Abnormal platelet activity in dogs and cats - impact and measurement.

Abnormal platelet activity can either lead to bleeding tendencies or inappropriate thrombus formation and can occur secondarily to a wide variety of disease processes, with a range of clinical consequences and severity. This article will discuss the pathophysiology of platelet function abnormalities and consider a logical diagnostic approach applicable to veterinary practice. Recent advances in platelet function testing will then be discussed, with regards to detection of platelet dysfunction and tailoring of pharmacological manipulation. Although many of these tests are still confined to research or academic institutions, techniques for indirectly assessing platelet function are starting to become more widely available. Although we still require further research to develop guidelines for the use of these tests in clinical decision-making, the recent advances in this field are an exciting step forward in being able to detect and manage platelet dysfunction in both primary care and referral practice.

A pilot study evaluating the prognostic utility of platelet indices in dogs with septic peritonitis.

OBJECTIVE: To characterize platelet indices at time of diagnosis of septic peritonitis in dogs and to assess the relationship between platelet parameter data and survival to discharge in dogs treated surgically. DESIGN: Retrospective, observational, descriptive pilot study from 2009 to 2014. SETTING: University teaching hospital. ANIMALS: Forty-eight dogs diagnosed with septic peritonitis were included in this study. Thirty-six dogs had surgical source control. Blood samples from 46 healthy control dogs were used for reference interval (RI) generation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Dogs with septic peritonitis had significantly increased mean values for mean platelet volume (MPV), plateletcrit (PCT), and platelet distribution width (PDW) with increased proportions of dogs having values above the RI compared to healthy dogs. A significantly increased proportion of dogs with septic peritonitis had platelet counts above (12.5%) and below (8.3%) the RI, with no significant difference in mean platelet count compared to healthy dogs. No significant differences in the mean platelet count, MPV, PCT, or PDW were found between survivors and nonsurvivors in dogs with surgical source control; however, dogs with MPV values above the RI had significantly increased mortality compared to dogs within the RI (P = 0.025). Values outside the RI for other platelet parameters were not associated with significant differences in mortality. CONCLUSIONS: Dogs with septic peritonitis have increased frequency of thrombocytosis and thrombocytopenia with increased MPV, PCT, and PDW. An increased MPV may be a useful indicator of increased risk of mortality in dogs treated surgically.

Infection-associated platelet dysfunction of canine platelets detected in a flow chamber model.

BACKGROUND: In the present study, the influence of bacterial infection, lipopolysacharides (LPS) and hydroxyethyl starch (HES) on platelet function in a parallel plate flow chamber were measured. Experiments were performed with non-activated and protease-activating-receptor (PAR) 4 agonist activated platelets. Comparative measurements were in vivo capillary bleeding time, platelet function analyzer and impedance aggregometry. RESULTS: PAR 4 agonist did not increase platelet adhesion of platelets from dogs with bacterial inflammation in the flow chamber in contrast to platelets of healthy dogs. Except from impedance aggregometry with lower sensitivity and specificity, PFA did not detect platelet dysfunctions in dogs with infection. In vitro addition of LPS or HES significantly reduced platelet covered area after PAR-activation. CONCLUSIONS: The flow chamber detects platelet dysfunctions in dogs with inflammatory diseases. In vitro addition of LPS highlights the inhibiting effect of bacterial wall components on platelet function. Platelet dysfunction induced by infection could possibly also be diagnosed after treatment of sepsis with colloids has commenced. The flow chamber could be a useful tool to detect sepsis associated platelet dysfunction given that larger prospective trials confirm these findings from a proof of concept study.

Platelet transfusions: treatment options for hemorrhage secondary to thrombocytopenia.

