R. Darren Wood

Characterization of Chicken Thrombocyte Responses to Toll-Like Receptor Ligands

Thrombocytes are the avian equivalent to mammalian platelets. In addition to their hemostatic effects, mammalian platelets rely in part on pattern recognition receptors, such as the Toll-like receptors (TLR), to detect the presence of pathogens and signal the release of certain cytokines. Ligands for TLRs include lipopolysaccharide (LPS), which is bound by TLR4, as well as unmethylated CpG DNA motifs, which are bound by TLR9 in mammals and TLR21 in chickens. Similar to mammalian platelets, avian thrombocytes have been shown to express TLR4 and secrete some pro-inflammatory cytokines in response to LPS treatment. However, the full extent of the contributions made by thrombocytes to host immunity has yet to be elucidated. Importantly, the mechanisms by which TLR stimulation may modulate thrombocyte effector functions have not been well characterized. As such, the objective of the present study was to gain further insight into the immunological role of thrombocytes by analyzing their responses to treatment with ligands for TLR4 and TLR21. To this end, we quantified the relative expression of several immune system genes at 1, 3, 8 and 18 hours post-treatment using real-time RT-PCR. Furthermore, production of nitric oxide and phagocytic activity of thrombocytes was measured after their activation with TLR ligands. We found that thrombocytes constitutively express transcripts for both pro- and anti-inflammatory cytokines, in addition to those associated with anti-viral responses and antigen presentation. Moreover, we found that both LPS and CpG oligodeoxynucleotides (ODN) induced robust pro-inflammatory responses in thrombocytes, as characterized by more than 100 fold increase in interleukin (IL)-1β, IL-6 and IL-8 transcripts, while only LPS enhanced nitric oxide production and phagocytic capabilities. Future studies may be aimed at examining the responses of thrombocytes to other TLR ligands.

Effects of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs

OBJECTIVE To determine effects of therapeutic dosages of aspirin, carprofen, deracoxib, and meloxicam on platelet function and systemic prostaglandin concentrations in healthy dogs. ANIMALS 10 hound-crossbred dogs. PROCEDURES Aspirin (10 mg/kg, PO, q 12 h), carprofen (4.4 mg/kg, PO, q 24 h), deracoxib (2 mg/kg, PO, q 24 h), meloxicam (0.1 mg/kg, PO, q 24 h), and a placebo were administered for 7 days in a random order to each of 10 healthy dogs; there was a 21-day washout period between subsequent treatments. One-stage prothrombin time (PT), activated partial thromboplastin time (aPTT), fibrinogen concentration, and plasma concentrations of thromboxane (TX)B(2) and 6-keto prostaglandin (PG)F(1alpha) were measured before and after treatment administration. Platelet function was assessed by use of a platelet-function analyzer and aggregation. RESULTS Aspirin, carprofen, and meloxicam did not significantly affect platelet function. Deracoxib caused a mild decrease in platelet aggregation induced by 50microM ADP. Platelet number, Hct, PT, aPTT, and plasma TXB(2) and 6-keto PGF(1alpha) concentrations were unchanged after NSAID administration. Meloxicam administration resulted in a significant decrease in fibrinogen concentration, but results remained within the laboratory reference interval. CONCLUSIONS AND CLINICAL RELEVANCE Oral administration of commonly used NSAIDs at therapeutic dosages in healthy dogs did not alter plasma TXB(2) and 6-keto PGF(1alpha) concentrations. Deracoxib administration resulted in a minor abnormality in platelet aggregation. Anti-inflammatory doses of aspirin did not affect platelet function as measured by use of optical aggregometry and a platelet-function analyzer. Further evaluation of the effects of aspirin and cyclooxygenase-2-selective inhibitors on hemostasis should be performed.

