procoagulant complex assembly. Through this mechanism of action, ARs cause hemorrhage and death in their target species. Various studies have documented the persistence of these contaminants in birds of prey but few have attempted to use affordable and accessible diagnostic tests to diagnose coagulopathy in free-ranging birds of prey. In our study free-ranging red-tailed hawks were found to be exposed to difethialone and brodifacoum. Eleven of sixteen (68%) livers tested for AR exposure had detectable residues. Difethialone was found in 1/16 (6%), and brodifacoum was detected in 10/16 (62%) liver samples that were tested for rodenticide residues. Difethialone was found at a concentration of 0.18 ug/g wet weight and brodifacoum concentrations ranged from 0.003-0.234 ug/g wet weight. Two out of 34 (6%) RTHA assessed for blood rodenticide had brodifacoum in serum with measured concentrations of 0.003 and 0.006 ug/g. The range of clotting times in the prothrombin time (PT) and Russell's viper venom time assays for control RTHA were 16.7 to 39.7 s and 11.5 to 91.8 s, respectively. One study bird was diagnosed with clinical AR intoxication with a brodifacoum levels in blood of 0.006 and 0.234 ug/g wet weight in blood and liver respectively, a packed cell volume (PCV) of 19%, and PT and RVVT times of >180 s. No correlation was found between PT and RVVT in the control or free-range RTHA, and there was no relationship found between the presence of liver anticoagulant residues and clotting times in the PT and RVVT.

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats.

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced >3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding.

Clinical features and posttreatment monitoring of dogs administered rivaroxaban (2018-2020): 19 cases.

OBJECTIVE: To describe a population of sick dogs administered rivaroxaban monitored with a rivaroxaban-calibrated anti-Xa activity assay (aXa). DESIGN: Descriptive retrospective study. SETTING: Two veterinary teaching hospitals. ANIMALS: Client-owned dogs administered rivaroxaban and monitored with aXa from January 2018 to January 2020 were eligible for study. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Medical records were reviewed and 19 dogs with a variety of underlying disease processes were identified. Rivaroxaban was administered to 12 of 19 dogs (63%) with confirmed thrombosis, 4 of 19 dogs (21%) with a strong clinical suspicion of thrombosis, and in 3 of 19 dogs (16%) with no current evidence of thrombosis. The median rivaroxaban dose administered was 0.96 mg/kg/day (0.62-1.58 mg/kg/day), with 15 of 19 dogs (79%) receiving rivaroxaban once daily. Clopidogrel was concurrently administered to 11 of 19 dogs (58%). Complete or partial thrombus resolution was identified in 5 of 12 (42%) and 3 of 12 (25%) dogs, respectively. Rivaroxaban appeared safe, with only 1 of 19 dogs (5%), concurrently administered clopidogrel, developing evidence of mild hematuria. Posttreatment monitoring revealed that 8 of 19 dogs (42%) had aXa below the target (aXa range of 150-250 ng/ml associated with effective treatment and prevention of venous thrombosis in people). The remaining 3 to 19 dogs (16%) achieved this range, and 8 of 19 dogs (42%) exceeded the range. No significant relationship between the initial rivaroxaban dose administered and the corresponding aXa result was identified. There were also no significant differences in baseline clinicopathological variables in dogs in which aXa fell within or outside this range. CONCLUSIONS: aXa was most commonly measured in dogs receiving rivaroxaban with confirmed or suspected thrombosis. Dogs in this study received a range of rivaroxaban dosages and attained variable aXa values that were not directly correlated with dosage.

Preliminary evaluation of a flow cytometric assay with microsphere controls for the detection of platelet-bound antibodies in canine immune thrombocytopenia.

