Platelet function in dogs treated for lymphoma and hemangiosarcoma and supplemented with dietary n-3 fatty acids

A prospective randomized, double-blind clinical trial was performed to test the hypothesis that dogs with malignancies that are supplemented with n-3 fatty acids do not have clinical or laboratory evidence of coagulation disorders or altered platelet function when compared with unsupplemented dogs with similar malignancies. Thirteen dogs with hemangiosarcoma and 66 dogs with lymphoma were evaluated. Coagulation status of the dogs with lymphoma and hemangiosarcoma was evaluated with prothrombin time, partial thromboplastin time, platelet count, and in vitro platelet aggregometry using the whole-blood method. These tests were performed at 5 time points: before beginning the diet (week 0), at weeks 3, 15, and 21, and at 1 year or when progressive disease was evident. Alterations in platelet function in dogs receiving a diet supplemented with dietary n-3 fatty acids were not identified when compared to dogs fed a control diet. Dietary n-3 fatty acid supplementation using this dosage and ratio in dogs with lymphoma or hemangiosarcoma did not induce clinically significant hemorrhage in these animals. Therefore, supplementation with n-3 fatty acids did not result in clinical or laboratory evidence relating to uncontrolled hemorrhage in these dogs.     

Platelet Dysfunction Associated With Immune-Mediated Thrombocytopenia in Dogs

The effect of antiplatelet antibody on in vitro platelet function was investigated in 15 dogs with immune-mediated thrombocytopenia (ITP). Platelet aggregation was assessed after addition of serum from healthy dogs (n = 5) or dogs with ITP (n = 15) to platelet-rich plasma from a healthy donor dog. The aggregation responses to adenosine diphosphate, thrombin, and collagen/epinephrine were measured as the maximum aggregation observed after 2 minutes. In 13 of 15 dogs with ITP, maximal aggregation was significantly inhibited in response to ADP, thrombin, or collagen/epinephrine. The slope of the aggregation curve was decreased after addition of serum from 9 of 15 patients. A polyclonal rabbit anti—dog platelet antiserum induced inhibition of aggregation with all 3 agonists.
Serum from control dogs neither inhibited nor activated platelet aggregation. Aggregation experiments were repeated with all 3 agonists after addition of patient immunoglobulin (lg)G or IgG from a healthy dog to platelet-rich plasma. The IgG fraction from 9 of 10 dogs with ITP suppressed platelet aggregation. The IgG fraction from polyclonal rabbit anti—dog platelet antiserum inhibited platelet aggregation with all agonists. These results suggest that many canine ITP patients have circulating antibodies that, in addition to causing platelet destruction, may cause platelet dysfunction.     

Platelet aggregation in dogs with mitral valve regurgitation

OBJECTIVE: To compare platelet aggregation in healthy dogs and dogs with mitral valve regurgitation (MVR) to determine whether regurgitation had an effect on platelet function. ANIMALS: 32 dogs with MVR and 43 healthy dogs. PROCEDURE: Platelet aggregation was measured with an aggregometer, using adenosine 5'-diphosphate as the aggregating agent, and the maximum aggregation and the enhancement of platelet sensitivity (EPS) values were calculated. RESULTS: Platelet count and maximum aggregation were not significantly different between healthy dogs and dogs with MVR. However, EPS values in dogs with MVR were significantly higher than values in healthy dogs. Platelet count and maximum aggregation were not significantly different between dogs classified as New York Heart Association functional class I or II and dogs classified as functional class III or IV; however, EPS values were significantly higher in dogs classified as functional class III or IV. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that platelet aggregation is decreased in dogs with MVR and that the EPS value may be more sensitive to differences in disease severity than in measurement of maximum aggregation.     

