Evaluation of clopidogrel response in healthy cats using a novel viscoelastic test and thromboelastography.

Introduction: Cats with cardiomyopathy face an increased risk of arterial thromboembolism (ATE). Although clopidogrel is frequently utilized to mitigate this risk, feline responses to this therapy exhibit variability. This study evaluated 2 viscoelastic devices, thromboelastography (TEG) and Viscoelastic Coagulation Monitor (VCM), for monitoring clopidogrel in cats in comparison to light transmission aggregometry (LTA). Methods: Twenty-eight healthy cats received clopidogrel for 7 days. Blood was collected at baseline and after treatment for analysis by TEG, VCM, and LTA. Results: On LTA, maximum amplitude, slope, and area under the curve (AUC) significantly decreased after treatment (p < 0.0001). On VCM, maximum clot firmness (MCF) significantly increased after treatment (p = 0.002). On TEG, R-time significantly prolonged (p = 0.024), while K and alpha angle significantly changed (p = 0.0002 and p = 0.0014, respectively). There was a moderate negative correlation between TEG R-time and LTA AUC (r = -0.39, p = 0.042). Eight cats were identified as non-responders to clopidogrel. Of the 8 non-responders, 6 (75%) had shortened R time after treatment. VCM appeared to be less discriminatory in identifying non-responders. Discussion: LTA remained the gold standard of monitoring clopidogrel treatment in cats. Unexpected changes on VCM and TEG were likely related to high interindividual and assay variability and increased sensitivity of feline platelets. R-time on TEG may have potential utility for point-of-care monitoring of clopidogrel response in cats.

Platelet hyperresponsiveness and increased platelet-neutrophil aggregates in dogs with myxomatous mitral valve disease and pulmonary hypertension.

BACKGROUND: Pulmonary hypertension (PH) in dogs with myxomatous mitral valve disease (MMVD) is caused by increased pulmonary venous pressure. Thrombosis, vascular remodeling, and vasoconstriction mediated by platelets could exacerbate PH. HYPOTHESIS: Dogs with PH will exhibit a hypercoagulable state, characterized by increased platelet activation, platelet-leukocyte, and platelet-neutrophil aggregate formation. ANIMALS: Eleven dogs (≥3.5 kg) diagnosed with MMVD and PH and 10 dogs with MMVD lacking PH. METHODS: Prospective cohort ex vivo study. All dogs underwent echocardiographic examination, CBC, 3-view thoracic radiographs, and heartworm antigen testing. Severity of PH and MMVD were assessed by echocardiography. Viscoelastic monitoring of coagulation was assessed using thromboelastography (TEG). Platelet activation and platelet-leukocyte/platelet-neutrophil interactions were assessed using flow cytometry. Plasma serotonin concentrations were measured by ELISA. RESULTS: Unstimulated platelets from dogs with MMVD and PH expressed more surface P-selectin than MMVD controls (P = .03). Platelets from dogs with MMVD and PH had persistent activation in response to agonists. The number of platelet-leukocyte aggregates was higher in dogs with MMVD and PH compared with MMVD controls (P = .01). Ex vivo stimulation of whole blood resulted in higher numbers of platelet-neutrophil aggregates in dogs with MMVD and PH (P = .01). Assessment of hypercoagulability based on TEG or plasma serotonin concentrations did not differ between groups. CONCLUSION AND CLINICAL IMPORTANCE: Platelet hyperresponsiveness and increased platelet-neutrophil interaction occur in dogs with MMVD and PH, suggesting that platelets play a role of in the pathogenesis of PH. Clinical benefits of antiplatelet drugs in dogs with MMVD and PH require further investigation.

A novel ITGA2B double cytosine frameshift variant (c.1986_1987insCC) leads to Glanzmann's thrombasthenia in a cat.

