Effects of peripheral active warming and passive insulation on core body temperature during feline ovariohysterectomy: a multi-arm randomized clinical trial.

OBJECTIVES: Hypothermia is a common complication of anesthesia, particularly in cats. Some veterinarians insulate the extremities of cats as a preventive measure, and there is evidence that heating the extremities of dogs decreases the rate of heat loss from the core. This study investigated whether active warming or passive insulation of the extremities of cats resulted in a slower decrease in rectal temperature during anesthesia. METHODS: Female cats were assigned via block randomization to passive (cotton toddler socks), active (heated toddler socks) or control group (uncovered extremities). Rectal temperature was monitored every 5 mins from induction through return to trap/carrier (final temperature). Multivariable linear regression models were used to compare temperature (rate change and final) between groups. RESULTS: There were 164 cats with 1757 temperature readings. Mean total duration of anesthesia was 53 ± 13 mins. The temperature of all groups decreased linearly over time (all P <0.0001), with the rates of temperature decrease being -0.039°F/min (95% confidence interval [CI] -0.043 to -0.035)/-0.022°C (95% CI -0.024 to -0.019), -0.039°F/min (95% CI -0.042 to -0.035)/-0.022°C (95% CI -0.023 to -0.019) and -0.029°F/min (95% CI -0.032 to -0.025)/-0.016°C (95% CI -0.018 to -0.014) for the control, passive and active groups, respectively. The control, passive and active groups had median final temperatures of 98.4°F (interquartile range [IQR] 97.6-99.4)/36.9°C (IQR 36.4-37.4), 98.0°F (IQR 97.2-98.7)/36.7°C (IQR 36.2-37.1) and 99.1°F (IQR 97.7-100.0)/37.3°C (IQR 36.5-37.8), respectively. After controlling for weight, postinduction temperature and duration of anesthesia, and as compared with controls, the final temperature of the active group was predicted to be 0.54°F (95% CI 0.03-1.01)/0.3°C (95% CI 0.02-0.56) greater (P = 0.023), while the passive group was not significantly different (P = 0.130). CONCLUSIONS AND RELEVANCE: The rate of rectal temperature decrease was significantly slower for the active group compared with the other groups. Although the cumulative difference in final temperature reading was modest, superior materials might enhance performance. Cotton toddler socks alone did not slow the rate of temperature decrease.

Assessment of Dilute Epinephrine and Mechanical Pressure for Prevention of Feline Postoperative Scrotal Hematoma: A Randomized Controlled Trial.

To determine whether two immediately postoperative preventive procedures, dilute epinephrine (1:400,000) as a scrotal wash or application of controlled mechanical pressure to the scrotum, reduce the risk or severity of scrotal hematoma following routine castration. Male cats with two descended testicles presenting to Midwestern University's Trap Neuter Return program were eligible for inclusion. Cats were assigned via block randomization to control, dilute epinephrine wash, or controlled mechanical pressure groups. For the epinephrine group, 0.2 ml (0.008 mg) of epinephrine diluted with sterile saline was instilled inside the scrotum. In the case of mechanical pressure, a broad-based clip generating less than 0.5 kg of pressure was applied for 10 minutes. Cats were evaluated for scrotal hematoma and the need for treatment by a veterinarian blinded to treatment group. Multivariable logistic regression was used to determine if the incidence of scrotal hematoma or scrotal hematoma requiring treatment was different between groups while controlling for other variables. There were 276 cats with a median age of 30 months (IQR 12,48) and a mean weight of 3.5 kg (SD 1.2). Scrotal hematomas were noted in 15 of the 92 (16%) control cats, as compared with 12 of the 92 (13%) epinephrine and nine of the 92 (10%) pressure cats. Treatment was required for 10 (67%) control, six (50%) epinephrine, and three (33%) pressure hematomas. Regression demonstrated a decreased risk of scrotal hematoma requiring treatment for cats in the pressure group (OR = 0.2, P = .044) controlling for weight (OR = 2.2, P = .006) and surgical duration (OR = 1.1, P = .026). Weight was the only significant variable for the presence of scrotal hematoma (OR = 2.2, P < .0001). Controlled mechanical pressure applied immediately after routine castration can help decrease the proportion of scrotal hematomas that require treatment.

