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Objective: To evaluate the effect of 6% hydroxyethyl starch (HES) 670/0.75 and 6% HES 130/0.4 dilution of canine whole blood on coagulation using dynamic viscoelastic coagulometry (DVC)., Animals: 56 healthy adult dogs., Procedures: 2 blood samples were obtained from each dog and randomized to 1 of 7 groups-undiluted or 2 dilutions (1:3 or 1:10) of 3 different fluids: saline (0.9% NaCl) solution, 6% HES 670/0.75, or 6% HES 130/0.4. Dilutions were calculated to simulate approximately a 10- or 30-mL/kg body weight IV bolus of each fluid. DVC was performed on each sample. Coagulation parameters compared between groups included clot rate (CR), platelet function (PF), and activated clotting time., Results: Dilution with saline solution did not significantly affect coagulation, while dilution with HES 670/0.75 and HES 130/0.4 caused a dose-dependent significant decrease in CR (1:3 HES 670/0.75, P = 0.007; 1:10 HES 670/0.75, P = 0.002; 1:3 HES130/0.4, P < 0.0001; and 1:10 HES 130/0.4, P = 0.0003) and PF (1:3 HES 670/0.75, P < 0.0001; 1:10 HES 670/0.75, P < 0.0001; 1:3 HES130/0.4, P < 0.0001; and 1:10 HES 130/0.4, P = 0.0015)., Clinical Relevance: Dilution of canine blood with HES 670/0.75 and HES 130/0.4, at clinically relevant doses (10 and 30 mL/kg), led to significant hypocoagulability beyond dilutional effect. This was, in part, due to impaired PF, which was significantly greater with HES 670/0.75. Further research using DVC to assess the effects of HES on coagulation in dogs, ideally with clinical conditions warranting HES administration, is needed.

Objective: To evaluate the effect of 1-Desamino-8-d-arginine vasopressin (DDAVP; desmopressin acetate) on platelet aggregation in healthy dogs receiving aspirin or clopidogrel., Animals: 7 healthy staff-owned dogs., Procedures: In this randomized double-blinded crossover study, impedance aggregometry was performed on samples of lithium-heparinized whole blood samples from dogs before (T0) treatment with aspirin (1 mg/kg, PO, q 24 h for 4 days; ASP group) or clopidogrel (1 mg/kg, PO, q 24 h for 4 days; CLP group) and then before (T1) and after (T2) treatment with DDAVP (0.3 μg/kg, IV, once). There was a 14-day washout period before the crossover component. Aggregometry was performed with 4 different assays, each of which involved a different agonist reagent to stimulate platelet function: ADP, thrombin receptor activating peptide-6, arachidonic acid, or collagen type 1., Results: Median results for platelet aggregometry with agonist reagents ADP, arachidonic acid, or thrombin receptor activating peptide-6 significantly decreased between T0 and T1 for the CLP group; however, no meaningful difference in platelet aggregation was detected in the ASP group. Results for platelet aggregometry did not differ substantially between T1 and T2 regardless of treatment group or assay., Conclusions and Clinical Relevance: Findings suggested that administration of DDAVP may have no effect on platelet aggregation (measured with platelet aggregometry) in healthy dogs treated with clopidogrel. Because no inhibition of platelet aggregation was detected for dogs in the ASP group, no conclusion could be made regarding the effects of DDAVP administered to dogs treated with aspirin.

Objective: To assess feasibility of the use of a dynamic viscoelastic coagulometer on chicken blood and compare coagulation variables for fresh whole blood and sodium citrate-preserved whole blood as well as effects of 3 coagulation activators on blood from chickens., Sample: Blood samples from 30 hens., Procedures: Chickens were allowed to rest undisturbed for 1 hour. A blood sample was collected from an ulnar vein; 1.4 mL was analyzed immediately, and 1.8 mL was mixed with sodium citrate and subsequently recalcified and analyzed. A separate coagulation activator (glass beads, kaolin clay, or tissue factor) was in each of the 2 channels of the analyzer. Chickens were allowed a 1-hour rest period, and another blood sample was collected from the contralateral ulnar vein; it was processed in the same manner as for the first sample, except both channels of the analyzer contained the same coagulation activator., Results: Compared with fresh samples, citrated samples had higher values for activated clotting time and platelet function and lower clotting rates. Intra-assay coefficients of variation of coagulation profiles for citrated samples were markedly greater than the limit of 10%, whereas values for fresh samples were close to or < 10%., Conclusions and Clinical Relevance: Results suggested that use of a dynamic viscoelastic coagulometer on chicken blood was feasible and that analysis of fresh whole blood from healthy chickens provided results with less variability than did analysis of citrated blood. Samples preserved with sodium citrate were associated with significant relative hypocoagulability, compared with results for fresh blood.