BACKGROUND: Glanzmann's thrombasthenia (GT) is a congenital platelet disorder affecting approximately 1:1 000 000 people globally and characterized by impaired platelet aggregation and clot retraction. Autosomal recessive, loss-of-function, variants in ITGA2B or ITGB3 of the αIIbß3 receptor cause the disease in humans. A cat affected by Glanzmann's and macrothrombocytopenia was presented to the UC Davis VMTH. HYPOTHESIS/OBJECTIVES: Severe thrombopathia in this cat has an underlying genetic etiology. ANIMALS: A single affected patient, 2 age-matched clinically healthy controls, and a geriatric population (n = 20) of normal cats. METHODS: Physical examination and clinical pathology tests were performed on the patient. Flow cytometry and platelet aggregometry analyses for patient phenotyping were performed. Patient and validation cohort gDNA samples were extracted for Sanger sequencing of a previously identified ITGA2B (c.1986delC) variant. Reverse transcriptase PCR was performed on patient and healthy control PRP samples to verify ITGA2B variant consequence. RESULTS: A novel c.1986_1987insCC autosomal recessive variant in ITGA2B was identified. This variant was absent in a population of 194 unrelated cats spanning 44 different breeds. Complete loss of ITGA2B transcript and protein expression was verified by RT-PCR and flow cytometry, explaining the underlying etiology of GT, and likely macrothrombocytopenia, in this cat. CONCLUSIONS AND CLINICAL IMPORTANCE: This study emphasizes the role of precision medicine in cardiovascular disease of cats and identified yet another variant that may be of utility for screening in the feline population. This study provides a small-volume, standardized, successful protocol for adequate platelet RNA isolation and subsequent molecular assessment of gene expression in cats.

Naturally occurring canine laminopathy leading to a dilated and fibrosing cardiomyopathy in the Nova Scotia Duck Tolling Retriever.

Dilated cardiomyopathy (DCM) is characterized by decreased systolic function and dilation of one or both ventricles, often leading to heart failure or sudden death. Two 10-month-old sibling Nova Scotia Duck Tolling Retrievers (NSDTR) died acutely with evidence of dilated cardiomyopathy with myocardial fibrosis. Association analysis using two cases and 35 controls identified three candidate regions homozygous in the two cases. Whole genome sequencing identified a frameshift deletion in the LMNA gene (NC_049228.1:g.41688530del, NP_001274080:p.(Asp576ThrfsTer124)). Three retrospectively identified NSDTRs with sudden death before 2 years of age and severe myocardial fibrosis were also homozygous for the deletion. One 5 year old with sudden death and myocardial fibrosis was heterozygous for the deletion. This variant was not identified in 722 dogs of other breeds, nor was it identified to be homozygous in 784 NSDTR. LMNA codes for lamin A/C proteins, which are type V intermediate filaments that provide structural support to the nuclear membrane. In humans, LMNA variants can cause DCM with sudden death as well as diseases of striated muscles, lipodystrophy, neuropathies, and accelerated aging disorders. This frameshift deletion is predicted to affect processing of prelamin A into lamin A. Pedigree analysis in the NSDTR and functional evaluation of heterozygotes is consistent with a predominantly recessive mode of inheritance and possibly low penetrance in heterozygotes in contrast to people, where most pathogenic LMNA variants are dominantly inherited.

Abnormal developmental trajectory and vulnerability to cardiac arrhythmias in tetralogy of Fallot with DiGeorge syndrome.

Tetralogy of Fallot (TOF) is the most common cyanotic congenital heart disease. Ventricular dysfunction and cardiac arrhythmias are well-documented complications in patients with repaired TOF. Whether intrinsic abnormalities exist in TOF cardiomyocytes is unknown. We establish human induced pluripotent stem cells (hiPSCs) from TOF patients with and without DiGeorge (DG) syndrome, the latter being the most commonly associated syndromal association of TOF. TOF-DG hiPSC-derived cardiomyocytes (hiPSC-CMs) show impaired ventricular specification, downregulated cardiac gene expression and upregulated neural gene expression. Transcriptomic profiling of the in vitro cardiac progenitors reveals early bifurcation, as marked by ectopic RGS13 expression, in the trajectory of TOF-DG-hiPSC cardiac differentiation. Functional assessments further reveal increased arrhythmogenicity in TOF-DG-hiPSC-CMs. These findings are found only in the TOF-DG but not TOF-with no DG (ND) patient-derived hiPSC-CMs and cardiac progenitors (CPs), which have implications on the worse clinical outcomes of TOF-DG patients.

Evaluation of thrombin generation in dogs administered clopidogrel.

