Role of platelet activating factor in development of thrombocytopenia and neutropenia in dogs with endotoxemia

OBJECTIVE: To determine the role of platelet activating factor (PAF) in lipopolysaccharide (LPS)-induced thrombocytopenia and neutropenia in dogs. ANIMALS: 42 dogs. PROCEDURES: Blood samples were obtained from dogs given LPS (40 microg/kg of body weight; n = 16), PAF (1 microg/kg; 6), PAF (5 microg/kg/h for 90 minutes; 4), or physiologic saline (0.9% NaCl) solution (0.1 ml/kg/h for 90 minutes; 3) IV to monitor changes in blood cell counts, using automated counters and blood smears stained with Giemsa. Blood samples were also obtained from dogs given LPS (40 microg/kg) that had (n = 5) or had not (6) been treated beforehand with TCV-309, a potent PAF antagonist. Concentration of PAF in blood was determined by use of 125I-radioimmunoassay in dogs given LPS at 1 mg/kg (n = 3) and 40 microg/kg (9). RESULTS: Thrombocytopenia and neutropenia were found in all dogs except those given saline solution. The LPS-induced thrombocytopenia was significantly suppressed by prior treatment with TCV-309. The PAF concentrations increased markedly 1 hour after injection of 1 mg/kg of LPS and increased slightly but significantly 10 minutes after injection of 40 microg/kg of LPS. CONCLUSION AND CLINICAL RELEVANCE: PAF plays an important role in the development of LPS-induced thrombocytopenia and neutropenia in dogs. Control of PAF production, PAF-induced effects, or both may be important in the treatment of dogs with gram-negative bacterial infections and associated thrombocytopenia and neutropenia.     

Effects of platelet clumping on platelet concentrations measured by use of impedance or buffy coat analysis in dogs.

OBJECTIVE: To determine whether platelet clumps are homogeneously distributed in blood samples, and whether platelet concentrations (PC) obtained by use of impedance and buffy coat analysis can be considered minimum values when platelet clumps are present. DESIGN: Prospective study. SAMPLE POPULATION: 50 blood samples obtained from 30 dogs. PROCEDURE: 10 blood samples containing platelet clumps were used and 10 smears were made from each sample; amount of platelet clumping was graded for all 100 smears. Blood from each of 20 healthy dogs was divided between 2 EDTA tubes before and after platelet clumping was induced by adenosine diphosphate (ADP). The PC for each ADP-treated and untreated sample were measured, using impedance and quantitative buffy coat analyzers. RESULTS: Platelet clumps were evident in all 100 blood smears, but the amount of clumping varied considerably within some samples. Using the impedance analyzer, the PC of ADP-treated samples were significantly lower and never higher than the PC of untreated samples. Using the buffy coat analyzer, some ADP-treated samples had increased PC; however, significant differences were not detected between treated and untreated samples. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet clumping was not homogeneous within blood samples. When platelet clumps were identified by direct examination of blood smears, the PC detected by use of the impedance analyzer could be considered minimum values. In contrast, the PC detected by use of the buffy coat analyzer were sometimes increased. Useful information can be obtained by measuring PC in blood with platelet clumps; values obtained by use of impedance can be considered minimums, and values obtained by use of buffy coat analysis may be either minimum values or reasonable estimates of PC.     

Evaluation of platelet function in dogs with cardiac disease using the PFA‐100 platelet function analyzer

Background: Cardiac disease has the potential to alter platelet function in dogs. Evaluation of platelet function using the PFA‐100 analyzer in dogs of multiple breeds and with a broad range of cardiac conditions would help clarify the effect of cardiac disease on platelets. Objectives: The objective of this study was to assess differences in closure time (CT) in dogs with cardiac disease associated with murmurs, when compared with that of healthy dogs. Methods: Thirty‐nine dogs with cardiac murmurs and turbulent blood flow as determined echocardiographically were included in the study. The dogs represented 23 different breeds. Dogs with murmurs were further divided into those with atrioventricular valvular insufficiency (n=23) and subaortic stenosis (n=9). Fifty‐eight clinically healthy dogs were used as controls. CTs were determined in duplicate on a PFA‐100 analyzer using collagen/ADP cartridges. Results: Compared with CTs in the control group (mean±SD, 57.6±5.9 seconds; median, 56.5 seconds; reference interval, 48.0–77.0 seconds), dogs with valvular insufficiency (mean±SD, 81.9±26.3 seconds; median, 78.0 seconds; range, 52.5–187 seconds), subaortic stenosis (71.4±16.5 seconds; median, 66.0 seconds; range, 51.5–95.0 seconds), and all dogs with murmurs combined (79.6±24.1 seconds; median, 74.0 seconds; range, 48.0–187 seconds) had significantly prolonged CTs (P<.01). Conclusions: The PFA‐100 analyzer is useful in detecting platelet function defects in dogs with cardiac murmurs, most notably those caused by mitral and/or tricuspid valvular insufficiency or subaortic stenosis. The form of turbulent blood flow does not appear to be an important factor in platelet hypofunction in these forms of cardiac disease.     