Chicken erythrocytes respond to Toll-like receptor ligands by up-regulating cytokine transcripts

Avian erythrocytes are nucleated cells of myeloid origin that are able to actively transcribe and translate proteins. Although their role in gas exchange and transportation has been well described, it has recently been shown that chicken erythrocytes produce cytokines in response to Toll-like receptor (TLR) ligands, which raises the possibility that they also contribute to host immunity. To this end, the objective of the study was to gain some further insight into the immunological role of erythrocytes by identifying the repertoire of TLRs that they express and to elucidate their responses to the TLR3 and TLR21 ligands poly I:C and CpG oligodeoxynucleotide (ODN), respectively. The results suggest that erythrocytes constitutively express transcripts for TLRs 2, 3, 4, 5, and 21, as well as for many immunological genes including type I interferons (IFN) and interleukin (IL)-8. Moreover, it was found that treatment with both poly I:C and CpG ODN up-regulated transcripts for type I IFNs, while only poly I:C up-regulated IL-8 transcripts and enhanced the production of nitrite. Future studies may be aimed at further characterizing the immunological role of erythrocytes.

Prospective multicenter evaluation of coagulation abnormalities in dogs following severe acute trauma

OBJECTIVES To describe coagulation abnormalities in dogs following severe acute trauma and to evaluate the relationship between coagulation, clinical, and laboratory variables, and disease and injury severity, as well as the ability of coagulation variables to predict the presence of body cavity hemorrhage (BCH), necessity of blood product administration, and outcome. DESIGN Prospective, multicenter, observational study. SETTING Two university teaching hospitals. ANIMALS Forty client-owned dogs sustaining severe blunt or penetrating trauma. INTERVENTIONS Blood samples were collected within 12 hours of the traumatic incident for measurement of blood gases, lactate concentration, platelet count, activated clotting time, prothrombin time, activated partial thromboplastin time (aPTT), fibrinogen concentration, antithrombin activity, D-dimer concentration, protein C activity, plasmin inhibition, plasminogen activity, and kaolin-activated thomboelastography. RESULTS Decreased platelet count was a risk factor for the presence of BCH (P = 0.006) and decreased platelet count (P < 0.001), protein C activity (P = 0.001), angle (α) (P = 0.001), maximum amplitude (MA) (P < 0.001), and clot strength (G) (P = 0.002) were risk factors for blood product administration. Nonsurviving dogs were hypocoagulable with prolonged aPTT (P = 0.008), decreased plasmin inhibition (P = 0.033), decreased α (P = 0.021), and decreased MA (P = 0.038) compared to surviving dogs. Multivariate analysis accounting for disease severity showed that prolonged aPTT (P = 0.004, OR = 1.74) was the strongest predictor of nonsurvival. Prolonged aPTT was positively correlated with APPLE-fast score (P < 0.001, r(2) = 0.35), lactate concentration (P < 0.001, r(2) = 0.35), and negative base excess (P = 0.001, r(2) = 0.27). Acute traumatic coagulopathy, as defined by 2 or more abnormal coagulation tests, was diagnosed in 15% of dogs at hospital admission and was more common in dogs with increased disease severity (P = 0.002), decreased systolic blood pressure (P = 0.002), and increased lactate concentration (P = 0.011). CONCLUSIONS In dogs with severe traumatic injuries and hypoperfusion, measurement of thromboelastography and aPTT should be considered to support clinical assessments in predicting the need for blood product administration and nonsurvival.

Comparing citrated native, kaolin-activated, and tissue factor–activated samples and determining intraindividual variability for feline thromboelastography