Introduction: The antiplatelet effect of clopidogrel can vary between patients. A modified thromboelastography (TEG) protocol (TEG-Platelet Mapping assay® [TEG-PM]) can be used for clopidogrel monitoring but is not widely available. Thrombin generation (TG) assays could offer a novel alternative. The main objective of this pilot study was to assess TG assay variables (lag time, peak, endogenous thrombin potential [ETP]) in dogs before and after 7 days of clopidogrel administration and compare with TEG-PM variables (maximum amplitude [MA]-ADP and percentage (%) inhibition). Methods: Six healthy mix-breed dogs were enrolled in this pilot study. Blood samples for platelet count, TG assays, and TEG-PM were obtained at two time points, corresponding to baseline, and after 7 days of clopidogrel administration (mean 2.3 +/- 0.3 mg/kg PO q24 hours). Data were then compared with a Student's t-test. Results: There was no significant change in TG assay variables performed on platelet poor plasma after 7 days of clopidogrel administration: lag time (Day 1: 1.8 +/- 0.2 min, Day 7: 1.8 +/- 0.2 min, p = 0.42); peak (Day 1: 76 +/- 7 nM, Day 7: 72 +/- 10 nM, p = 0.49); and ETP (Day 1: 399 +/- 27 nM*min, Day 7: 392 +/- 32 nM*min; p = 0.49). There were significant changes in TEG MA-ADP (Day 1: 19 +/- 8 mm, Day 7: 9 +/- 6 mm, p = 0.04) and % inhibition (Day 1: 58 +/- 27, Day 7: 99 +/- 0.3, p = 0.02). Discussion: Clopidogrel administration did not lead to changes in TG assay variables performed on platelet poor plasma samples, despite concomitant changes in TEG-PM variables consistent with platelet inhibition. Based on this pilot study, thrombin generation performed on platelet poor plasma may not be a useful antiplatelet monitoring tool in dogs.

Preventing Cardiogenic Thromboembolism in Cats: Literature Gaps, Rational Recommendations, and Future Therapies.

Cardiogenic arterial thromboembolism (CATE) is a devastating complication in cats with cardiomyopathies with significant morbidity and mortality. Despite recent advances in the diagnosis and treatment of CATE, its recurrence and mortality remain high. This highlights the urgent need for a greater understanding of CATE pathophysiology so that novel diagnostic tests and therapeutics can be developed. This comprehensive review aims to summarize existing literature on pathophysiology, clinical diagnosis, and current recommendations on the prevention and treatment of CATE. It also identifies and describes knowledge gaps and research priorities in the roles of immunothrombosis and procoagulant platelets in the pathogenesis of CATE.

Synergistic inhibitory effects of clopidogrel and rivaroxaban on platelet function and platelet-dependent thrombin generation in cats.

BACKGROUND: Dual antithrombotic treatment (DAT) with clopidogrel and rivaroxaban sometimes is prescribed to cats with hypertrophic cardiomyopathy at risk of thromboembolism. To date, no studies have evaluated their combined effects on platelet function. OBJECTIVES/HYPOTHESIS: Evaluate the safety of DAT in healthy cats and compare, ex vivo, platelet-dependent thrombin generation and agonist-induced platelet activation and aggregation in cats treated with clopidogrel, rivaroxaban, or DAT. We hypothesized that DAT would safely modulate agonist-induced platelet activation and aggregation more effectively than single agent treatment. ANIMALS: Nine apparently healthy 1-year-old cats selected from a research colony. METHODS: Unblinded, nonrandomized ex vivo cross-over study. All cats received 7 days of rivaroxaban (0.6 ± 0.1 mg/kg PO), clopidogrel (4.7 ± 0.8 mg/kg PO), or DAT with defined washout periods between treatments. Before and after each treatment, adenosine diphosphate (ADP)- and thrombin-induced platelet P-selectin expression was evaluated using flow cytometry to assess platelet activation. Platelet-dependent thrombin generation was measured by fluorescence assay. Platelet aggregation was assessed using whole blood impedance platelet aggregometry. RESULTS: No cats exhibited adverse effects. Of the 3 treatments, only DAT significantly decreased the number of activated platelets (P = .002), modulated platelet activation in response to thrombin (P = .01), dampened thrombin generation potential (P = .01), and delayed maximum reaction velocity (P = .004) in thrombin generation. Like clopidogrel, DAT inhibited ADP-mediated platelet aggregation. However, rivaroxaban alone resulted in increased aggregation and activation in response to ADP. CONCLUSION AND CLINICAL IMPORTANCE: Treatment combining clopidogrel and rivaroxaban (DAT) safely decreases platelet activation, platelet response to agonists, and thrombin generation in feline platelets more effectively than monotherapy with either clopidogrel or rivaroxaban.

Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues.

Cardiac in vitro models have become increasingly obtainable and affordable with the optimization of human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) differentiation. However, these CMs are immature compared to their in vivo counterparts. Here we study the cellular phenotype of hPSC-CMs by comparing their single-cell gene expression and functional profiles in three engineered cardiac tissue configurations: human ventricular (hv) cardiac anisotropic sheet, cardiac tissue strip, and cardiac organoid chamber (hvCOC), with spontaneously aggregated 3D cardiac spheroids (CS) as control. The CM maturity was found to increase with increasing levels of complexity of the engineered tissues from CS to hvCOC. The contractile components are the first function to mature, followed by electrophysiology and oxidative metabolism. Notably, the 2D tissue constructs show a higher cellular organization whereas metabolic maturity preferentially increases in the 3D constructs. We conclude that the tissue engineering models resembling configurations of native tissues may be reliable for drug screening or disease modeling.

Circulating neutrophil extracellular traps in cats with hypertrophic cardiomyopathy and cardiogenic arterial thromboembolism.

BACKGROUND: Cats with hypertrophic cardiomyopathy (HCM) are at risk of cardiogenic arterial thromboembolism (CATE). Neutrophil extracellular traps (NETs) may be a potential biomarker and therapeutic target for cardiomyopathy in cats. HYPOTHESIS/OBJECTIVES: Characterize NETs in cats with HCM or CATE. We hypothesized that circulating NETs assessed in the form of cell-free DNA (cfDNA) and citrullinated histone H3 (citH3) are increased in cats with HCM and CATE and associated with reported predisposing factors for thrombus formation. ANIMALS: Eighty-five cats including client-owned cats with HCM and CATE and staff- and student-owned clinically healthy cats without HCM. METHODS: After echocardiographic evaluations, NETs were measured as cfDNA and citH3. RESULTS: Cats with CATE had significant increases in cfDNA (11.2 ng/µL; interquartile range [IQR], 8.1 to 29.6) compared to those without HCM (8.2 ng/µL; IQR, 5.7 to 11.7 µL; P = .01) and were responsible for 75% to 83% of cases with cfDNA fragments sized 100 to 2000 base pairs. Citrullinated histone 3, detected in 52% of cats with HCM (31.1 ng/mL; IQR, 16.9 to 29.8), was significantly lower than in those with CATE (48.2 ng/mL; IQR, 34.2 to 60.2; P = .007). The citH3 concentrations correlated significantly with reported risk factors of CATE, such as left atrial auricular velocity. CONCLUSIONS AND CLINICAL IMPORTANCE: Neutrophil extracellualr traps, especially citH3, are increased in cats with HCM and CATE. They may serve as a novel therapeutic target and biomarker of thrombosis in cats with HCM.

Evaluation of autoantibodies to desmoglein-2 in dogs with and without cardiac disease.

Autoantibodies to desmoglein-2 have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC) in people. ARVC is a common disease in the Boxer dog. The role of anti-desmoglein-2 antibodies in Boxers with ARVC and correlation with disease status or severity is unknown. This prospective study is the first to evaluate dogs of various breeds and cardiac disease state for anti-desmoglein-2 antibodies. The sera of 46 dogs (10 ARVC Boxers, 9 healthy Boxers, 10 Doberman Pinschers with dilated cardiomyopathy, 10 dogs with myxomatous mitral valve disease, and 7 healthy non-Boxer dogs) were assessed for antibody presence and concentration via Western blotting and densitometry. Anti-desmoglein-2 antibodies were detected in all dogs. Autoantibody expression did not differ between study groups and there was no correlation with age or body weight. In dogs with cardiac disease, there was weak correlation with left ventricular dilation (r = 0.423, p = 0.020) but not left atrial size (r = 0.160, p = 0.407). In ARVC Boxers there was strong correlation with the complexity of ventricular arrhythmias (r = 0.841, p = 0.007) but not total number of ectopic beats (r = 0.383, p = 0.313). Anti-desmoglein-2 antibodies were not disease specific in the studied population of dogs. Correlation with some measures of disease severity requires further study with larger populations.