Platelet size,platelet surface-associated IgG,and reticulated platelets in dogs with immune-mediated thrombocytopenia

Abstract: Immune-mediated thrombocytopenia (IMT) is a disorder in which bound IgG on the surface of platelets results in platelet removal and alterations in mean platelet volume. Using flow cytometry, alterations in platelet size, platelet surface-associated IgG (PSAIgG), and numbers of reticulated platelets were determined in 13 dogs with primary IMT and 4 dogs with secondary IMT induced by experimental infection with Babesia gibsoni . Effects of sample age on platelet parameters also were determined, using samples from 20 dogs with normal platelet counts analyzed within 4 hours and after 24, 48, and 72 hours of storage in EDTA. No significant changes in platelet count, platelet size, or reticulated platelet percentage were observed in samples assayed within 4 and 24 hours of blood collection; whereas PSAIgG values increased 3 to 7 fold in samples stored for 24–72 hours. Using reference values for freshly collected or 24-hour-old samples, 10 of 13 (77%) dogs with primary IMT and all B gibsoni-inf ected dogs had increased PSAIgG levels. In 12 (75%) of the 16 dogs with thrombocytopenia the percentage of reticulated platelets was increased; however, absolute numbers of reticulated platelets were within reference values. Moreover, PSAIgG level and the percentage of reticulated platelets were not always increased concurrently in dogs with primary and secondary IMT. Platelet microparticles were detected in all B gibsoni-infected dogs, 8 of 13 (62%) dogs with primary IMT, and transiently in a dog that responded to immunosuppressive treatment. The results of this study indicate that sample age and time of sampling during disease affect interpretation of platelet parameters in dogs with IMT.     

Platelet distribution width and mean platelet volume in the interpretation of thrombocytopenia in dogs

Objectives : To investigate the use of platelet volume indices in the interpretation of thrombocytopenia in dogs with systemic disease. Methods : Case records of 80 control dogs and 159 thrombocytopenic dogs with systemic disease were reviewed retrospectively. The relationships between mean platelet volume, platelet distribution width and platelet count in systemically well dogs were established. Knowledge of these relationships was used to interpret thrombocytopenia in terms of the underlying disease process. Results : There was a positive relationship between platelet distribution width and mean platelet volume. Both platelet distribution width and mean platelet volume varied in a negative relationship with platelet count, which was unaffected by signalment, underlying disease, variations in haematological parameters or serum proteins. Clinical Significance : The interpretation of mean platelet volume and platelet distribution width in dogs requires simultaneous reference to the platelet count. Platelet volume indices did not aid interpretation of thrombocytopenia in terms of underlying pathological processes in the population studied.     

Escherichia coli associated endotoxemia in dogs with parvovirus infection

Escherichia coli bacteremia and endotoxemia were observed in 3 adult mongrel dogs which had been prediagnosed as canine parvoviral disease. The endotoxin level was 46.5 pg/ml in the plasma of clinical cases, while 2.3 pg/ml in healthy controls. The microflora of the feces was confused in the clinical cases. The percentage of E. coli was major in the feces. Serologically similar strains were isolated from the blood. These strains did not produce enterotoxins such as heat-stable enterotoxin (ST) and heat-labile enterotoxin (LT). Histopathologically, the lesions in the small intestine consisted of epithelial degeneration and necrosis. Viral inclusion bodies were frequently observed in the epithelial cells. Disseminated intravascular coagulation was observed in various tissues including the liver and small intestinal submucosa. After experimental infection with CPV, all dogs showed various clinical signs. CPV was positive in the feces. Endotoxin level in the plasma gradually increased and high level continued for long period from 10 to 30 days. Mean maximum level of endotoxin in the experimental dogs was 73.6 pg/ml. These results indicate that intestinal flora plays a important role in the pathogenesis of CPV infection and that endotoxin is one of the factors which predispose to severe disease after the infection.     

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.     

