Kira L. Epstein

Thrombelastography in 26 healthy horses with and without activation by recombinant human tissue factor

OBJECTIVES To develop a standardized technique for thrombelastography (TEG) analysis in healthy adult horses, with and without the ex vivo addition of tissue factor (TF) as an activator. To determine reference intervals for TEG parameters in the horse, and to determine if traditional coagulation tests correlate with TEG. DESIGN Prospective, observational. SETTING Veterinary teaching hospital. ANIMALS Twenty-six healthy adult horses. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Thrombelastography with (TF-TEG) and without (TEG) the addition of TF performed by 4 operators. Coagulation profiles (prothrombin time, activated partial thromboplastin time, platelet count, fibrinogen, antithrombin, and fibrinogen degradation products) were assessed in a subset of horses. Mean values (SD) for TEG parameters in healthy horses were: reaction time (R)=17.0 minutes (3.0 min), K time (K)=5.8 minutes (2.3 min), clotting rate (Ang)=42 degrees (14 degrees ), maximum clot strength (maximum amplitude [MA])=60.3 mm (5.7 mm), CL30=97.0% (2.0%), LY30=0.8% (0.6%), CL60=92% (5.9%), LY60=3.2% (2.5%). Mean values (SD) for TF-TEG parameters were: R-TF=6.6 minutes (1.4 min), K-TF=3.1 minutes (1.0 min), Ang-TF=50.9 degrees (9 degrees ), MA-TF=62.3 mm (5.1 mm), CL30-TF=97.8% (1.6%), LY30-TF=0.6% (0.5%), CL60-TF=90.8% (4.2%), and LY60-TF=3.6% (1.9%). The addition of TF decreased R and K and increased Ang. TF-TEG had a narrower SD for R, K, Ang, CL60 and LY60 compared with TEG. Interoperator differences were reduced by the addition of TF. Regression analysis indicated a positive relationship between MA and fibrinogen concentrations (P=0.02) and R-TF time and prothrombin time (P=0.03). CONCLUSION TF-TEG using the described protocol may minimize variability in data obtained across institutions or users. However, due to the variability associated with different operators, it is recommended that each laboratory set up individual reference intervals with the personnel who will perform the assay, and that the assay protocols and data obtained are compared on a regular basis.

Thrombelastography in Horses with Acute Gastrointestinal Disease

BACKGROUND Coagulopathies in horses with gastrointestinal disease are frequently identified and associated with morbidity and fatality. OBJECTIVE Determine if thrombelastography (TEG) identifies abnormalities associated with lesion type, presence of systemic inflammatory response syndrome (SIRS), morbidity, and fatality more consistently than traditional coagulation testing. ANIMALS One-hundred and one horses examined for gastrointestinal disease and 20 healthy horses. METHODS TEG, tissue factor (TF)-TEG, and traditional coagulation panels parameters and percentages of horses with coagulopathies were compared for lesion type, presence of SIRS, complications, and survival. RESULTS Changes in individual parameters and increased incidence of coagulopathies were associated with fatality (R, P= .007; k-value [K], P= .004; clot lysis [CL]30, P= .037; CL60, P= .050; angle [Ang], P= .0003; maximum amplitude [MA], P= .006; lysis [Ly]30, P= .042; Ly60, P= .027; CI, P= .0004; ≥ 2 TEG coagulopathies, P= .013; ≥ 3 TEG coagulopathies, P= .038; TF-R, P= .037; TF-K, P= .004; TF-CL30, P < .0001; TF-CL60, P < .0001; TF-Ang, P= .005; TF-Ly30, P= .0002; TF-Ly60, P < .0001; TF-CI, P= .043; ≥ 1 TF-TEG coagulopathies, P= .003; ≥ 2 TF-TEG coagulopathies, P= .0004; prothrombin tme [PT], P < .0001; activated partial throboplastin time [aPTT], P= .021), inflammatory lesions (MA, P= .013; TF-CL30, P= .033; TF-CL60, P= .010; TF-Ly60, P= .011; ≥ 1 TF-TEG coagulopathy, P= .036; ≥ 2 TF-TEG coagulopathy, P= .0007; PT, P= .0005; fibrinogen, P= .019), SIRS (MA, P= .004; TF-CL30, P= .019; TF-CL60, P= .013; TF-Ly30, P= .020; TF-Ly60, P= .010; PT, P < .0001; aPTT, P= .032; disseminated intravascular coagulation, P= .005), and complications (ileus: aPTT, P= .020; diarrhea: TF-CL30, P= .040; TF-Ly30, P= .041; thrombophlebitis: ≥ 1 TF-TEG coagulopathy, P= .018; laminitis: MA, P= .004; CL60, P= .045; CI, P= .036; TF-MA, P= .019; TF-TEG CI, P= .019). Abnormalities in TEG and TF-TEG parameters were indicative of hypocoagulation and hypofibrinolysis. CONCLUSIONS AND CLINICAL IMPORTANCE TEG identifies changes in coagulation and fibrinolysis associated with lesion type, SIRS, morbidity, and fatality in horses with gastrointestinal disease.