Case report: Disseminated intravascular coagulation in a dog following treatment with melarsomine for Dirofilaria immitis.

Disseminated intravascular coagulation following melarsomine therapy for Dirofilaria immitis (D. immitis) is reported in a 9-year-old female intact pit bull-type dog. The dog had been diagnosed with D. immitis (antigen and microfilaria positive) and treated with imidacloprid, moxidectin, doxycycline and 3 doses of melarsomine over a 92-day period. Seven days after the third melarsomine injection, the patient was presented to her family veterinarian due to right pelvic limb swelling. Prothrombin and activated partial thromboplastin times were prolonged beyond the detectable range. Treatment included vitamin K1 and fresh frozen plasma (FFP) prior to referral to the authors' institution. At this time the patient remained coagulopathic. Further investigations included thoracic radiographs, abdominal ultrasound and an echocardiogram. The patient was administered multiple units of packed red blood cells and FFP, sildenafil, dexamethasone SP, aminocaproic acid and vitamin K1. Repeat CBC approximately 20 h after admission showed persistent anemia and thrombocytopenia. Despite ongoing administration of FFP, a repeat coagulation panel showed worsening of the coagulopathy with prothrombin time of 84.2s [reference interval (RI) 7.0-9.3s], activated partial thromboplastin time >140s (RI 10.4-12.9s) and fibrinogen <50 mg/dL (RI 109-311 mg/dL). Following discussion with the owners, the patient was euthanized. Necropsy was performed and confirmed heartworm infection with severe pulmonary arterial thrombosis, vascular remodeling, and intraluminal degenerate nematodes. Multifocal subcutaneous and organ hemorrhage was apparent. Although coagulopathy has been described in caval syndrome associated with heartworm disease and is listed as a potential side effect of melarsomine administration, this is the first report of documented disseminated intravascular coagulation following melarsomine treatment for D. immitis. Potential mechanisms for the coagulopathy are discussed and the case report highlights a rare, but serious complication of adulticide therapy.

Platelet Priming and Activation in Naturally Occurring Thermal Burn Injuries and Wildfire Smoke Exposure Is Associated With Intracardiac Thrombosis and Spontaneous Echocardiographic Contrast in Feline Survivors.

Wildfires pose a major health risk for humans, wildlife, and domestic animals. We previously discovered pathophysiologic parallels between domestic cats with naturally occurring smoke inhalation and thermal burn injuries and human beings with similar injuries; these were characterized by transient myocardial thickening, cardiac troponin I elevation and formation of intracardiac thrombosis. While the underlying mechanisms remain unclear, results from murine models suggest that platelet priming and activation may contribute to a global hypercoagulable state and thrombosis. Herein, we evaluated and compared the degree of platelet activation, platelet response to physiologic agonists and levels of platelet-derived microvesicles (PDMV) in 29 cats with naturally occurring wildfire thermal injuries (WF), 21 clinically healthy cats with subclinical hypertrophic cardiomyopathy (HCM) and 11 healthy cats without HCM (CC). We also quantified and compared circulating PDMVs in WF cats to CC cats. In addition, we examined the association between thrombotic events, severity of burn injuries, myocardial changes, and the degree of platelet activation in cats exposed to wildfires. Flow cytometric detection of platelet surface P-selectin expression showed that WF cats had increased platelet response to adenosine diphosphate (ADP) and thrombin compared to the two control groups indicating the presence of primed platelets in circulation. In addition, cats in the WF group had increased circulating levels of PDMV, characterized by increased phosphatidylserine on the external leaflet. Cats in the WF group with documented intracardiac thrombosis had elevated platelet activation and platelet priming in the presence of ADP. While high dose arachidonic acid (AA) mostly resulted in platelet inhibition, persistent response to AA was noted among cats in the WF group with intracardiac thrombosis. Univariate and multiple logistic regression analyses demonstrated that increased platelet response to AA was independently associated with thrombotic events. This is the first study reporting the significant association between platelet priming and intracardiac thrombosis in domestic cats with naturally occurring wildfire-related injuries and smoke inhalation. Further studies are required to delineate additional mechanisms between inflammation and thrombosis, especially regarding platelet primers and the cyclooxygenase pathway. One Sentence Summary: Platelet activation and shedding of platelet-derived microvesicles due to platelet priming is present following naturally occurring wildfire smoke exposure and thermal burn injuries in a population of domestic cats.