Platelet aggregation and haemostatic response in dogs naturally co-infected by Leishmania infantum and Ehrlichia canis

Haemostatic alterations in dogs naturally infected by ehrlichiosis and/or leishmaniasis were studied. Platelet count, ADP and collagen-induced platelet aggregation, prothrombin time, activated partial thromboplastin time (APTT) and plasma fibrinogen concentration were measured. An evident reduction of platelet aggregation response was shown for Leishmania-Ehrlichia co-infected dogs where platelet aggregation was lower in comparison with control and leishmaniotic dogs (ADP and collagen, P < or = 0.01) and ehrlichiotic dogs (ADP 10 and 7.5 microm, P < or = 0.05). Moreover, a significant increase in APTT as well as a reduction of the albumin/globulin rate (A/G) for leishmaniotic and co-infected dogs versus control and ehrlichiotic dogs was detected. The hypothesis of a synergism between leishmaniosis and ehrlichiosis in altering platelet function by different pathways is discussed.     

The effects of prednisone on haemostasis in leishmaniotic dogs treated with meglumine antimoniate and allopurinol

Thirty dogs naturally infected with Leishmania infantum were studied in order to determine the effects of treatment on haemostatic function. The animals were divided randomly into two treatment groups: Group 1 received meglumine antimoniate and allopurinol; Group 2 dogs were given the same treatment plus prednisone. Ten healthy animals were used as untreated controls. Clinical examination and determination of platelet aggregation, coagulation factors and biochemical parameters were undertaken before treatment and after 15, 30 and 60 days. A significant improvement in platelet aggregation was detected after 60 days in Group 1, but only after 15 days in Group 2. In both treated groups, platelet aggregation was lower than in the control group at the end of the study. The results suggest that prednisone may be a useful tool in the treatment of haemostatic disorders during canine leishmaniosis. The potential benefits and risks due to the use of corticosteroids in the treatment of leishmaniosis are discussed.     

Thrombopathies causing bleeding in a boxer and mixed-breed dog

Hereditary platelet function disorders are clinically characterized by recurrent surface bleeding and prolonged bleeding time, despite normal platelet count and coagulation tests. The authors describe persistent thrombopathies in two young dogs with increased bleeding tendencies but with normal plasma coagulation times and von Willebrand factor (vWf) concentrations. Buccal mucosal bleeding times were prolonged in both dogs. In aggregation studies, platelets underwent only a shape change or minimal aggregation in response to adenosine diphosphate and collagen. Whole-platelet adenine nucleotide concentrations were normal. Electron microscopic evaluation of fibrinogen and vWf binding to the platelets of case no. 1 demonstrated the presence of glycoprotein IIb/IIIa and Ib receptors. Thus, the intrinsic platelet function defects may be different in these two dogs and may likely represent secretion/signal transduction disorders.     

Influence of a cyclic combination chemotherapeutic protocol on primary haemostasis in dogs suffering from malignant lymphoma

The purpose of this study was to examine the influence of cyclic combination chemotherapy on primary haemostasis in dogs with malignant lymphoma. Seventeen dogs receiving cytostatic treatment for high-grade lymphoma were included in the study. The dogs were treated with a Madison–Wisconsin derived protocol, which included asparaginase, vincristine, doxorubicin and prednisolone. At different time points during the first 4 weeks of induction, platelet count, capillary bleeding time, analysis of the platelet function using the platelet function analyser PFA-100, and platelet aggregation by the Born-method were measured.The most obvious changes were found for median values of the platelet count, which increased significantly from 210,000/μL before induction to 349,000/μL during the second week of induction (P = 0.0010). Median platelet count subsequently decreased by the fourth week of treatment (Friedman-test: P < 0.0001). None of the parameters of platelet function (capillary bleeding time, automatic platelet function analysis, aggregation maximum) showed significant changes with time (P > 0.05, Friedman-test). The results did not suggest that significant platelet dysfunction was induced by the chemotherapeutic protocol used in the study.     

Effects of low-dose aspirin and specific thromboxane synthetase inhibition on whole blood platelet aggregation and adenosine triphosphate secretion in healthy dogs.