Coagulopathy of the critically ill equine patient.

OBJECTIVE To review the hemostasis literature relevant to development of coagulopathy in the critically ill equine patient. DATA SOURCE Original scientific and review articles. HUMAN DATA SYNTHESIS Inflammation plays a critical role in the activation and amplification of clot formation, as well as the impairment of physiologic anticoagulant mechanisms, and fibrinolysis. Earlier identification of coagulopathy in patients at risk and restoration of physiologic hemostasis may result in better outcome. Development of scoring systems based on information other than coagulation markers alone may better identify patients with subclinical coagulopathy. VETERINARY DATA SYNTHESIS Critically ill equine patients commonly at risk for coagulopathy include those with severe gastrointestinal disease, septic foals, and adults subjected to severe systemic inflammatory response syndrome. Publications provide information regarding coagulation markers helpful for identification of hemostatic dysfunction in specific patient populations, as well as information regarding the influence of coagulopathy on outcome. Data regarding clinically relevant information on therapeutic intervention are lacking. CONCLUSIONS The relationship between inflammation and endotoxemia and development of coagulopathy is better understood in both human patients and the critically ill equine patient. Prospective clinical trials evaluating clinically relevant and financially feasible approaches to treatment are still needed.

Effect of experimentally induced synovitis on amikacin concentrations after intravenous regional limb perfusion.

OBJECTIVES To determine the effects of experimentally induced synovitis of the radiocarpal joint on the intra-articular pharmacokinetics and pharmacodynamics of amikacin after intravenous regional limb perfusion (IVRLP). STUDY DESIGN Randomized crossover experimental design. ANIMALS Adult horses (n = 8). METHODS Horses were randomly assigned into 2 trials: synovitis and no-synovitis. Radiocarpal joint synovitis was induced with lipopolysaccharide 6 hours before IVRLP. IVRLP (5-mg/kg amikacin qs 60 mL) was performed with a pneumatic tourniquet under general anesthesia. Synovial fluid was obtained before and 0.5, 1, 3, 5, 12, 24, 48 hours after IVRLP. Amikacin concentrations at each time point and pharmacokinetic values were compared between synovitis and no-synovitis trials with Students t-test. RESULTS Amikacin synovial fluid concentrations indicated suspected tourniquet failure on 3 occasions (2 synovitis, 1 no-synovitis) on 3 different horses. Data from both trials in these 3 horses were excluded from further analysis. Observed time to maximal concentration (T(max); mean ± SD = 54 ± 13.42 min) was reached earlier in synovitis joints (5/5, 30 min) than in no-synovitis joints (1/5, 30 min and 4/5, 1 h; P = .0476) (P = .0161). Mean observed maximal concentration (C(max)) was higher in synovitis joints (144.48 ± 43.17 μg/mL) than in no-synovitis joints (60.02 ± 28.81 μg/mL; P = .0301). The recommended C(max): minimum inhibitory concentration ratio of 8 was achieved in 3/5 of the successfully perfused joints with induced synovitis, but this ratio was not achieved in any of the clinically normal joints. CONCLUSION Synovitis of the radiocarpal joint resulted in an earlier observed T(max) and higher observed C(max) of intra-articular amikacin after IVRLP compared with normal joints.