Thromboelastography (TEG) is a point-of-care whole blood test of hemostasis. While TEG is becoming more widely used in veterinary medicine, few studies describe the use of TEG in cats. The objectives of the current study were to: 1) document the range of TEG variables produced in healthy cats using 3 sample types (citrated native, kaolin-activated, and tissue factor–activated), and 2) determine if there was a significant difference between 2 separate samples obtained from individual healthy cats on the same day. Jugular venipuncture was performed in 20 cats, and citrated blood collected for TEG. TEG analysis was performed on citrated native, kaolin-activated, and tissue factor–activated blood for each sample. Two hours later, the procedure was repeated from the opposite jugular vein, yielding a total of 120 analyses. Reaction time (R), alpha angle (α), kappa value (κ), and maximum amplitude (MA) were recorded from each tracing. No significant differences were found between TEG tracings from the first and second venipuncture samples. Significant differences were found between sample types for R, α, κ, and MA. Means for citrated native/kaolin-activated/tissue factor–activated methods were R = 4.1/3.7/0.6 min; κ = 2.5/1.8/2.2 min; α = 59.9/65.1/70.4 degrees; MA = 47.4/49.9/44.7 mm. A limitation of this study was the small number of cats used. Thromboelastography analysis may be a suitable method of evaluating hemostasis in cats.

Independent and combined effects of prednisone and acetylsalicylic acid on thromboelastography variables in healthy dogs

OBJECTIVE To describe the effects of prednisone and acetylsalicylic acid (ASA) on results of thromboelastography in healthy dogs. ANIMALS 16 male mixed-breed dogs. PROCEDURES Dogs were randomly assigned to 3 treatment groups (4 dogs/group) that received prednisone (median dose, 2.07 mg/kg), ASA (median dose, 0.51 mg/kg), or both drugs, PO, every 24 hours from days 0 through 6. Another group received no treatment (control dogs; n = 4). Thromboelastography variables (reaction time, clotting time, α-angle, maximum amplitude [MA], global clot strength, coagulation index, and percentage of clot lysis at 60 minutes [CL(60)]) were evaluated in blood samples collected (prior to drug administration in treated dogs) on days 0 (baseline), 2, 4, and 6. RESULTS Administration of ASA alone did not alter TEG variables. For treatment effect, mean global clot strength was increased in the prednisone and drug combination groups, compared with values for control dogs; MA was also increased in the prednisone and drug combination groups, compared with that of controls. For treatment-by-time effect, median CL(60) was increased in the prednisone group on day 6, compared with baseline value in the same dogs and with median CL(60) of the control group on day 6. Median CL(60) was also increased in the drug combination group on day 6, compared with the baseline value and with that of the control group on day 6. CONCLUSIONS AND CLINICAL RELEVANCE Prednisone administered at approximately 2 mg/kg/d, PO, for 7 days with or without concurrently administered ASA increased clot strength and decreased clot lysis in healthy dogs.

Effect of synthetic colloid administration on hemodynamic and laboratory variables in healthy dogs and dogs with systemic inflammation.