Platelet aggregation and adenosine triphosphate (ATP) secretion in response to arachidonic acid (10 microM) or collagen (5 micrograms/ml) were compared in healthy, adult female Beagles treated with low-dosage aspirin (3.5 mg/kg of body weight, PO, q 12 h for 7 treatments) or with CGS 12970, a specific thromboxane synthetase inhibitor (10 mg/kg, PO, q 8 h for 10 treatments). Platelet aggregation was assessed in whole blood by use of an electrical impedance method. Baseline values obtained prior to treatment served as controls. Addition of arachidonic acid to blood from nontreated dogs resulted in significantly (P less than 0.001) increased impedance, but had no effect in blood from dogs treated with either aspirin or CGS 12970. Treatment with CGS 12970 or aspirin significantly (P less than 0.001) decreased platelet ATP secretion in response to arachidonic acid, compared with baseline values; however ATP secretion in aspirin-treated dogs was significantly (P less than 0.01) less than ATP secretion in CGS 12970-treated dogs. Differences in platelet aggregation were not observed between control dogs and aspirin- or CGS 12970-treated dogs in response to collagen as an aggregant, however, collagen-induced platelet ATP secretion was significantly (P less than 0.001) decreased in dogs treated with aspirin, compared with control values and values from dogs treated with CGS 12970. In dogs treated orally with 0.1, 0.2, 1.0, or 10 mg of CGS 12970/kg, dose-dependent inhibition of arachidonic acid-induced platelet aggregation was observed, with impedance changes not observed at the 10-mg/kg dosage and normal platelet aggregation associated with the 0.1-mg/kg dosage.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dogs with heart diseases causing turbulent high-velocity blood flow have changes in platelet function and von Willebrand factor multimer distribution

The purpose of this prospective study was to investigate platelet function using in vitro tests based on both high and low shear rates and von Willebrand factor (vWf) multimeric composition in dogs with cardiac disease and turbulent high-velocity blood flow. Client-owned asymptomatic, untreated dogs were divided into 4 groups: 14 Cavalier King Charles Spaniels (Cavaliers) with mitral valve prolapse (MVP) and no or minimal mitral regurgitation (MR), 17 Cavaliers with MVP and moderate to severe MR, 14 control dogs, and 10 dogs with subaortic stenosis (SAS). Clinical examinations and echocardiography were performed in all dogs. PFA100 closure times (the ability of platelets to occlude a hole in a membrane at high shear rates), platelet activation markers (plasma thromboxane B2 concentration, platelet surface P-selectin expression), platelet aggregation (in whole blood and platelet-rich plasma with 3 different agonists), and vWf multimers were analyzed. Cavaliers with moderate to severe MR and dogs with SAS had longer closure times and a lower percentage of the largest vWf multimers than did controls. Maximal aggregation responses were unchanged in dogs with SAS but enhanced in Cavaliers with MVP (regardless of MR status) compared with control dogs. No significant difference in platelet activation markers was found among groups. The data suggest that a form of platelet dysfunction detected at high shear rates was present in dogs with MR and SAS, possibly associated with a qualitative vWf defect. Aggregation results suggest increased platelet reactivity in Cavaliers, but the platelets did not appear to circulate in a preactivated state in either disease.     

Increased platelet aggregation response in Cavalier King Charles Spaniels with mitral valve prolapse

Mitral valve prolapse (MVP) is a fundamental feature of myxomatous mitral valve disease in the dog. In humans, primary MVP is associated with increased platelet reactivity. In Cavalier King Charles Spaniels (CKCS), a breed predisposed to myxomatous mitral valve disease, there is a high prevalence of hypomagnesemia and platelet anomalies, such as thrombocytopenia and macrothrombocytosis. The objective of this study was to evaluate platelet aggregation responses in CKCS and to determine the relationship between the platelet aggregation response and serum magnesium concentration, MVP, mitral regurgitation (MR), and platelet count. In 19 CKCS with MVP and 7 control dogs (not CKCS), the platelet aggregation response to 3 different agonists was compared. The CKCS with >100,000 platelets/microL (n = 10) had a significantly higher maximum aggregation response with regard to all tested agonists than the CKCS with <100,000 platelets/microL (n = 9) and control dogs (n = 7). The CKCS with <100,000 platelets/microL had a platelet aggregation response similar to the control dogs. There was no correlation between degree of MVP and platelet aggregation response. Platelet diameter increased (P = .006) and serum magnesium concentration decreased (P = .04) with lower platelet concentration. In conclusion, CKCS with MVP appeared to separate into 2 groups--1 group with <100,000 platelets/microL, normal platelet aggregation, low serum magnesium concentration, and enlarged platelets, and another group with >100,000 platelets/microL, increased platelet aggregation, and normal serum magnesium concentration and platelet size.     

