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Abstract 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.

IntroductionCardiogenic arterial thromboembolism (CATE) is a life-threatening complication of hypertrophic cardiomyopathy (HCM) with a high mortality rate. As the primary responders in hemostasis, platelets play a crucial role in the progression of CATE. Procoagulant platelets are a subpopulation of activated platelets that facilitate thrombin generation to strengthen thrombus structure. Since their discovery, procoagulant platelets have gained a considerable amount of attention due to their potential role in thromboembolic complications. Uncovering the key phenotypic markers and agonists underlying the procoagulant response in feline platelets may provide diagnostic and therapeutic insights in the treatment of CATE. However, species differences in platelet physiology and the sensitive nature of feline platelets pose some significant challenges in studying procoagulant platelets in cats.ObjectivesTo first devise a flow cytometric method to sequentially assess procoagulant platelet markers and to identify agonists that could induce procoagulant platelet phenotypes. Furthermore, a novel scoring system was established to evaluate the procoagulant tendency of platelets in cats.MethodsPlatelets were isolated from eight healthy cats and activated by thrombin in the presence or absence of collagen (COL) and convulxin (CVX). The following markers were measured sequentially by flow cytometry: (1) loss of mitochondrial membrane potential (ΔΨm), (2) phosphatidylserine (PS) externalization, and (3) P-selectin upregulation.ResultsThrombin in the presence of CVX significantly reduced ΔΨm and induced P-selectin upregulation (p = 0.0078, p = 0.0128, respectively). In addition, thrombin-treated platelets in the presence of COL and CVX augmented PS externalization significantly (p = 0.043, p = 0.0172, respectively). Of the agonists tested, thrombin and CVX resulted in the highest procoagulant tendency score with 75% cats reaching a score ≥2/3. The number of cats with a procoagulant tendency score of 2 or 3 for thrombin and thrombin + COL was 4/8 (50%) and 5/8 (62.5%), respectively.DiscussionSequential analysis of procoagulant markers via flow cytometry may be an effective technique for studying procoagulant platelets in cats.

Abstract 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 αIIbβ3 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.

Abstract 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.

IntroductionCats 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).MethodsTwenty-eight healthy cats received clopidogrel for 7 days. Blood was collected at baseline and after treatment for analysis by TEG, VCM, and LTA.ResultsOn LTA, maximum amplitude, slope, and area under the curve (AUC) significantly decreased after treatment (p

Abstract 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.

Abstract 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.

Abstract 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.

IntroductionThe 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).MethodsSix 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.ResultsThere 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).DiscussionClopidogrel 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.

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

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 SummaryPlatelet 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.

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.