Clinical, Diagnostic, and Imaging Findings in Three Juvenile Dogs With Paraspinal Hyperesthesia or Myelopathy as a Consequence of Hemophilia A: A Case Report.

Three juvenile dogs presented with an acute onset of paraspinal hyperesthesia and/or neurologic deficits. These dogs underwent anesthesia for MRI and additional diagnostics. The thoracolumbar MRI in Dog 1 revealed an accumulation of T2-weighted (T2W) hyperintense, T1-weighted (T1W) iso- to hyperintense, contrast enhancing extradural material. The differential diagnoses were meningitis with secondary hemorrhage or empyema or late subacute hemorrhage. The initial cervical MRI in Dog 2 revealed T1W meningeal contrast enhancement suspected to be secondary to meningitis. A repeat MRI following neurologic decline after CSF sampling revealed a large area of T2W and T1W hyperintensity between fascial planes of the cervical musculature as well as T2W iso- to hyperintense and T1W iso- to hypointense extradural material at the level of C1 consistent with hemorrhage. The cervical MRI in Dog 3 revealed T2W hyperintense and T1W iso- to hypointense extradural compressive material consistent with hemorrhage. Dogs 1 and 2 underwent CSF sampling and developed complications, including subcutaneous hematoma and vertebral canal hemorrhage. Dog 3 underwent surgical decompression, which revealed a compressive extradural hematoma. In each case, a hemophilia panel including factor VIII concentration confirmed the diagnosis of hemophilia A. Dog 1 had a resolution of clinical signs for ~5 months before being euthanized from gastrointestinal hemorrhage. Dog 2 was euthanized due to neurologic decompensation following CSF sampling. Dog 3 did well for 2 weeks after surgery but was then lost to follow-up. This case series provides information on clinical signs, MRI findings, and outcome in 3 juvenile dogs with hemophilia A that developed neurologic deficits or paraspinal hyperesthesia secondary to spontaneous or iatrogenic vertebral canal hemorrhage. Hemophilia A should be considered as a differential in any young dog presenting with an acute onset of hyperesthesia with or without neurologic deficits. This diagnosis should be prioritized in young male dogs that have other evidence of hemorrhage on physical exam.

Reducing meningo-cerebral adhesions by implanting an interpositional subdural polyesterurethane graft after high-grade glioma resection.

BACKGROUND: Meningo-cerebral adhesions are frequently encountered during recurrent high-grade glioma resections. Adhesiolysis not only lengthens operation times, but can also induce focal cortical tissue injury that could affect overall survival. METHODS: Immediately after the primary resection of a high-grade glioma, a polyesterurethane interpositional graft was implanted in the subdural space covering the entire exposed cortex as well as beneath the dural suture line. No postoperative complications were documented. All patients received adjuvant radiotherapy. Upon repeat resection for focal tumor recurrence, the graft was shown to effectively reduce meningo-cerebral adhesion development. CONCLUSION: The implantation of a synthetic subdural graft is a safe and effective method for preventing meningo-cerebral adhesions.

Dual therapy with clopidogrel and rivaroxaban in cats with thromboembolic disease.