OBJECTIVE: To review current human and veterinary protocols for platelet transfusion triggers, available platelet transfusion products to support veterinary thrombocytopenic patients, and the advantages and disadvantages of each product. DATA SOURCES: Data from human and veterinary literature. HUMAN DATA SYNTHESIS: Prophylactic and therapeutic platelet transfusions are instrumental in managing human patients with thrombocytopenia. The platelet transfusion products used in human medicine consist of platelet concentrates, derived from pooled random donor platelets, or single-donor apheresis platelets. Historically, platelet transfusions in human medicine have been prophylactic in nature; however, recent research suggests changing from a prophylactic transfusion strategy to a therapeutic transfusion strategy may be safe for most patients. The optimal platelet transfusion trigger and the use of prophylactic verses therapeutic platelet transfusions are ever changing in human medicine. VETERINARY DATA SYNTHESIS: There have been many advances in platelet transfusion products, but fresh whole blood remains the most commonly used platelet transfusion product in veterinary medicine. New products such as lyophilized platelets and cryopreserved platelets offer the benefits of long shelf life, immediate availability, and higher concentration of platelets at smaller doses. Veterinary platelet transfusion guidelines are mostly extrapolated from human literature because data on veterinary platelet transfusions are lacking. CONCLUSIONS: In veterinary medicine the most commonly available product for platelet transfusions is fresh whole blood, because of availability of blood donors and lack of a cost effective easily obtainable alternative. Cryopreserved and lyophilized platelets are promising new products being used in the treatment of hemorrhaging patients with thrombocytopenia. These products offer increased platelet concentrations at decreased volumes, longer storage shelf life, and decreased exposure to whole blood products. With the development of newer readily available products, platelet transfusion parameters, to include dose, platelet count trigger, presence of disease, and clinical signs, should be further evaluated in veterinary medicine.

Assessment of platelet function.

OBJECTIVE: To review the current in vivo and in vitro tests of platelet function (PF) currently available and applicable to companion animals. DATA SOURCES: Scientific reviews, case reports, original clinical and laboratory research publications, and recent veterinary research conference proceedings. HUMAN DATA SYNTHESIS: Disorders of primary hemostasis are very common in human medicine. These include inborn errors of PF and granule storage contents, primary disease mechanisms that alter PF, disorders secondary to surgical interventions, and the effects of anticoagulant medications. Knowledge of PF disorders and the optimal method for assessment must be known to understand the mechanism and to monitor the process or drug therapy. VETERINARY DATA SYNTHESIS: Interest in the study and treatment of primary coagulopathies in clinical veterinary patients has resulted in a surge of recent publications and scientific research presentations. A translational approach that uses laboratory and point-of-care tools to uncover the pathophysiologic mechanisms in the patient with defects in primary hemostasis allows the clinician to plan the diagnosis and treatment more effectively. SUMMARY: Primary hemostatic disorders are being more commonly recognized in clinical veterinary practice. The diagnosis of platelet dysfunction may be obtained via point-of-care analyzers that use relatively small blood samples and have a quick turnaround time. Recent investigations may lead to a better understanding of the pathophysiology of PF disorders and potentially the optimization, or discovery, of novel treatments. CONCLUSIONS: The assessment of PF can be completed through in vivo and in vitro point-of-care techniques as well as by submission of blood samples to more specialized platelet biology laboratories. The information obtained including the physical examination and clinical manifestations of a hemostatic disorder, as well as the benefits of each testing modality, must be known prior to the diagnostic investigation of a patient with a coagulopathy.

Evaluation and clinical application of platelet function testing in small animal practice.

Tests that evaluate many aspects of platelet function have been applied in both human and veterinary medicine for the monitoring of treatment with platelet function inhibitors and for detection of platelet function abnormalities (inherited or acquired). Interspecies variation in the response to various platelet agonists is an important consideration when methods that have been developed for people are applied in other species. At the present time, many of these assays are not readily available in standard veterinary practice. Advanced platelet function testing for veterinary patients is offered at select academic institutions. Discussion with a specialist is recommended when considering the use of these tests, and the relative strengths and limitations of each assay should be considered in the interpretation of test results.