Pharmacokinetics of fluoxetine in horses following oral administration.

This study aimed to investigate pharmacokinetics of fluoxetine in horses and validate a method for liquid chromatography mass spectrometry analysis of serum levels. Fluoxetine pharmacokinetics were determined using 10 healthy, adult horses. Fluoxetine pharmacokinetics following a single oral dose (0.25 mg/kg) were determined using blood samples collected prior to and at several time points over 7 days following administration. Serum concentrations of fluoxetine and its bioactive metabolite norfluoxetine were measured using liquid chromatography coupled to an accurate mass/high-resolution mass spectrometer. Pharmacokinetic parameters were estimated using a noncompartmental model. Time to maximum serum concentration and serum half-life of fluoxetine was 1.5 and 15.6 h, respectively. Steady-state serum concentrations were evaluated using five horses each receiving fluoxetine (0.25 mg/kg, PO, q24hrs) for 8 weeks and were found to be 62.9 ± 25.5 ng/ml on average. Norfluoxetine was not detected in any sample.

Pharmacokinetics and competitive pharmacodynamics of ADP-induced platelet activation after oral administration of clopidogrel to horses.

OBJECTIVE: To determine pharmacokinetics and pharmacodynamics after oral administration of a single dose of clopidogrel to horses. ANIMALS: 6 healthy adult horses. PROCEDURES: Blood samples were collected before and at various times up to 24 hours after oral administration of clopidogrel (2 mg/kg). Reactivity of platelets from each blood sample was determined by optical aggregometry and phosphorylation of vasodilator-stimulated phosphoprotein (VASP). Concentrations of clopidogrel and the clopidogrel active metabolite derivative (CAMD) were measured in each blood sample by use of liquid chromatography-tandem mass spectrometry, and pharmacokinetic parameters were determined with a noncompartmental model. RESULTS: Compared with results for preadministration samples, platelet aggregation in response to 12.5µM ADP decreased significantly within 4 hours after clopidogrel administration for 5 of 6 horses. After 24 hours, platelet aggregation was identical to that measured before administration. Platelet aggregation in response to 25µM ADP was identical between samples obtained before and after administration. Phosphorylation of VASP in response to ADP (20µM) and prostaglandin E1 (3.3µM) was also unchanged by administration of clopidogrel. Time to maximum concentration of clopidogrel and CAMD was 0.54 and 0.71 hours, respectively, and calculated terminal-phase half-life of clopidogrel and CAMD was 1.81 and 0.97 hours, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Clopidogrel or CAMD caused competitive inhibition of ADP-induced platelet aggregation during the first 24 hours after clopidogrel administration. Because CAMD was rapidly eliminated from horses, clopidogrel administration may be needed more frequently than in other species in which clopidogrel causes irreversible platelet inhibition. (Am J Vet Res 2019;80:505-512).

Seasonal influences on CAPs exposures: differential responses in platelet activation, serum cytokines and xenobiotic gene expression.