OBJECTIVES: Feline arterial thromboembolism (ATE), an often devastating outcome, was recently shown to affect 11.3% of cats with hypertrophic cardiomyopathy over 10 years. Current American College of Veterinary Internal Medicine guidelines recommend the use of clopidogrel in cats at risk for ATE, with addition of a factor Xa inhibitor in very high risk or post-ATE cases. To date, no studies have examined the safety or efficacy of this combined antithrombotic therapy. This retrospective case series aimed to assess the frequency and type of adverse events that occurred in cats prescribed dual clopidogrel and rivaroxaban therapy. Secondary aims were to evaluate indications for dual therapy and clinical outcome. METHODS: The study included 32 cats prescribed clopidogrel (18.75 mg PO q24h) and rivaroxaban (2.5 mg PO q24h) on an outpatient basis over a 5-year period. RESULTS: Cats were prescribed dual therapy for at least one of the following: ATE event (n = 18), presence of an intracardiac thrombi (n = 17) or presence of spontaneous echocardiographic contrast (SEC) (n = 16). Five cats experienced adverse effects that could be attributed to medications, a median of 13 days from initiation (epistaxis, hematemesis, hematochezia or hematuria). No cat required hospitalization as a result of these events. Median survival time from onset of therapy was 257 days (interquartile range [IQR] = 38-497) for all cats, 502 days (IQR = 171-663) for ATE cats, 725 days (IQR = 133-856) for cats with an ATE to two or more limbs and 301 days (IQR = 221-431) for cats with only one limb affected. Recurrence rate of ATE while on dual therapy was 16.7%; no cat newly developed an ATE while on dual therapy. CONCLUSIONS AND RELEVANCE: Dual antithrombotic therapy with clopidogrel and rivaroxaban resulted in a low reported incidence of adverse events. Cats placed on dual therapy for an ATE event experienced a low rate of recurrence and effective thromboprophylaxis was achieved in cats with intracardiac thrombi or SEC.

Neutrophil extracellular traps in feline cardiogenic arterial thrombi: a pilot study.

OBJECTIVES: The aim of this study was to investigate the spatial distribution of neutrophil extracellular traps (NETs) in cardiogenic arterial thromboembolism (CATE). Specifically, we aimed to examine the related structural features of NETs in feline arterial thrombi in relation to their arterial locations. METHODS: Paraffin-embedded aortic bifurcations from nine cats with hypertrophic cardiomyopathy (four with CATE and five without) were deparaffinized, and NETs were identified by immunodetection based on colocalization of cell-free DNA, citrullinated histone H3 and neutrophil elastase. The distribution of NETs in thrombi within the aortic bifurcations and common iliac arteries (CIAs) was compared based on their proximity to the descending aorta (proximal, mid, distal). Ten random fields per section were captured at × 10 and × 20 magnification for each section of the clot and analyzed. RESULTS: The distributions of NETs in thrombi within the aortic bifurcation and CIAs were found to differ in relation to their assigned zones (proximal, mid, distal; P = 0.04); NETs were concentrated mostly in the proximal region in the aortic bifurcations (47.56%, interquartile range [IQR] 14.07-77.95) and CIAs (44.69%, IQR 24.65-85.28), compared with the distal regions (2.69%, IQR 0.10-50.04 [P = 0.027]; 7.08%, IQR 1.27-59.33 [P = 0.02]). CONCLUSIONS AND RELEVANCE: The variation in NET distribution within arterial thrombi may shed light on the pathogenesis of thrombus growth. This may be due to possible neutrophil entrapment or variations in shear stress.

Arrhythmic Risk Assessment of Hypokalaemia Using Human Pluripotent Stem Cell-Derived Cardiac Anisotropic Sheets.

Introduction: Hypokalaemia, defined as an extracellular concentration of K+ below 3.5 mM, can cause cardiac arrhythmias by triggered or re-entrant mechanisms. Whilst these effects have been reported in animal and human stem cell-based models, to date there has been no investigation in more complex structures such as the human ventricular cardiac anisotropic sheet (hvCAS). Here, we investigated arrhythmogenicity, electrophysiological, and calcium transient (CaT) changes induced by hypokalaemia using this bioengineered platform. Methods: An optical mapping technique was applied on hvCAS derived from human pluripotent stem cells to visualize electrophysiological and CaT changes under normokalaemic (5 mM KCl) and hypokalaemic (3 mM KCl) conditions. Results: Hypokalaemia significantly increased the proportion of preparations showing spontaneous arrhythmias from 0/14 to 7/14 (Fisher's exact test, p = 0.003). Hypokalaemia reduced longitudinal conduction velocity (CV) from 7.81 to 7.18 cm⋅s-1 (n = 9, 7; p = 0.036), transverse CV from 5.72 to 4.69 cm⋅s-1 (n = 12, 11; p = 0.030), prolonged action potential at 90% repolarization (APD90) from 83.46 to 97.45 ms (n = 13, 15; p < 0.001), increased action potential amplitude from 0.888 to 1.195 ΔF (n = 12, 14; p < 0.001) and CaT amplitude from 0.76 to 1.37 ΔF (n = 12, 13; p < 0.001), and shortened effective refractory periods from 242 to 165 ms (n = 12, 13; p < 0.001). Conclusion: Hypokalaemia exerts pro-arrhythmic effects on hvCAS, which are associated with alterations in CV, repolarization, refractoriness, and calcium handling. These preparations provide a useful platform for investigating electrophysiological substrates and for conducting arrhythmia screening.