BACKGROUND: Canine immune thrombocytopenia (ITP) ranges from a mild to severe bleeding disorder, and platelet counts do not reliably predict clinical disease course. The detection of platelet autoantibodies may further define the disease phenotype, but variability in assay configurations and a lack of well-characterized controls limit the diagnostic utility of anti-platelet antibody assays. OBJECTIVES: We aimed to develop control reagents to facilitate the characterization of canine platelet surface-associated immunoglobulin (PSAIg) in flow cytometric assays. METHODS: Silica microspheres were coated with canine IgG and IgM to assess the reactivity of goat and rabbit origin anti-canine immunoglobulin reagents. They were also used as positive controls in the PSAIg assay. Preliminary assay evaluation and determination of sample stability used PRP isolated from seven healthy dogs and 26 dogs newly diagnosed with thrombocytopenia. RESULTS: Blood sample stability was established for up to a 48-hour storage time. The conjugated positive control microspheres demonstrated stable fluorescent labeling over a 2-year observation period. Rabbit and goat origin anti-dog IgM fluorescent antibody labels reacted nonspecifically with canine IgG. Rabbit origin anti-dog IgG antibody demonstrated greater class specificity for canine IgG than a goat origin antibody. Thrombocytopenic dogs had a broad range of membrane-bound immunoglobulin. Median PSAIgG for dogs with primary or secondary ITP (18.4%, 34.1%, respectively) were significantly higher than controls (3.8%, P < .05). CONCLUSIONS: The described assay reagents and procedures provide positive controls and allow consistent thresholding to define a positive test result, suitable for any flow cytometer. A rabbit anti-dog IgG fluorescent label demonstrated specificity for canine IgG and was useful for the detection of PSAIgG in thrombocytopenic dogs.

HYPOCOAGULABILITY EFFECT OF ADEQUAN IN DOMESTIC CHICKENS (GALLUS GALLUS) AND CHILEAN FLAMINGOS (PHOENICOPTERUS CHILENSIS).

Effective management of articular injuries in avian species is a known and frequent challenge. Potential treatments include many domestic animal therapeutics, such as Adequan®, which is used widely in dogs and horses. However, clinical reports have described hemorrhagic diatheses in a variety of avian species treated with varying doses and administration frequency of Adequan. This study investigated the hypocoagulability associated with parenteral administration of Adequan in avian species. Following a pilot dosing study in domestic chickens (Gallus gallus), citrated plasma from Chilean flamingos (Phoenicopterus chilensis) (n = 42) was spiked with Adequan to represent three dosing regimens (1 mg/kg, 5 mg/kg, 10 mg/kg). The fibrinogen content of plasma samples was determined and thrombin-clotting times (TCTs) were compared for the untreated (control) and spiked flamingo samples. The TCT for control and 1-mg/kg spiked plasma were not significantly different; however, both 5 mg/kg and 10 mg/kg spiked samples demonstrated significantly prolonged TCT (P-value < 0.0001) indicating hypocoagulability. These results support that Adequan given parenterally at 1 mg/kg can be utilized safely in clinical case management as an adjunctive treatment for osteoarthritis in flamingos and potentially other avian species.

Anticoagulant profile of subcutaneous enoxaparin in healthy dogs.

Our study objective was to identify a subcutaneous enoxaparin dosage that provided a consistent anticoagulant intensity in dogs. Our hypotheses were that a dose of 0.8 mg/kg would provide inconsistent anticoagulation, a higher dose would provide consistent anticoagulation over a greater duration of time, and viscoelastometry would effectively monitor the anticoagulant status. Six healthy dogs received two subcutaneous enoxaparin doses (0.8 and 2 mg/kg) for anti-Xa activity determinations and pharmacokinetic modeling. Based on calculations derived from these results, 1.3 mg/kg, SC, q8 h was administered for seven doses. Target ranges for anticoagulant intensity were defined as anti-Xa activity of 0.5-1 U/ml, and change from baseline of two viscoelastometric parameters: activated clotting time (ΔACT; ≥40 s), and clot rate (CRpost; ≤20 U/min). Following an initial injection at 1.3 mg/kg, anti-Xa activity of 5/6 dogs reached or exceeded the target range. Following the final dose, anti-Xa activity reached or exceeded the target range in all dogs, and ΔACT and CRpost values exceeded target for 2-6 and 4-12 h, respectively. At an enoxaparin dosage of 1.3 mg/kg, SC, q8 h, anti-Xa activity was consistently above the minimum threshold of the target range; however, the safety of this dosage remains to be determined.

Pharmacokinetics and Biologic Activity of Apixaban in Healthy Dogs.