Whole blood platelet aggregation in uremic dogs

Whole blood platelet aggregation responses to collagen, arachidonic acid, and adenosine diphosphate were determined by use of the impedance method in 22 dogs with serum urea concentrations greater than or equal to 20 mmol/L, which was attributable to renal disease, and in 25 healthy control dogs. The median changes in impedance for the control dogs were 23 ohms for collagen, 18 ohms for arachidonic acid, and 6 ohms for adenosine diphosphate. The median changes in impedance in uremic dogs were 25 ohms for collagen, 21 ohms for arachidonic acid, and 15 ohms for adenosine diphosphate. There were no significant differences in platelet aggregation responses to collagen, arachidonic acid, and adenosine diphosphate between uremic and control dogs. Hemorrhagic tendencies were not detected in uremic dogs by use of whole blood platelet aggregation. Results of this study suggest that platelet aggregation by use of the whole blood platelet aggregometer is not abnormal in uremic dogs, but does not exclude the possibility of a platelet aggregation defect undetected by the whole blood system.     

Platelet function in dogs: breed differences and effect of acetylsalicylic acid administration

BACKGROUND: Clinical studies investigating platelet function in dogs have had conflicting results that may be caused by normal physiologic variation in platelet response to agonists. OBJECTIVES: The objective of this study was to investigate platelet function in clinically healthy dogs of 4 different breeds by whole-blood aggregometry and with a point-of-care platelet function analyzer (PFA-100), and to evaluate the effect of acetylsalicylic acid (ASA) administration on the results from both methods. METHODS: Forty-five clinically healthy dogs (12 Cavalier King Charles Spaniels [CKCS], 12 Cairn Terriers, 10 Boxers, and 11 Labrador Retrievers) were included in the study. Platelet function was assessed by whole-blood aggregation with ADP (1, 5, 10, and 20 microM) as agonist and by PFA-100 using collagen and epinephrine (Col + Epi) and Col + ADP as agonists. Plasma thromboxane B(2) concentration was determined by an enzyme immunoassay. To investigate the effect of ASA, 10 dogs were dosed daily (75 or 250 mg ASA orally) for 4 consecutive days. RESULTS: A higher platelet aggregation response was found in CKCS compared to the other breeds. Longer PFA-100 closure time (Col + Epi) was found in Cairn Terriers compared to Boxers. Plasma thromboxane B(2) concentration was not statistically different between groups. Administration of ASA prolonged the PFA-100 closure times, using Col + Epi (but not Col + ADP) as agonists. Furthermore, ASA resulted in a decrease in whole-blood platelet aggregation. CONCLUSIONS: Platelet function is influenced by breed, depending upon the methodology applied. However, the importance of these breed differences remains to be investigated. The PFA-100 method with Col + Epi as agonists, and ADP-induced platelet aggregation appear to be sensitive to ASA in dogs.     

Whole blood platelet aggregation in dogs with liver disease

Whole blood platelet aggregation was determined in response to collagen, arachidonic acid, and adenosine diphosphate in 20 dogs with liver disease and in 20 control dogs. Platelet aggregation in response to collagen and arachidonic acid was reduced in dogs with liver disease, compared with control dogs (P less than 0.05), whereas there was no significant difference in platelet response to adenosine diphosphate between the 2 groups of dogs. Adenosine diphosphate was found not to be a reliable aggregation agent for determination of whole blood platelet aggregation in dogs. Dogs whose platelets did not aggregate in response to collagen and/or arachidonic acid manifested bleeding tendencies that could be attributed to platelet dysfunction.     