Evaluation of the effect of ketoprofen and carprofen on platelet function in dogs studied by PFA-100 point-of-care analyser

The effect of two nonsteroidal anti-inflammatory drugs (carprofen and ketoprofen) on platelet adhesion and aggregation functions was evaluated by the PFA-100 analyser (Dade-Behring, CA, U.S.A.) using its collagen-adenosine diphosphate (ADP) and collagen-epinephrine (EPI) cartridges. The function of platelets was evaluated in 55 healthy dogs, in 7 dogs treated with ketoprofen and in 31 dogs treated with carprofen in a therapeutic dose for minimum 5 days. The therapeutic doses of carprofen had no effect on the closure time of PFA-100 (which is the marker of platelet function) but ketoprofen caused a significant increase when using collagen-EPI stimulation The closure times for both the healthy (control) and the treated dogs using EPI cartridges were often longer than the upper default cut-off point (300 sec) of the device. The PFA-100 analyser with collagen-ADP cartridges could be a useful tool for veterinary applications including the evaluation of platelet aggregation in dogs treated with NSAIDs. The upper cut-off point of PFA-100 might be extended.     

Platelet aggregation in dogs experimentally infected with Rickettsia rickettsii

Platelet aggregation studies were performed on nine Beagle dogs experimentally infected with Rickettsia rickettsia. Platelets from dogs with Rocky Mountain spotted fever tended to be more aggregable than controls.     

Activated coagulation times of whole blood in normal dogs and dogs with coagulopathies

The activated coagulation time (ACT) of whole blood was determined at 37C and at room temperature for 42 normal dogs and eight dogs with naturally–occurring or experimentally–induced coagulation defects.
Normal ACT values ranged from 64 to 95 seconds at 37C, and 83 to 129 seconds at room temperature. In abnormal dogs, ACT was increased on 14 of 17 occasions that a prolonged activated partial thromboplastin time (APTT) was recorded: the ACT failed to detect an abnormality on three occasions the APTT was slightly increased. ACT determination at 37C correlated better with APTT than did ACT testing at room temperature.
The ACT test is simple, inexpensive and convenient. It is a useful screening test for intrinsic coagulation defects in the dog. It is suggested that the test be performed at 37C: at this temperature an ACT of 95 seconds or more in a dog warrants further investigation.     

Vincristine impairs platelet aggregation in dogs with lymphoma

Platelet aggregation before and after administration of 0.5 mg/m2 of vincristine (VCR) was evaluated in 7 dogs with spontaneously occuring lymphoma. Aggregation on platelet-rich plasma separated from blood collected in 3.8% sodium citrate was performed using adenosine diphosphate (ADP), arachidonic acid (AA), and collagen (COL) as agonists. The slope for aggregation in response to ADP was significantly lower after administration of VCR (P = .032). Maximal aggregation after administration of VCR was significantly lower in response to ADP, COL, and AA (P = .03, P = .04, and P = .03, respectively).     

Platelet aggregation studies in dogs with acute Ehrlichia platys infection

Ten adult dogs (5 Beagles and 5 mixed-breed dogs) were inoculated IV with canine platelets containing Ehrlichia platys. Inclusions and morulae of E platys developed in platelets of infected dogs at 10 to 14 days after inoculation, followed by marked thrombocytopenia at 14 to 21 days. Parasitemia and marked thrombocytopenia recurred at 24 to 28 days after inoculation. Increased numbers of megakaryocytes were observed in marrow aspirate smears from infected dogs, indicative of regenerative thrombocytopenia. Prior to infection, platelet-rich plasma from these dogs was determined to have similar aggregatory response to arachidonate. After infection with E platys, the aggregatory response of platelet-rich plasma to collagen or 3 dilutions of adenosine diphosphate was evaluated. A statistically significant (P less than 0.05) inhibition of platelet aggregatory response to the lowest dilution of adenosine diphosphate was detected for mixed-breed dogs, whereas aggregation responses were unchanged in Beagles. Results indicate that platelet activation may occur in dogs with acute ehrlichial infection.     