OBJECTIVE To assess the effect of decreased platelet and WBC counts on platelet aggregation as measured by a multiple-electrode impedance aggregometer in dogs. ANIMALS 24 healthy dogs. PROCEDURES From each dog, 9 mL of blood was collected into a 10-mL syringe that contained 1 mL of 4% sodium citrate solution to yield a 10-mL sample with a 1:9 citrate-to-blood ratio. Each sample was then divided into unmanipulated and manipulated aliquots with progressively depleted buffy-coat fractions such that 2 to 3 blood samples were evaluated per dog. The Hct for manipulated aliquots was adjusted with autologous plasma so that it was within 2% of the Hct for the unmanipulated aliquot for each dog. All samples were analyzed in duplicate with a multiple-electrode impedance aggregometer following the addition of ADP as a platelet agonist. The respective effects of platelet count, plateletcrit, Hct, and WBC count on platelet aggregation area under the curve (AUC), aggregation, and velocity were analyzed with linear mixed models. RESULTS WBC count was positively associated with platelet AUC, aggregation, and velocity; blood samples with leukopenia had a lower AUC, aggregation, and velocity than samples with WBC counts within the reference range. Platelet count, plateletcrit, and Hct did not have an independent effect on AUC, aggregation, or velocity. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that WBC count was positively associated with platelet aggregation when ADP was used to activate canine blood samples for impedance aggregometry. That finding may be clinically relevant and needs to be confirmed by in vivo studies.

OBJECTIVE To compare platelet function and viscoelastic test results between healthy dogs and dogs with chronic kidney disease (CKD) to assess whether dogs with CKD have platelet dysfunction and altered blood coagulation. ANIMALS 10 healthy control dogs and 11 dogs with naturally occurring CKD. PROCEDURES Blood and urine were collected once from each dog for a CBC, serum biochemical analysis, urinalysis, and determination of the urine protein-to-creatinine ratio, prothrombin time, activated partial thromboplastin time, plasma fibrinogen concentration, and antithrombin activity. Closure time was determined by use of a platelet function analyzer and a collagen-ADP platelet agonist. Thromboelastography (TEG) variables (reaction time, clotting time, α angle, maximum amplitude, and global clot strength [G value]) were determined by use of recalcified nonactivated TEG. Platelet expression of glycoprotein Ib (GPIb; receptor for von Willebrand factor), integrin αIIbβ3 (αIIbβ3; receptor for fibrinogen), and P-selectin (marker for platelet activation) was assessed by flow cytometry. RESULTS Compared with healthy control dogs, the median closure time was prolonged, the median maximum amplitude and G value were increased, and the median clotting time was decreased for dogs with CKD. Platelet expression of both αIIbβ3 and P-selectin was also significantly increased for dogs with CKD, compared with that for control dogs. Platelet expression of GPIb, αIIbβ3, and P-selectin was not correlated with closure time or any TEG variable. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that dogs with CKD frequently had evidence of platelet dysfunction and hypercoagulability that were not totally attributable to alterations in platelet surface expression of GPIb, αIIbβ3, and P-selectin.

OBJECTIVE To assess platelet closure time (CT), mean platelet component (MPC) concentration, and platelet component distribution width (PCDW) in dogs with subclinical chronic valvular heart disease. ANIMALS 89 Cavalier King Charles Spaniels (CKCSs) and 39 control dogs (not CKCSs). PROCEDURES Platelet count, MPC concentration, PCDW, and Hct were measured by use of a hematology analyzer, and CT was measured by use of a platelet function analyzer. Murmur grade and echocardiographic variables (mitral valve regurgitant jet size relative to left atrial area, left atrial-to-aortic diameter ratio, and left ventricular internal dimensions) were recorded. Associations between explanatory variables (sex, age, murmur grade, echocardiographic variables, platelet count, and Hct) and outcomes (CT, MPC concentration, and PCDW) were examined by use of multivariate regression models. RESULTS A model with 5 variables best explained variation in CT (R(2), 0.74), with > 60% of the variance of CT explained by mitral valve regurgitant jet size. The model of best fit to explain variation in MPC concentration included only platelet count (R(2), 0.24). The model of best fit to explain variation in PCDW included platelet count and sex (R(2), 0.25). CONCLUSIONS AND CLINICAL RELEVANCE In this study, a significant effect of mitral valve regurgitant jet size on CT was consistent with platelet dysfunction. However, platelet activation, as assessed on the basis of the MPC concentration and PCDW, was not a feature of subclinical chronic valvular heart disease in CKCSs.