Thrombosis is common in critically ill dogs and causes considerable morbidity and mortality. The direct factor Xa inhibitor apixaban is safe, efficacious, and convenient in humans. This study aimed to determine the pharmacokinetics (PK), bioactivity, protein binding, and bioavailability of apixaban following intravenous (IV) and oral (PO) administration to healthy dogs. Six healthy, adult, mixed-breed dogs were administered apixaban 0.18 mg/kg IV and then following a minimum 2-week washout period administered apixaban 0.2 mg/kg PO. Dogs were monitored using an apixaban-calibrated anti-Xa bioassay, prothrombin time (PT) and activated partial thromboplastin time (aPTT) and tissue-factor thromboelastography (TF-TEG). Plasma apixaban concentrations were measured using liquid chromatography-tandem mass spectrometry. Concentration-time plots were constructed, and PK modeling performed using compartmental methods. Administration of IV and PO apixaban was well-tolerated. Following IV administration, mean half-life was 4.1 h, and volume of distribution was 177 ml/kg. Apixaban was highly protein bound (98.6%). Apixaban concentrations and anti-Xa activity were highly correlated (R2 0.994, P < 0.0001). Intravenous apixaban significantly prolonged PT at time points up to 1 h, and aPTT at time points up to 0.25 h post-administration. Coagulation times were positively correlated with apixaban concentrations (PT R2 0.599, P < 0.0001; aPTT R2 0.430, P < 0.0001) and TF-TEG R-time was significantly prolonged 0.25 h post-administration. Following oral administration, mean bioavailability was 28.4%, lag time was 2 h, time to Cmax was 5 h and the apparent elimination half-life was 3.1 h. Oral apixaban significantly prolonged PT at 4, 6, and 8 h but aPTT and TF-TEG were not consistently affected by oral apixaban. Apixaban concentrations are best monitored using anti-Xa activity. Future studies should determine PK and bioactivity of other doses using commercial tablets and following multidose administration and establish safe, effective dosing ranges in sick dogs.

An in vitro study of canine cryopoor plasma to correct vitamin K-dependent coagulopathy in dogs.

OBJECTIVE: To compare the efficacy of fresh frozen plasma (FFP) with cryopoor plasma (CPP) to treat vitamin K-dependent factor deficiency in a canine in vitro setting. DESIGN: In vitro laboratory study. SETTING: University veterinary medical teaching hospital. ANIMALS: Seven units of FFP and 6 units of CPP from unique canine donors from the university veterinary blood bank. INTERVENTIONS: Canine FFP was adsorbed by oral barium sulfate suspension to mimic vitamin K-dependent coagulopathy. A sequential mixing study was completed by adding FPP or CPP to the adsorbed plasma. Measurements of prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen, and factor activities of factors II, VII, and IX (FII, FVII, and FIX) were compared between the 2 treatment groups. MEASUREMENTS AND MAIN RESULTS: When comparing the sequential addition of CPP or FPP to adsorbed plasma, the following had no statistical significance: PT (P = 0.94), aPTT (P = 0.66), FII (P = 0.05), and FIX (P = 0.90). There was a dose-dependent decrease with PT and aPTT and a dose-dependent increase with FII and FIX. In contrast, after the addition of either CPP or FFP, there was a significant difference between the treatment groups for the concentration of fibrinogen (P = 0.005) and activity of FVII (P = 0.044), with FFP resulting in a greater concentration of fibrinogen and CPP resulting in a greater concentration of FVII. Measurements of factor X (FX) were initially included in the study but were later excluded because FX appeared to be continually adsorbed even after the addition of CPP or FFP. CONCLUSIONS: CPP partially corrected the coagulation times and concentration of vitamin K-dependent coagulation factors to the same degree as FFP. CPP, generally less expensive than FFP, may provide an alternative treatment option for vitamin K-dependent coagulopathies, although in vivo testing is needed.

The canine activated platelet secretome (CAPS): A translational model of thrombin-evoked platelet activation response.