Lipopolysaccharide-Activated Canine Platelets Upregulate High Mobility Group Box-1 via Toll-Like Receptor 4.

High mobility group box-1 (HMGB1) and the toll-like receptor 4 (TLR4) axis is a key mediator of inflammation. Platelet-derived high mobility group box-1 (HMGB1) may also play a critical role in sepsis-mediated thrombosis resulting in complications like disseminated intravascular coagulation and multiple organ failure. While elevated levels of HMGB1 have been documented in humans and dogs with systemic inflammatory response syndrome and sepsis, a better understanding of how platelet agonists and lipopolysaccharide (LPS) mediate platelet HMGB1 expression would open doors to novel therapies for sepsis-mediated thrombosis. Herein, we sought to determine if canine platelets express HMGB1 in the presence or absence of LPS and agonists (ADP or thrombin) and if surface expression of HMGB1 is dependent on platelet TLR4. Canine platelets were unstimulated (resting) or activated with thrombin or adenosine diphosphate (ADP) in the presence or absence of Escherichia coli LPS prior to flow cytometric and western blot analyses for HMGB1 expression. We also treated canine platelets with or without TLR4 function blocking antibody or its isotype control. We discovered that while thrombin upregulated both surface and cellular HMGB1 expression, LPS-mediated activation in the presence of ADP priming led to upregulation of surface HMGB1 expression. This expression was found to be most prominent in platelets that had undergone alpha-granule secretion. Inhibition of TLR4 attenuated LPS-induced HMGB1 expression indicating that exteriorization of HMGB1 may be dependent on the non-genomic pathway of platelet TLR4. Our findings indicate that upregulation of platelet-derived HMGB1 occurs as a result of thrombin or TLR4-mediated activation in dogs. Future studies should explore the translational implication of platelet-derived HMGB1 as novel therapeutic targets in humans and dogs with sepsis.

A genetic polymorphism in P2RY1 impacts response to clopidogrel in cats with hypertrophic cardiomyopathy.

Clopidogrel is converted to its active metabolite by cytochrome P450 isoenzymes and irreversibly inhibits platelet activation by antagonizing the adenosine-diphosphate (ADP) receptor. It is frequently used in cats with hypertrophic cardiomyopathy (HCM) to prevent thromboembolic complications. However, significant interpatient variability of the response to clopidogrel therapy has been suspected. In this study, we assessed the impact of single nucleotide polymorphisms (SNPs) within ADP receptor (P2RY1, P2RY12) and cytochrome P450 isoenzyme (CYP2C41) genes on platelet inhibition by clopidogrel administration in cats with HCM. Forty-nine cats completed the study, and blood samples were obtained before and after clopidogrel therapy to assess the degree of platelet inhibition based on flow cytometry and whole blood platelet aggregometry. Plasma concentrations of clopidogrel metabolites were measured after the last dose of clopidogrel. Whole blood platelet aggregometry revealed a significant reduction of platelet inhibition by clopidogrel in cats with the P2RY1:A236G and the P2RY12:V34I variants. The association with the P2RY1:A236G variant and clopidogrel resistance remained significant after adjustment for multiple comparisons. This study demonstrated that a genetic polymorphism in the P2RY1 gene altered response to clopidogrel therapy and suggests that clinicians may consider alternative or additional thromboprophylactic therapy in cats with the P2RY1:A236G variant.