Objective: To determine whether soybean oil emulsion has an in vitro effect on platelet aggregation and thromboelastography in blood samples obtained from healthy dogs., Animals: 12 healthy adult dogs., Procedures: Blood samples were collected from each dog into tubes containing EDTA, hirudin, or sodium citrate for a CBC, collagen- and ADP-induced impedance aggregometry, or thromboelastography, respectively. Whole blood platelet aggregation, determined with ADP or collagen agonists, was measured in blood samples containing hirudin and final lipid concentrations of 0, 1, 10, and 30 mg/mL. The thromboelastographic variables R (reaction time), K (clotting time), α angle, and maximum amplitude were evaluated in blood samples containing sodium citrate and final lipid concentrations equivalent to those used for assessment of platelet aggregation., Results: Median maximum ADP- and collagen-induced platelet aggregation in blood samples containing 1, 10, or 30 mg of lipid/mL did not differ significantly from the value for the respective lipid-free blood sample. Maximum amplitude determined via thromboelastography was significantly reduced in blood samples containing 10 and 30 mg of lipid/mL, compared with findings for lipid-free blood samples. Values of other thromboelastographic variables did not differ, regardless of lipid concentrations., Conclusions and Clinical Relevance: Maximum amplitude determined via thromboelastography in canine blood samples was significantly affected by the addition of lipid to final concentrations that are several orders of magnitude higher than clinically relevant lipid concentrations in dogs. Lipid treatment appears to have no significant effect on hemostatic variables in dogs, although clinical studies should be performed to confirm these in vitro findings.

Objective: To evaluate the effect of 6% hydroxyethyl starch (HES) solution, with a molecular weight of 130 kDa and a degree of substitution of 0.42, on canine platelet function in vitro., Samples: Blood samples from 31 healthy adult dogs., Procedures: Citrated blood was diluted with saline (0.9% NaCl) solution or HES 130/0.42 in ratios of 1:9 (ie, 1 part saline solution or HES 130/0.42 and 9 parts blood) and 1:3. Platelet plug formation time (closure time [Ct]) was measured with a platelet function analyzer and cartridges coated with collagen and ADP., Results: Median baseline Ct with citrated blood was 84.0 seconds (interquartile range, 74.5 to 99.5 seconds). Results obtained with 1:9 dilutions with saline solution and HES 130/0.42 were not significantly different from baseline results. The 1:3 dilutions with saline solution and HES 130/0.42 resulted in median Cts of 96.0 seconds (interquartile range, 85.5 to 110.8 seconds) and 112.0 seconds (92.0 to 126.0 seconds), respectively. Results obtained with both 1:3 dilutions were significantly different from baseline results. The Ct obtained with the HES dilution was also significantly different from that of the 1:3 dilution with saline solution., Conclusions and Clinical Relevance: Saline solution and HES 130/0.42 in a 1:3 dilution affected canine platelet function by prolonging Cts. The HES 130/0.42 had a significantly greater effect on canine platelets than did saline solution.

Objective: To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation., Animals: 5 mature healthy horses., Procedure: Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 microM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody., Results: Released platelet proteins in the presence of beta-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean +/- SEM) 194 +/- 2, 159 +/- 2, 151 +/- 2, 104 +/- 2, and 95 +/- 1 kd. Immunoblots of released platelet proteins had a prominent 180 +/- 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 +/- 1 and 155 +/- 1 kd) were observed., Conclusions: Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets., Clinical Relevance: An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses.

A prolonged mean template bleeding time of 13 minutes was present in nine Aleutian mink affected with Chediak-Higashi syndrome (CHS) compared with 4 minutes in dark control mink. The concentrations of blood platelets in normal and affected animals did not differ significantly. However, in mink with CHS, a marked disturbance of platelet response to collagen was present. Administration of aspirin and indomethacin completely blocked CH platelet response to collagen. Blood platelet adenosine triphosphate and adenosine diphosphate values from mink with CHS were significantly less than those of normal mink, and the platelet adenosine triphosphate/adenosine diphosphate ratios were 10.31 in affected mink and 2.74 in normal mink. These findings are consistent with our previous investigations in affectd cattle and persons and indicate that a "storage pool disease" of platelets exist in the mink with CHS.