Platelet function and association of bovine viral diarrhea virus with platelets of persistently infected cattle

OBJECTIVE: To determine whether viral involvement with platelets obtained from cattle persistently infected (PI) with bovine viral diarrhea virus (BVDV) is associated with altered platelet function or decreased platelet counts. SAMPLE POPULATION: Platelets obtained from 8 cattle PI with BVDV and 6 age-, sex-, and breed-matched uninfected control cattle. PROCEDURE: Manual platelet counts were determined, and platelet function was assessed through optical aggregometry by use of the aggregation agonists ADP and platelet-activating factor. Identification of BVDV in serum and preparations of purified platelets was determined by use of virus isolation tests. RESULTS: No significant difference in platelet counts was detected between cattle PI with BVDV and control cattle. In response to the aggregation agonists, maximum aggregation percentage and slope of the aggregation curve were not significantly different between cattle PI with BVDV and control cattle. We isolated BVDV from serum of all PI cattle and from purified platelets of 6 of 8 PI cattle, but BVDV was not isolated from serum or platelets of control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Isolation of BVDV from platelets in the peripheral circulation of cattle immunotolerant to BVDV does not result in altered platelet function or decreases in platelet counts.     

A comparison of platelet parameters in EDTA- and citrate-anticoagulated blood in dogs

BACKGROUND: Platelet aggregates are a common artifact in canine blood. Aggregates may affect the accuracy of platelet counts, with important consequences for patient care. OBJECTIVES: The purpose of this study was to determine if platelet counts in dogs were more accurate if blood was collected into citrate instead of EDTA as an anticoagulant. METHODS: Blood was collected from 50 dogs with neoplasia admitted to the oncology service at Cornell University. EDTA and citrate Vacutainer tubes were filled with blood in random order. Platelet counts and parameters (mean platelet volume [MPV], platelet distribution width [PDW], mean platelet component concentration [MPC], platelet component distribution width [PCDW], and automated platelet clump count [APCC]) were determined using an optical-based hematology analyzer (ADVIA 120). Blood smears from each anticoagulated sample were scored visually for platelet aggregates. RESULTS: The median platelet count was significantly lower (median decrease, 27 x 10(9)/L) in citrate-anticoagulated blood compared with EDTA-anticoagulated blood. This was attributed to platelet activation and aggregation: significantly more aggregates were seen in smears of citrate- than of EDTA-anticoagulated blood. Aggregates were typically small and not detected by the analyzer. Also, the MPV and MPC (or density) were significantly higher (median increase, 3 fL) and lower (median decrease, 33 g/L) in citrate-anticoagulated samples, respectively. CONCLUSIONS: Platelets aggregate, likely from activation, when blood from dogs with neoplasia is anticoagulated with citrate for hematology testing, resulting in lower platelet counts. Citrate also yields inaccurate results for MPV and MPC, likely because of inadequate sphering of platelets. Thus, we recommend that citrate not be used as an anticoagulant when accurate platelet counts are desired in dogs.     

Assessment of neutrophil function in canine cancer patients undergoing chemotherapy and correlation with neutrophil numbers