BACKGROUND: Domestic dogs represent a translational animal model to study naturally occurring human disease. Proteomics has emerged as a promising tool for characterizing human platelet pathophysiology; thus a detailed characterization of the core canine activated platelet secretome (CAPS) will enhance utilization of the canine model. The objectives of this study were development of a robust, high throughput, label-free approach for proteomic identification and quantification of the canine platelet (i) thrombin releasate proteins, and (ii) the protein subgroup that constitutes CAPS. METHODS: Platelets were isolated from 10 healthy dogs and stimulated with 50 nmol/L of γ-thrombin or saline. Proteins were in-solution trypsin-digested and analyzed by nano-liquid chromatography-tandem spectrometry. Core releasate proteins were defined as those present in 10 of 10 dogs, and CAPS defined as releasate proteins with a significantly higher abundance in stimulated versus saline controls (corrected P < .05). RESULTS: A total of 2865 proteins were identified; 1126 releasate proteins were present in all dogs, 650 were defined as CAPS. Among the differences from human platelets were a canine lack of platelet factor 4 and vascular endothelial growth factor C, and a 10- to 20-fold lower concentration of proteins such as haptoglobin, alpha-2 macroglobulin, von Willebrand factor, and amyloid-beta A4. Twenty-eight CAPS proteins, including cytokines, adhesion molecules, granule proteins, and calcium regulatory proteins have not previously been attributed to human platelets. CONCLUSIONS: CAPS proteins represent a robust characterization of a large animal platelet secretome and a novel tool to model platelet physiology, pathophysiology, and to identify translational biomarkers of platelet-mediated disease.

A Translational Model for Venous Thromboembolism: MicroRNA Expression in Hibernating Black Bears.

BACKGROUND: Hibernating American black bears have significantly different clotting parameters than their summer active counterparts, affording them protection against venous thromboembolism during prolonged periods of immobility. We sought to evaluate if significant differences exist between the expression of microRNAs in the plasma of hibernating black bears compared with their summer active counterparts, potentially contributing to differences in hemostasis during hibernation. MATERIALS AND METHODS: MicroRNA sequencing was assessed in plasma from 21 American black bears in summer active (n = 11) and hibernating states (n = 10), and microRNA signatures during hibernating and active state were established using both bear and human genome. MicroRNA targets were predicted using messenger RNA (mRNA) transcripts from black bear kidney cells. In vitro studies were performed to confirm the relationship between identified microRNAs and mRNA expression, using artificial microRNA and human liver cells. RESULTS: Using the bear genome, we identified 15 microRNAs differentially expressed in the plasma of hibernating black bears. Of these microRNAs, three were significantly downregulated (miR-141-3p, miR-200a-3p, and miR-200c-3p), were predicted to target SERPINC1, the gene for antithrombin, and demonstrated regulatory control of the gene mRNA expression in cell studies. CONCLUSIONS: Our findings suggest that the hibernating black bears' ability to maintain hemostasis and achieve protection from venous thromboembolism during prolonged periods of immobility may be due to changes in microRNA signatures and possible upregulation of antithrombin expression.

The influence of feeding and gastroprotectant medications on the Factor Xa inhibitory activity of orally administered rivaroxaban in normal dogs.

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.

Use of the ZDF rat to model dietary fat induced hypercoagulability is limited by progressive and fatal nephropathy.

INTRODUCTION: Zucker diabetic fatty (ZDF) rats are used widely as an animal model of metabolic syndrome and insulin resistance. Our study focused on the effects of high versus low dietary fat on the development of Type 2 diabetes in obese male ZDF rats (fa/fa), including biomarkers to detect early signs of hypercoagulability and vascular injury in the absence of overt thrombosis. METHODS: In this study, male (5/group) 10-week-old CRL:ZDF370(obese) rats were fed low (LFD, 16.7% fat) or high fat (HFD, 60% fat) diet for 12 or 15 weeks. Cohorts of 5 rats within diet groups were scheduled for sample collection after weeks 12 and 15. RESULTS: HFD-fed ZDF rats had oily coats, lower rates of food consumption, more accelerated weight gain and increased serum cholesterol (+15%) and triglyceride concentrations (+75%) vs. LFD-fed ZDF rats. Urinary ketones were observed only in HFD-fed ZDF rats and greater urine glucose and protein concentrations in HFD-fed ZDF vs. LFD-fed ZDF rats were seen. Hemostasis testing showed ~2-fold greater fibrinogen concentration, increased von Willebrand factor concentration, and high thrombin generation in HFD-fed ZDF vs LFD-fed ZDF rats. Increased mortality in the HFD-fed ZDF rat was attributed to exacerbations of altered carbohydrate metabolism as evidenced by ketonuria and nephropathy leading to renal failure. DISCUSSION: This characterization shows that the ZDF rat at the age, sex and weight used in this study is highly sensitive to dietary fat content that can exacerbate prothrombotic, metabolic and renal disturbances and increase mortality.