Effects of Clopidogrel and Aspirin on Platelet Aggregation, Thromboxane Production, and Serotonin Secretion in Horses

BACKGROUND Critically ill horses are susceptible to thrombotic disease, which might be related to increased platelet reactivity and activation. OBJECTIVES To compare the effect of oral clopidogrel and aspirin (ASA) on equine platelet function. ANIMALS Six healthy adult horses. METHODS Horses received clopidogrel (2 mg/kg p.o. q24h) or ASA (5 mg/kg p.o. q24h) for 5 days in a prospective randomized cross-over design. Platelet aggregation responses to adenosine diphosphate (ADP) and collagen via optical aggregometry, and platelet secretion of serotonin (5HT) and production of thromboxane B(2) (TXB(2) ) by ELISA were evaluated. In horses receiving clopidogrel, high-performance liquid chromatography analysis for clopidogrel and its carboxylic-acid metabolite SR 26334 was performed. RESULTS SR 26334 was identified in all clopidogrel-treated horses, although the parent compound was not detected. Clopidogrel resulted in decreases in ADP-induced platelet aggregation persisting for 120 hours after the final dose. ADP-induced platelet aggregation decreased from a baseline of 70.2 ± 14.7% to a minimum of 15.9 ± 7.7% 24 hours after the final dose (P < .001). Collagen-induced aggregation decreased from a baseline of 93 ± 9.5% to a minimum of 70.8 ± 16.9% 48 hours after the final dose (P < .001). ASA did not decrease platelet aggregation with either agonist. ASA decreased serum TXB(2) from a baseline value of 1310 ± 1045 to 128 ± 64 pg/mL within 24 hours (P < .01). CONCLUSIONS AND CLINICAL IMPORTANCE Clopidogrel effectively decreases ADP-induced platelet aggregation in horses, and could have therapeutic applications for equine diseases associated with platelet activation.

Cardiovascular, Colloid Osmotic Pressure, and Hemostatic Effects of 2 Formulations of Hydroxyethyl Starch in Healthy Horses

Background Lower molecular weight and molar substitution formulations of hydroxyethyl starch (HES) solutions might maximize cardiovascular function and colloid osmotic pressure (COP) and minimize adverse effects on coagulation. Hypothesis/Objectives To compare effects of 1 low and 1 high molecular weight and molar substitution HES solution on cardiovascular variables, COP, and hemostasis in normal horses. Animals Eight healthy adult horses. Methods Randomized, crossover designed study: 10 mL/kg bolus of 6% HES (600/0.75) (hetastarch) (HS), 6% HES (130/0.4) tetrastarch (TS), and 0.9% NaCl (NS). Variables recorded included central venous pressure (CVP), noninvasive arterial blood pressure, packed cell volume (PCV), COP, and automated platelet analysis (CT). Results Central venous pressure was increased for 8 hours after all treatment (baseline = 8.4 ± 3.8; 8 hours = 10.3 ± 3.5 cm H2O; P < .001). HS and TS produced an increase in systolic arterial pressure (HS = 109.1 ± 11.9; TS = 109.5 ± 10.9 mmHg) and mean arterial pressure (HS = 80.4 ± 13.0; TS = 82.3 ± 10.1 mmHg) compared to NS (SAP = 103.2 ± 13.2 [P = .023]; MAP = 74.2 ± 11.4 mmHg [P = .048]). PCV decreased transiently with HS (baseline = 37.1 ± 4.4%; 1.5 hours = 31.6 ± 3.9%) and TS (baseline = 38.4 ± 3.9%; 1.5 hours = 32.2 ± 3.3%), but not NS (P = .007). COP was greater with HS (1 hour; 24.0 ± 2.1 mmHg) and TS (8 hours; 25.9 ± 2.1 mmHg) than NS (1 hour = 20.8 ± 2.6; 8 hours = 22.9 ± 3.1 mmHg; P < .001). CT was greater at 8 (HS = 178.6 ± 36.9; TS = 121.9 ± 33.3; NS = 108.3 ± 23.6 seconds) and 24 hours (HS = 174.2 ± 41.7; TS = 100.8 ± 26.0; NS = 118.7 ± 38.7 seconds; P < .001) in horses receiving HS than TS or NS. Conclusion and Clinical Importance Both TS and HS resulted in more effective volume expansion and arterial pressure support than NS. TS produced a more sustained effect on COP with shorter duration of adverse effects on platelet function than HS.