Objective To compare the effects of administering equal volumes of isotonic crystalloids and synthetic colloids on hemodynamic and laboratory variables in healthy dogs and dogs with systemic inflammation. Design Randomized, placebo-controlled, blinded study. Setting Comparative clinical research facility. Animals Sixteen adult purpose-bred Beagles. Interventions Dogs were first randomized to receive either lipopolysaccharide (LPS; 5 μg/kg, IV) or an equal volume of placebo (0.9% NaCl, IV). Dogs were then randomized into 1 of 2 groups receiving fluid resuscitation with either 40 mL/kg IV isotonic crystalloid (0.9% NaCl) or synthetic colloid (tetrastarch). After a 14-day washout, the study was repeated such that dogs received the opposite treatment (LPS or placebo) and the same resuscitation fluid regimen. Vital signs (heart rate (HR), oscillometric blood pressure) were measured and blood samples were collected for PCV, total plasma protein (TPP), serum lactate concentration, and colloid osmotic pressure (COP) measurements. Measurements and Main Results Healthy (placebo) dogs had similar decreases in PCV and TPP after administration of either fluid. Tetrastarch administration was associated with a larger increase in HR, systolic blood pressure, and mean blood pressure. Dogs with systemic inflammation had similar increases in systolic blood pressure and decreases in PCV, TPP, and lactate after administration of either fluid. Tetrastarch administration caused greater immediate increase in HR and mean blood pressure compared to 0.9% NaCl. In all dogs, 0.9% NaCl administration decreased COP and tetrastarch administration increased COP. Conclusions Resuscitation with equal volumes of 0.9% NaCl and tetrastarch caused similar changes in hemodynamic and laboratory variables in dogs with LPS-induced systemic inflammation; however, larger increases in HR and blood pressure were seen within the first 2 hours following tetrastarch administration compared to 0.9% NaCl. Tetrastarch administration increased COP in all dogs, despite a decrease in TPP.OBJECTIVE To compare the effects of administering equal volumes of isotonic crystalloids and synthetic colloids on hemodynamic and laboratory variables in healthy dogs and dogs with systemic inflammation. DESIGN Randomized, placebo-controlled, blinded study. SETTING Comparative clinical research facility. ANIMALS Sixteen adult purpose-bred Beagles. INTERVENTIONS Dogs were first randomized to receive either lipopolysaccharide (LPS; 5 μg/kg, IV) or an equal volume of placebo (0.9% NaCl, IV). Dogs were then randomized into 1 of 2 groups receiving fluid resuscitation with either 40 mL/kg IV isotonic crystalloid (0.9% NaCl) or synthetic colloid (tetrastarch). After a 14-day washout, the study was repeated such that dogs received the opposite treatment (LPS or placebo) and the same resuscitation fluid regimen. Vital signs (heart rate (HR), oscillometric blood pressure) were measured and blood samples were collected for PCV, total plasma protein (TPP), serum lactate concentration, and colloid osmotic pressure (COP) measurements. MEASUREMENTS AND MAIN RESULTS Healthy (placebo) dogs had similar decreases in PCV and TPP after administration of either fluid. Tetrastarch administration was associated with a larger increase in HR, systolic blood pressure, and mean blood pressure. Dogs with systemic inflammation had similar increases in systolic blood pressure and decreases in PCV, TPP, and lactate after administration of either fluid. Tetrastarch administration caused greater immediate increase in HR and mean blood pressure compared to 0.9% NaCl. In all dogs, 0.9% NaCl administration decreased COP and tetrastarch administration increased COP. CONCLUSIONS Resuscitation with equal volumes of 0.9% NaCl and tetrastarch caused similar changes in hemodynamic and laboratory variables in dogs with LPS-induced systemic inflammation; however, larger increases in HR and blood pressure were seen within the first 2 hours following tetrastarch administration compared to 0.9% NaCl. Tetrastarch administration increased COP in all dogs, despite a decrease in TPP.

Comparison of citrated native and kaolin-activated samples for thrombelastographic analysis in healthy dogs.

BACKGROUND Thrombelastographic (TEG) analysis is a test of global hemostasis in veterinary medicine; however, there have been limited comparisons of analysis of citrated native and kaolin-activated samples. OBJECTIVES The purpose of this study was to determine the variation in TEG variables between citrated native and kaolin-activated whole blood samples and to establish reference intervals for both sample types. METHODS Citrated whole blood samples were obtained from 40 healthy dogs. Thirty minutes after collection, TEG analysis was performed simultaneously on samples with and without kaolin-activation. Reaction time (R), clotting time (K), angle (α), maximum amplitude (MA), global clot strength (G), and clot lysis at 30 minutes (LY30) were recorded, and the concordance correlation coefficient (ρ(c)) was calculated for each sample type. RESULTS Significant differences between results obtained for kaolin-activated and native samples were obtained for R (mean difference -1.3 minute, P = .0009), K (-0.7 minute, P = .0003), α (+5.1º, P = .002), MA (+2.4 mm, P = .002), and G (+568 dyn/cm(2), P = .0009). LY30 was not different between methods. There was substantial agreement between methods for G (ρ(c) = .69) and MA (ρ(c) = .65), moderate agreement for R (ρ(c) = .45) and α (ρ(c) = .44), fair agreement for K (ρ(c) = .29), and slight agreement for LY30 (ρ(c) = .04). CONCLUSIONS The TEG variables were significantly altered by kaolin activation; however, some agreement between sample types suggests a consistent bias. In citrated whole blood activated with kaolin, clot formation time is shortened and the amplitude of the tracing is increased, resulting in a TEG tracing that appears to indicate relative hypercoagulability compared with that obtained using native citrated whole blood samples.