Increasing evidence suggests a role for a systemic pro-coagulant state in the pathogenesis of cardiac dysfunction subsequent to inhalation of airborne particulate matter (PM). We evaluated platelet activation, systemic cytokines and pulmonary gene expression in mice exposed to concentrated ambient particulate matter (CAPs) in the summer of 2008 (S08) and winter of 2009 (W09) from the San Joaquin Valley of California, a region with severe PM pollution episodes. Additionally, we characterized the PM from both exposures including organic compounds, metals, and polycyclic aromatic hydrocarbons. Mice were exposed to an average of 39.01 µg/m(3) of CAPs in the winter and 21.7 µg/m3 CAPs in the summer, in a size range less than 2.5 µm for 6 h/day for 5 days per week for 2 weeks. Platelets were analyzed by flow cytometry for relative size, shape, CD41, P-selectin and lysosomal associated membrane protein-1 (LAMP-1) expression. Platelets from W09 CAPs-exposed animals had a greater response to thrombin stimulation than platelets from S08 CAPs-exposed animals. Serum cytokines were analyzed by bead based immunologic assays. W09 CAPs-exposed mice had elevations in IL-2, MIP-1α, and TNFα. Laser capture microdissection (LCM) of pulmonary vasculature, parenchyma and airways all showed increases in CYP1a1 gene expression. Pulmonary vasculature showed increased expression of ICAM-1 and Nox-2. Our findings demonstrate that W09 CAPs exposure generated a greater systemic pro-inflammatory and pro-coagulant response to inhalation of environmentally derived fine and ultrafine PM. Changes in platelet responsiveness to agonists, seen in both exposures, strongly suggests a role for platelet activation in the cardiovascular and respiratory effects of particulate air pollution.

Effects of preparation method, shear force, and exposure to collagen on release of growth factors from equine platelet-rich plasma.

OBJECTIVE: To test the hypotheses that preparation method, exposure to shear force, and exposure to collagen affect the release of growth factors from equine platelet-rich plasma (PRP). SAMPLE POPULATION: PRP obtained from 6 horses. PROCEDURES: PRP was prepared via 2 preparation methods (tube and automated) and subjected to 6 treatment conditions (resting, detergent, exposure to shear via 21- and 25-gauge needles, and exposure to collagen [10 and 20 µg/mL]). Concentrations of platelet-derived growth factor, isoform BB (PDGF-BB); transforming growth factor ß, isoform 1 (TGFß1); and insulin-like growth factor, isoform 1 (IGF-1) were quantified by use of ELISAs. Statistical analysis was conducted via repeated-measures ANOVA. RESULTS: Platelet numbers were significantly higher in tube-prepared PRP than in automated-prepared PRP Growth factor concentrations did not differ significantly between preparation methods. Mean PDGF-BB concentration ranged from 134 to 7,157 pg/mL, mean TGFß1 concentration ranged from 1,153 to 22,677 pg/mL, and mean IGF-1 concentration ranged from 150 to 280 ng/mL. Shear force did not affect growth factor concentrations. Dose-dependent increases in PDGF-BB and TGFß1 were detected in response to collagen, but equalled only 10% of the estimated total platelet content. Concentrations of IGF-1 were not significantly different among treatments and negative or positive control treatments. Serum concentrations of PDGF-BB and TGFß1 exceeded concentrations in PRP for most treatment conditions. CONCLUSIONS AND CLINICAL RELEVANCE: Release of growth factors from equine PRP was negligible as a result of the injection process alone. Investigation of platelet-activation protocols is warranted to potentially enhance PRP treatment efficacy in horses.

Analysis of a commercial dimethyl-sulfoxide-stabilized frozen canine platelet concentrate by turbidimetric aggregometry.

OBJECTIVES: To assess platelet function of a commercial dimethyl-sulfoxide (DMSO)-stabilized frozen platelet concentrate (PC) using turbidimetric aggregometry. DESIGN: In vitro analysis. SETTING: Research laboratory in a school of veterinary medicine. ANIMALS: Five units of frozen PC in 6% DMSO were studied. Fresh platelet-rich plasma (PRP), with and without 6% DMSO, from 6 healthy dogs were used as controls. INTERVENTIONS: Turbidimetric platelet aggregation was measured after initiation of platelet aggregation by addition of adenosine diphosphate (ADP), collagen, or thrombin at concentrations of 30 µM, 20µg/mL, and 0.5U/mL, respectively. Measures were performed at thaw and repeated 2 hours after thaw for the frozen PC. MEASUREMENTS AND MAIN RESULTS: Compared with PRP, the frozen PC showed decreased aggregation in response to thrombin (amplitude of 84% versus 25%, P=0.01), and collagen (amplitude of 13% versus 3%, P=0.05) but not ADP (6.5% versus 18%, P=0.2). Compared with frozen PC at thaw, the frozen PC at 2 hours after thaw showed decreased aggregation in response to thrombin, collagen, and ADP (P<0.05). There was no difference in aggregation between PRP in 6% DMSO and frozen PC. CONCLUSIONS: These in vitro data suggest there is a decrease in platelet response to agonists associated with the freeze-thaw process in the commercially available 6% DMSO canine frozen PC.