Therapeutic Plasma Concentrations of Epsilon Aminocaproic Acid and Tranexamic Acid in Horses

BACKGROUND Antifibrinolytic drugs such as epsilon aminocaproic acid (EACA) and tranexamic acid (TEA) are used to treat various bleeding disorders in horses. Although horses are hypofibrinolytic compared to humans, dosing schemes have been derived from pharmacokinetic studies targeting plasma concentrations in humans. HYPOTHESIS/OBJECTIVES We hypothesized therapeutic plasma concentrations of antifibrinolytic drugs in horses would be significantly lower than in humans. Our objective was to use thromboleastography (TEG) and an in vitro model of hyperfibrinolysis to predict therapeutic concentrations of EACA and TEA in horses and humans. ANIMALS Citrated plasma collected from 24 random source clinically healthy research horses. Commercial pooled human citrated plasma with normal coagulation parameters was purchased. METHODS Minimum tissue plasminogen activator (tPA) concentration to induce complete fibrinolysis within 10 minutes was determined using serial dilutions of tPA in equine plasma. Results used to create an in vitro hyperfibrinolysis model with equine and human citrated plasma, and the minimum concentrations of EACA and TEA required to completely inhibit fibrinolysis for 30 minutes (estimated therapeutic concentrations) determined using serial dilutions of the drugs. RESULTS Estimated therapeutic concentrations of EACA and TEA were significantly lower in horses (5.82; 95% CI 3.77-7.86 μg/mL and 0.512; 95% CI 0.277-0.748 μg/mL) than in humans (113.2; 95% CI 95.8-130.6 μg/mL and 11.4; 95% CI 8.62-14.1 μg/mL). CONCLUSIONS AND CLINICAL IMPORTANCE Current dosing schemes for EACA and TEA in horses may be as much as 20× higher than necessary, potentially increasing cost of treatment and risk of adverse effects.

Coagulopathies in Horses

Although primary coagulopathies are rare in horses, changes in coagulation and fibrinolysis are commonly associated with inflammatory diseases. A clear understanding of the pathophysiology of normal and abnormal hemostasis is required to be able to choose and interpret diagnostic tests evaluating coagulation and fibrinolysis. After diagnosis, treatment of the underlying disease must occur regardless of whether clinical manifestations (excessive bleeding or thrombosis) of the coagulopathy are present or not. Specific treatment may be initiated if there are clinical signs of coagulopathy.

Serial viscoelastic and traditional coagulation testing in horses with gastrointestinal disease