Effect of Synthetic Colloid Administration on Coagulation in Healthy Dogs and Dogs with Systemic Inflammation

Background Synthetic colloids are often used during fluid resuscitation and affect coagulation. Objective To compare the effects of an isotonic crystalloid and synthetic colloid on coagulation in healthy dogs and dogs with systemic inflammation. Animals Sixteen adult purpose‐bred Beagles. Methods Randomized, placebo‐controlled, blinded study. Dogs were randomized into one of two groups receiving fluid resuscitation with either 40 mL/kg IV 0.9% NaCl or tetrastarch after administration of lipopolysaccharide or an equal volume of placebo. After a 14‐day washout period, the study was repeated such that dogs received the opposite treatment (LPS or placebo) but the same resuscitation fluid. Blood samples were collected at 0, 1, 2, 4, and 24 hours for measurement of coagulation variables. Results Administration of either fluid to healthy dogs and dogs with systemic inflammation resulted in similar increases in prothrombin time and activated clotting time. In comparison to saline administration, tetrastarch administration resulted in significantly decreased R (P = .017) in healthy dogs, as well as significantly increased activated partial thromboplastin time (P ≤ .016), CL30% (P ≤ .016), and K (P < .001) and significantly decreased platelet count (P = .019), α (P ≤ .001), MA (P < .001), and von Willebrand factor antigen (P < .001) and collagen binding activity (P ≤ .003) in both healthy dogs and dogs with systemic inflammation. Conclusions and Clinical Importance Tetrastarch bolus administration to dogs with systemic inflammation resulted in a transient hypocoagulability characterized by a prolonged activated partial thromboplastin time, decreased clot formation speed and clot strength, and acquired type 1 von Willebrand disease.

Assessment of platelet function in healthy sedated cats using three whole blood platelet function tests.

The objectives of this study were to establish feline references intervals for 3 commercial whole blood platelet function test analyzer systems: Multiplate analyzer (MP; Roche Diagnostics International Ltd., Rotkreuz, Switzerland), Platelet Function Analyzer-100 (PF: Siemens Canada, Mississauga, Ontario, Canada), and Plateletworks Combo-25 kit (PW; Helena Laboratories, Beaumont, TX). Venipuncture was performed on 55 healthy sedated cats, and platelet aggregation in response to adenosine diphosphate (ADP), collagen (COL), and arachidonic acid (AA; MP only) was assessed using citrated blood. For the MP analyzer, median (95% confidence intervals [CIs]) area under curve (Units) for ADP, COL, and AA agonists were 87 (11–176), 81 (32–129), and 91 (59–129), respectively. For the PF analyzer, median (95% CIs) closure time, using COL–ADP cartridges, was 69 (46–89) sec. For the PW assay, median (95% CIs) percent aggregations for ADP and COL agonists were 71 (18–92) and 49 (9–96), respectively, using impedance hematology analyzer platelet counts, and 94 (25–98) and 68 (14–119), respectively, using flow cytometry hematology analyzer platelet counts. There were low correlations between the PF analyzer (COL–ADP cartridge) and MP analyzer (COL agonist; ρ = 0.11), and between the PF analyzer (COL–ADP cartridge) and PW assay (COL agonist using impedance platelet counts; ρ = 0.14). The PW assay percent aggregations using impedance and flow cytometric platelet counts were correlated for both ADP (ρ = 0.64) and COL (ρ = 0.64) agonists. Platelet function testing using these tests are feasible in cats, but 95% CIs are wide, so single results may be difficult to interpret. Platelet counting by impedance or flow cytometry may be used for the PW assay but are not interchangeable.