Assessment of a dimethyl sulfoxide-stabilized frozen canine platelet concentrate.

OBJECTIVE: To assess platelet count, mean platelet volume (MPV), metabolic characteristics, and platelet function in a dimethyl sulfoxide (DMSO)-stabilized canine frozen platelet concentrate (PC). SAMPLE POPULATION: 11 units of a commercial frozen PC in 6% DMSO and fresh platelet-rich plasma from 6 healthy control dogs. PROCEDURES: PCs were thawed, and the following data were collected: thaw time, platelet count, MPV, pH, PCO2, and PO2 and HCO3-, glucose, and lactate content. Phosphatidylserine translocation was determined by use of flow cytometry. Fresh platelet-rich plasma from healthy dogs served as a source of control platelets for flow cytometric analysis. RESULTS: At thaw, the platelet count in the frozen PC ranged from 243,000 to 742,000 platelets/microL. Median platelet count of paired samples was 680,000 platelets/microL and decreased significantly to 509,000 platelets/microL at 2 hours after thaw. Median MPV at thaw was 11.15 femtoliters and was stable after 2 hours. Compared with fresh platelets, frozen PC had increased amounts of phosphatidylserine in the outer leaflet of the platelet membrane in the resting (ie, not treated with thrombin) state (19% vs 99%, respectively) and alterations in cellular morphology, all of which were consistent with platelet activation. CONCLUSIONS AND CLINICAL RELEVANCE: Results of this in vitro study indicated that there was a decrease in platelet quantity and function as well as an increase in platelet activation during the freeze-and-thaw process in DMSO-stabilized canine frozen PC. In vivo effects on PC remain to be determined.

Platelet function in clinically healthy cats and cats with hypertrophic cardiomyopathy: analysis using the Platelet Function Analyzer-100.

BACKGROUND: There is currently no simple analytical tool for the evaluation of hypercoagulability in cats. The Platelet Function Analyzer-100 (PFA-100; Dade Behring Inc., Deerfield, IL, USA) is a bench-top machine that evaluates platelet function by measuring closure time (CT) in citrated whole blood under high shear conditions. We hypothesized that cats with hypertrophic cardiomyopathy (HCM) have up-regulated platelet function, which shortens their CT and increases their risk for thromboembolic events. OBJECTIVES: The goals of this study were to: (1) establish a feline reference interval for CT using the PFA-100, (2) measure CT in blood from cats with HCM, and (3) determine if there is a measurable difference between the CT of healthy cats compared with cats with HCM. METHODS: Citrated blood samples from 42 clinically healthy cats and 30 cats with HCM were analyzed according to manufacturer's specifications. CT was measured in triplicate and the mean value was used for analysis. Transformed data were compared between clinically healthy cats and cats with HCM using a Student's t-test, and among cats with mild, moderate, or severe HCM using ANOVA. RESULTS: The median CT of clinically healthy cats was 64 seconds (range 43-176 seconds). The median CT of cats with HCM was 74 seconds (range 48-197 seconds). There was no significant difference in CT between cats with HCM and clinically healthy cats. There also were no significant differences in cats with mild, moderate, or severe HCM. CONCLUSIONS: A feline reference interval for PFA-100 CT will be useful in future studies of platelet function in cats. Cats with HCM do not have shorter CTs when compared with clinically healthy cats.

Assessment of platelet growth factors in supernatants from rehydrated freeze-dried equine platelets and their effects on fibroblasts in vitro.