OBJECTIVE Objectives of this study were to compare the ability of serial thromboelastography, Sonoclot, and traditional coagulation panels to detect coagulopathies associated with disease category, systemic inflammatory response syndrome (SIRS), complications, and nonsurvival in horses with gastrointestinal disease. DESIGN Prospective clinical evaluation. SETTING University referral hospital. ANIMALS One hundred twenty-one horses admitted as emergencies for gastrointestinal disease and 28 healthy adult horses. INTERVENTION Blood samples were collected ≤4 times from emergency horses (admission and if surviving and hospitalized on days 2-4) and once from healthy horses. Thromboelastography (with and without tissue factor activation), Sonoclot, and a traditional coagulation panel were performed on each sample. MEASUREMENTS AND MAIN RESULTS Emergency horses were grouped based on disease category (ie, nonstrangulating medical, nonstrangulating surgical, strangulating, and inflammatory), survival to discharge, SIRS at admission, requirement for exploratory celiotomy, ileus, diarrhea, fever, thrombophlebitis, and laminitis. Changes over time were evaluated individually and compared between disease groups. Horses with gastrointestinal disease had dynamic changes in coagulation and fibrinolysis during the first 4 days of hospitalization that were correlated with disease category, SIRS, complications, and fatality. The multivariate logistic regression model for nonsurvival included activated partial thromboplastin time on day 2 and LY30 on day 3 (overall model significance P < 0.0001). The odds of nonsurvival were 23.75 times higher if activated partial thromboplastin time was >85.6 s on day 2 and 9.38 times higher if LY30 was >1% on day 3. CONCLUSIONS Horses with gastrointestinal disease have activation of coagulation and fibrinolysis. Magnitude of change in these parameters is small and predictive value moderate, making application of these tests to direct therapy in clinical patients difficult. Effect of specific treatments (eg, surgery) on these tests and coagulation has not been determined. Further studies are required to determine if these tests could be used to help monitor response to treatment in individual animals or specific disease states.Objective Objectives of this study were to compare the ability of serial thromboelastography, Sonoclot, and traditional coagulation panels to detect coagulopathies associated with disease category, systemic inflammatory response syndrome (SIRS), complications, and nonsurvival in horses with gastrointestinal disease. Design Prospective clinical evaluation. Setting University referral hospital. Animals One hundred twenty-one horses admitted as emergencies for gastrointestinal disease and 28 healthy adult horses. Intervention Blood samples were collected ≤4 times from emergency horses (admission and if surviving and hospitalized on days 2–4) and once from healthy horses. Thromboelastography (with and without tissue factor activation), Sonoclot, and a traditional coagulation panel were performed on each sample. Measurements and Main Results Emergency horses were grouped based on disease category (ie, nonstrangulating medical, nonstrangulating surgical, strangulating, and inflammatory), survival to discharge, SIRS at admission, requirement for exploratory celiotomy, ileus, diarrhea, fever, thrombophlebitis, and laminitis. Changes over time were evaluated individually and compared between disease groups. Horses with gastrointestinal disease had dynamic changes in coagulation and fibrinolysis during the first 4 days of hospitalization that were correlated with disease category, SIRS, complications, and fatality. The multivariate logistic regression model for nonsurvival included activated partial thromboplastin time on day 2 and LY30 on day 3 (overall model significance P 85.6 s on day 2 and 9.38 times higher if LY30 was >1% on day 3. Conclusions Horses with gastrointestinal disease have activation of coagulation and fibrinolysis. Magnitude of change in these parameters is small and predictive value moderate, making application of these tests to direct therapy in clinical patients difficult. Effect of specific treatments (eg, surgery) on these tests and coagulation has not been determined. Further studies are required to determine if these tests could be used to help monitor response to treatment in individual animals or specific disease states.

Ex vivo comparison of three hand sewn end-to-end anastomoses in normal equine jejunum.

REASONS FOR PERFORMING STUDY Although single layer techniques are preferred in man and small animals for small intestinal end-to-end anastomoses, double layer techniques are more popular in equine surgery. This study was undertaken to evaluate the ex vivo characteristics of 2 single layer anastomoses in comparison to the traditionally performed double layer anastomosis in equine jejunum. OBJECTIVES To compare ex vivo: 1) construction time; 2) bursting pressure; and 3) lumen size reduction of 3 suture patterns (double layer simple continuous/Cushing pattern [2C]; single layer Lembert pattern [1L]; and single layer Cushing pattern [1C]) for end-to-end equine jejunojejunostomies. METHODS End-to-end jejunojejunostomies were constructed using 2C (n = 7), 1L (n = 7) and 1C (n = 7) in harvested equine jejunum and construction times were recorded. Anastomosed and control segments were distended with gas until failure. Intraluminal pressure at failure and mode of failure were recorded. Lumen size reduction was calculated as a percentage decrease from control jejunum. Results were compared using an ANOVA and P<0.05 was considered significant. RESULTS The 1C anastomoses were faster to construct than the 1L anastomoses, which were faster to construct than the 2C anastomoses. There were no differences in bursting pressures between the different anastomoses and control jejunum. All anastomoses decreased lumen size from control values but there were no differences in lumen reduction between types of anastomoses. CONCLUSIONS Single layer anastomoses are faster to construct than double layer anastomoses, with the 1C being fastest. Single layer anastomoses are as strong and result in comparable lumen size reduction as traditional 2C anastomoses. POTENTIAL RELEVANCE As the 1C anastomosis results in less exposed potentially adhesiogenic suture material than the 1L while providing adequate strength and similar luminal size reduction, the 1C may be better for equine small intestine anastomosis and further in vivo studies are warranted.