Decreased neutrophil function following administration of chemotherapy has been reported in dogs with lymphoma. The first objective of our study was to determine if neutrophil oxidative burst and phagocytic activity are affected by chemotherapy 7 to 10 days following initiation of treatment in dogs with lymphoma and non-lymphoma malignancies. The second objective was to determine if there is a correlation between neutrophil numbers and neutrophil function before or after initiation of chemotherapy. Flow cytometric assessment of neutrophil oxidative burst and phagocytosis following stimulation with Escherichia coli was performed in 9 dogs diagnosed with lymphoma and 17 non-lymphoma tumor-bearing dogs pre- and post-chemotherapy, as well as 14 tumor-free control dogs. Spearman rank correlation was performed to determine if blood neutrophil numbers and neutrophil function were significantly correlated. Lymphoma patients showed significantly reduced percentage neutrophil oxidative burst post-chemotherapy compared to healthy controls as well as compared to pre-chemotherapy values (P = 0.0022 and P = 0.0020, respectively). Lymphoma patients also exhibited significantly reduced neutrophil phagocytosis activity post-chemotherapy compared to controls and pre-chemotherapy values (P = 0.0016 and P = 0.014, respectively). Dogs with non-lymphoma malignancies also showed a significant decrease in both percentage oxidative burst and phagocytosis post-chemotherapy compared to pre-chemotherapy values (P = 0.00040 and P = 0.029, respectively). Neutrophil numbers and function were not significantly correlated. The results of the study suggest that chemotherapeutic treatment decreases neutrophil oxidative burst and phagocytic activity 7 to 10 days post-treatment in dogs with various malignancies. Furthermore, neutrophil numbers cannot be used to predict neutrophil function.     

Enhanced platelet reactivity in heartworm-infected dogs

Platelet number, mean platelet volume, and platelet function were evaluated in 34 clinically normal dogs and 28 heartworm-infected (HWI) dogs. Mean platelet numbers for dogs of the HWI group was not significantly lower than those for dogs of the control group (214,000 vs 254,000 cells/microliter); however, 6 (21%) HWI dogs had platelet numbers less than 150,000/microliter, compared with only 2 (6%) heartworm-negative dogs. The mean platelet volume was not significantly different (7.8 vs 7.7 fl) between the 2 groups of dogs. Mean platelet aggregation responses to intermediate and low concentrations of collagen (3.0 and 1.5 micrograms) and to high, intermediate, and low concentrations of ADP (25, 10, and 5 microM) were greater in dogs of the HWI group. Mean platelet 14C-serotonin release was also greater in HWI dogs in response to high concentration of ADP (25 microM) and to intermediate concentration of collagen (3.0 micrograms).     

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.     

Identification of an Intrinsic Platelet Function Defect in Spitz Dogs

A recently identified intrinsic platelet function defect in 2 Spitz dogs is described. Both affected dogs had a history of chronic intermittent bleeding primarily from the nasal, oral, and gastrointestinal mucosa. Platelet aggregation in response to adenosine diphosphate (ADP), collagen, and platelet activating factor (PAF) was absent; however, platelet shape change did occur. Platelets aggregated in response to gamma thrombin, although a delayed onset and a reduced velocity of aggregation were present. Platelet ¹⁴C-serotonin release was diminished in response to collagen and PAF. Glycoprotein Illa was detected on the surface of platelets by flow cytometry. Platelets were morphologically normal under light and electron microscopy. Two male Spitz dogs, related to one of the affected dogs, did not have a bleeding diathesis. Collagen-induced platelet aggregation, however, was diminished in these 2 dogs. This platelet defect most closely resembles the defect described in Basset hounds.     

Changes in platelet life span in dogs with mitral valve regurgitation

Platelet life span was measured in dogs with experimentally-induced mitral regurgitation (MR) by using an in vitro whole-blood biotinylation technique to evaluate the effects of shear stress induced by MR. Mitral regurgitation was created in healthy dogs by ablation of the mitral valve chordae tendineae. Mitral regurgitation was identified by auscultation and postoperative color Doppler echocardiography. Platelets were biotinylated. and derivatized blood was reinfused into the dogs. Biotinylated platelet disappearance was measured over time to determine platelet life span. Pre- and postablation platelet life span was compared in each dog. Platelet life span was shorter in dogs with experimentally induced MR. Shear stress from MR was postulated to shorten platelet life span. Alternations in platelet function and life span may contribute to the progression of cardiovascular disease in dogs with MR. Further studies are required to determine if platelet dysfunction is related to disease progression in these patients.