OBJECTIVE: To determine whether platelet growth factors are preserved in supernatants obtained from rehydrated trehalose-stabilized, freeze-dried (lyophilized) equine platelets and whether those growth factors stimulate fibroblast proliferation and migration and enhance fibroblast-associated contraction in a collagen gel assay. ANIMALS: 6 clinically normal adult horses. PROCEDURES: Blood samples were obtained from 6 horses, and washed platelets were prepared via differential centrifugation. Washed platelets were freeze-dried in a physiologic buffer with a mixture of trehalose and polyethylene glycol 4000. Rehydrated platelet supernatants and releasates prepared from fresh washed platelets stimulated with thrombin or platelet-activating factor were evaluated for transforming growth factor beta1 and platelet-derived growth factor-BB by use of ELISAs. Effects of rehydrated freeze-dried platelet supernatants on fibroblast proliferation, migration, and collagen gel contraction were compared with effects of 1%, 2.5%, or 10% fetal bovine serum (FBS). RESULTS: Supernatants from freeze-dried platelets contained similar amounts of growth factors as thrombin- and platelet-activating factor-stimulated platelet releasates. The supernatants significantly enhanced fibroblast proliferation and migration in a scratch assay, compared with FBS-free control or low (1%) FBS conditions. Additionally, supernatants from freeze-dried platelets enhanced contraction of fibroblast-seeded collagen gels, compared with the effect of 1% FBS. CONCLUSIONS AND CLINICAL RELEVANCE: The preparation technique preserved platelet growth factors, enhanced fibroblast proliferation and migration, and improved fibroblastseeded collagen gel contraction under conditions of low FBS concentration; these platelet supernatant preparations may prove useful as an aid to conventional wound management.

Prevalence of reduced fibrinogen binding to platelets in a population of Thoroughbreds.

OBJECTIVE: To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm. ANIMALS: 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males. PROCEDURES: Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen. RESULTS: Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 horses, fibrinogen binding to platelets was reduced from 67.6% to 83.4%, compared with normal platelets, which indicated an inability of platelets to aggregate in response to thrombin (0.1 U/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Platelet fibrinogen binding of the affected horses identified in this study was characteristic of a reported heritable bleeding disorder in which the reduction in fibrinogen binding correlated with prolonged bleeding times in template bleeding assays. The bleeding disorder is distinct from Glanzmann thrombasthenia, in which platelets fail to bind fibrinogen because of lack of alphallb-beta3 integrin on their surface. The prevalence of affected horses within the small sample population studied here (0.7% [n = 3]) is considerably higher than the prevalence of bleeding disorders within more genetically diverse groups.

Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay.

BACKGROUND: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. HYPOTHESIS: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors. ANIMALS: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring. METHODS: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry. RESULTS: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin. CONCLUSIONS AND CLINICAL IMPORTANCE: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.

Structural characterization of the ADAM 16 disintegrin loop active site.

ADAM's have various roles in intercellular adhesion and are thought to function by binding integrins through a 13 amino acid motif called the disintegrin loop. Xenopus laevis sperm express the protein ADAM 16, and peptides with the sequence of its disintegrin loop cause downstream events in eggs that require a rise in intracellular calcium similar to that occurring at fertilization. We characterized the portion of the ADAM 16 disintegrin loop responsible for causing egg activation. A peptide based on the C-terminal half of the motif, which includes a known integrin-binding sequence, is a partial agonist of calcium release. A peptide with the N-terminal sequence of the motif activates eggs in a manner virtually identical to the full-length peptide but lacks a recognized integrin-binding sequence. None of these peptides alter the permeability or fluidity of liposomes made from membrane lipids of X. laevis eggs. This result reflects the fact that the peptides do not cause calcium to leak across the egg membrane and indirectly provides evidence that they act through a receptor on the egg surface. The infrared spectrum of the full-length peptide has a strong absorption peak corresponding to a beta-turn. We predict this structure occurs at the N-terminal sequence MPKT. A rearranged peptide lacking any turns fails to activate eggs. These results provide the first structural information about the active site of an ADAM disintegrin loop. We interpret these results in terms of active site sequences from other ADAM's and the role of integrins during fertilization.