Changes in platelet concentrates from dogs due to storage. I. Platelet count in in vitro function]

The possibility of storage of canine platelet concentrates (PC) was investigated using PC from dogs which were obtained with an automatic cell separator in C4-cell separation sets with low gasdiffusionable Polyvinylchlorid (PVC) storage containers or in C4L-sets developed for storage with high gasdiffusionable Polyolefin (PO) containers, respectively. The storage was carried out for a period of 10 days under permanent agitation at 22 degrees C (C4/22 degrees C, n = 10; C4L/22 degrees C, n = 11) or at 4 degrees C (C4L/4 degrees C, n = 6), respectively. Measurements were done directly after production of the PC, after 6 hours and then daily during the 10-day storage period. In the first part of this paper the results of platelet count (determined automatically with a blood cell differentiation automat and visually), the number of platelet aggregates, the mean platelet volume (MPV) as well as the platelet function with regard to the platelet aggregation induced by collagen or ADP and the resonance-thrombogram (RTG) are presented. The platelet count, measured automatically as well as visually, remained preponderantly constant over the complete storage time in all storage conditions. Dependent on the storage conditions--especially under storage at 22 degrees C--an increase of the number of platelet aggregates and a decrease of MPV was determined. In addition, the loss of platelet function measured by aggregation induced by collagen as well as by ADP showed a significant dependency of storage conditions. The stored platelets lost their ability to aggregate under C4/22 degrees C-conditions after a storage period of 2 days, under C4L/22 degrees C-conditions after 4 days and under C4L/4 degrees C-conditions not before 8 days of storage. Previous resuspending of platelets in fresh plasma delayed the loss of platelet function. Because the loss of platelet function described in the RTG became significant at nearly the same point in time, a storage of canine PC under corresponding conditions can be recommended for upto 2 days (C4/22 degrees C), for 4 days (C4L/22 degrees C) or 8-10 days (C4L/4 degrees C), respectively.     

Preclinical studies for the use of the platelet function analyser PFA-100 with the collagen/ADP cartridge in dogs

In this study, the following three aspects of platelet function analyser were investigated in dogs, using a collagen/ADP cartridge: precision, influence of the cartridge batch and of the sample storage time. Closure time and total volume of blood flow until closure of the capillary were measured. Based on several series of 5 repeated measurements mean coefficients of variation were 5% (3-6%; closure time) or 3% (1-5%; total volume). Neither closure time, nor total volume showed significant differences (p > 0.05) when comparing the results of 6 different batches of the collagen/ADP cartridge. Closure time (p = 0.0211, analysis of variance) and total volume (p = 0.0310) were significantly influenced by storage time, based on the sample material of 6 healthy dogs which was stored for 24 hours. Shortening of the closure time and decrease of the total volume observed in the time interval 1-2 hours after blood collection was followed by a significant prolongation of closure time and increase of the total volume (p < 0.05) starting 8 hours after blood collection. This study shows sufficient reproducibility which is not affected by reagent batch number. The results of the studies on storage indicated nearly identical recommendations for storage time before measurement of canine (0.5-2 hours) and human (0.5-3 hours) sample material.     

Effects of flunixin meglumine in dogs following experimentally induced endotoxemia

Twelve dogs were randomly divided into three groups. Group 1 dogs were given Escherichia coli endotoxin and then treated with flunixin meglumine. Group 2 dogs were given endotoxin as group 1, but untreated. Group 3 dogs were given flunixin meglumine alone. The dogs were monitored clinically and urine and serum samples were collected at regular intervals for 72 hours. All surviving dogs were humanely killed after 72 hours and examined for gross and histologic lesions. Group 1 dogs all survived 72 hours, but showed prerenal azotemia, hepatocellular damage, hemorrhagic enteritis, and numerous gastric ulcerations. Three of the four dogs in group 2 died before 72 hours. Group 2 dogs showed many of the same chemical and hemodynamic changes as group 1. They had severe hemorrhage into the intestinal lumen; however, there were no gastric ulcerations. Group 3 dogs all survived and showed little physical or hematologic change. The study suggested the following: 1) flunixin meglumine was an effective drug in ameliorating the fatal effects of canine endotoxemia, 2) the effects of endotoxin in combination with flunixin meglumine, at 1.1 mg/kg body weight, caused gastric ulcerations, and 3) in normal dogs flunixin meglumine at 1.1 mg/kg body weight did not cause severe side effects or gross lesions.     

Hypoalbuminemia-related platelet hypersensitivity in two dogs with nephrotic syndrome

Platelet aggregation studies in 2 dogs with nephrotic syndrome disclosed increased platelet sensitivity to a low dose of adenosine diphosphate. Subsequent studies with isolated platelets and plasma indicated that a plasma factor was responsible primarily for inducing platelet hypersensitivity. The increased platelet aggregation response was corrected by increasing the albumin concentration of the plasma. The study suggested an important role for albumin in modulating platelet aggregation and may partially explain the tendency toward thrombosis noted in hypoalbuminemic dogs with nephrotic syndrome.