Influencia del concentrado de plaquetas sobre la reconstrucción de defectos de cartílago articular en la rodilla de cordero / Influence of concentrated platelets on the reconstruction of cartilage defects in the lamb knee joint

Objetivo. Analizar el efecto de las plaquetas sobre el crecimiento de cartílago en los defectos articulares provocados en la rodilla ovina. Material y método. Se provocó un defecto de 4mm de diámetro y 3mm de profundidad en la tróclea femoral de ambas rodillas en corderos macho de 6 meses de edad. La distribución de los grupos fue: grupo A (n=6): el defecto de la rodilla derecha se rellenó con concentrado de plaquetas 5min después de ser activado con ClCa. Grupo B (n=6): el defecto se rellenó con colágeno y plaquetas. Material y método. Las plaquetas se obtuvieron por centrifugación de 10ml de sangre arterial obtenida de cada animal antes de la cirugía. En los defectos de la rodilla izquierda no se administraron plaquetas. Las ovejas fueron sacrificadas 10 semanas después de la cirugía. Se realizaron estudios macro y microscópicos. Resultados. En el grupo A, se observó cartílago hialino en 4 de los defectos de la rodilla derecha a las 10 semanas de la cirugía. Ninguno de los defectos de la rodilla izquierda mostró crecimiento de cartílago hialino. En el grupo B, no se observó cartílago hialino en nigún defecto. No obstante, todos los defectos presentaron mejor celularidad condral y menor fibrosis en los defectos tratados con plaquetas que en los no tratados. Conclusiones. Esta técnica para la reconstrucción con plaquetas de defectos articulares de oveja ha mostrado en nuestro estudio resultados esperanzadores que empeoran combinadas con un andamiaje de colágeno (AU)

Objective. To study the influence of platelets on cartilage growth in articular defects in the sheep knee. Material and methods. Male Rasa Aragonesa sheep (6 months) were operated under general anaesthesia. A 4mm diameter and 3mm deep defect was made in the femoral trochlea in both knees. The right knee defect was filled with platelet concentrate 5min after being activated with ClCa in group A (n=6), and similarly activated platelets + collagen scaffold in group B (n=6). Platelets were obtained by centrifuging 10ml arterial blood from the sheep prior to the surgical procedure. The left knee defect was not filled. The sheep were sacrificed 10 weeks after surgery. Macroscopic and microscopic studies were performed. Results. In group A, hyaline cartilage was observed in the right knee defect at the end of the experiment in four cases. None of the defects of the left knees showed hyaline cartilage growth. In group B, hyaline cartilage was not observed in any right knee defect. However, in group B, all sheep showed better chondral cellularity and regeneration and lower fibrosis in the defects treated with platelets than in non-treated ones. Conclusions. This technique for articular defect reconstruction with platelets has shown satisfactory results in our study. However, collagen scaffolds may decrease this positive effect (AU)

Characteristics, diagnosis, and treatment of inherited platelet disorders in mammals.

Inherited intrinsic platelet disorders have been identified in dogs, cattle, horses, and cats as well as other animals. The prevalence of mutations in some breeds is high, making these disorders potentially as common as von Willebrand disease in certain breed lineages.

Plateletcrit is superior to platelet count for assessing platelet status in Cavalier King Charles Spaniels.

BACKGROUND: Many Cavalier King Charles Spaniel (CKCS) dogs are affected by an autosomal recessive dysplasia of platelets resulting in fewer but larger platelets. The IDEXX Vet Autoread (QBC) hematology analyzer directly measures the relative volume of platelets in a blood sample (plateletcrit). We hypothesized that CKCS both with and without hereditary macrothrombocytosis would have a normal plateletcrit and that the QBC results would better identify the total circulating volume of platelets in CKSC than methods directly enumerating platelet numbers. OBJECTIVES: The major purpose of this study was to compare the QBC platelet results with platelet counts from other automated and manual methods for evaluating platelet status in CKCS dogs. METHODS: Platelet counts were determined in fresh EDTA blood from 27 adult CKCS dogs using the QBC, Sysmex XT-2000iV (optical and impedance), CELL-DYN 3500, blood smear estimate, and manual methods. Sysmex optical platelet counts were reanalyzed following gating to determine the number and percentage of normal- and large-sized platelets in each blood sample. RESULTS: None of the 27 CKCS dogs had thrombocytopenia (defined as <164 x 10(9) platelets/L) based on the QBC platelet count. Fourteen (52%) to 18 (66%) of the dogs had thrombocytopenia with other methods. The percentage of large platelets, as determined by regating the Sysmex optical platelet counts, ranged from 1% to 75%, in a gradual continuum. CONCLUSIONS: The QBC may be the best analyzer for assessing clinically relevant thrombocytopenia in CKCS dogs, because its platelet count is based on the plateletcrit, a measurement of platelet mass.

Calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) gene mutations in a thrombopathic Simmental calf.

Simmental thrombopathia is an inherited platelet disorder that closely resembles the platelet disorders described in Basset Hounds and Eskimo Spitz dogs. Recently, two different mutations in the gene encoding calcium diacylglycerol guanine nucleotide exchange factor I (CalDAG-GEFI) were described to be associated with the Basset Hound and Spitz thrombopathia disorders, and a third distinct mutation was identified in CalDAG-GEFI in thrombopathic Landseers of European Continental Type. The gene encoding CalDAG-GEFI was sequenced using DNA obtained from normal cattle and from a thrombopathic calf studied in Canada. The affected calf was found to have a nucleotide change (c.701 T>C), which would result in the substitution of a proline for a leucine within structurally conserved region two (SCR2) of the catalytic domain of the protein. This change is likely responsible for the thrombopathic phenotype observed in Simmental cattle and underscores the critical nature of this signal transduction protein in platelets.

Prevalence of reduced fibrinogen binding to platelets in a population of Thoroughbreds.

OBJECTIVE: To measure the frequency and magnitude of reduced fibrinogen binding in a population of horses from a Thoroughbred breeding farm. ANIMALS: 444 Thoroughbred horses, 1 to 27 years old, including 316 females, 72 geldings, and 56 sexually intact males. PROCEDURES: Blood was collected from horses into tubes containingacid citrate dextrose adenine, and washed platelets were examined by use of flow cytometry for their ability to bind fibrinogen. RESULTS: Data regarding fibrinogen binding to activated platelets were normally distributed, with nearly identical amounts of variation regardless of sex. In 3 horses, fibrinogen binding to platelets was reduced from 67.6% to 83.4%, compared with normal platelets, which indicated an inability of platelets to aggregate in response to thrombin (0.1 U/mL). CONCLUSIONS AND CLINICAL RELEVANCE: Platelet fibrinogen binding of the affected horses identified in this study was characteristic of a reported heritable bleeding disorder in which the reduction in fibrinogen binding correlated with prolonged bleeding times in template bleeding assays. The bleeding disorder is distinct from Glanzmann thrombasthenia, in which platelets fail to bind fibrinogen because of lack of alphallb-beta3 integrin on their surface. The prevalence of affected horses within the small sample population studied here (0.7% [n = 3]) is considerably higher than the prevalence of bleeding disorders within more genetically diverse groups.

Platelet dysfunction in Chediak-Higashi syndrome-affected cattle.

A serious symptom of cattle affected with Chediak-Higashi syndrome (CHS) is a bleeding tendency. This diathesis is characterized by insufficient platelet aggregation as a result of depressed response to collagen. One possible cause for the depression is a decrease in contribution of endogenous agonists such as ADP or thromboxane A(2), which are released following collagen stimulation. However, these endogenous agonists play only a minor role in collagen-induced aggregation of bovine platelets. More importantly, activation of phospholipase C as a result of a direct action of collagen is depressed, leading to a depression of Ca(2+) mobilization, in platelets from CHS-affected cattle. Several types of collagen receptor are proposed to work in concert to induce aggregation. Among them, glycoprotein VI (GPVI) and GPIa/IIa (integrin alpha2 beta1) have been supposed to play dominant roles in collagen-induced aggregation. However, there are arguments about the role of each receptor, especially the role of GPIa/IIa, and the crosstalk between receptors. Recently, we reported that the Ca(2+) signaling produced by rhodocytin, which had been first reported to be an agonist for the collagen receptor GPIa/IIa, produced much less Ca(2+) signaling in CHS platelets than in normal ones, whereas that produced by GPVI activators was normal. These suggest that GPIa/IIa or the rhodocytin-associated pathway is impaired in CHS platelets. CHS platelets are valuable to reassess the mechanism of collagen-dependent signal transduction system and to delineate the inter-relationship among collagen receptors.

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.

Thrombocytopathia and light-chain proteinuria in a dog naturally infected with Ehrlichia canis.

A 6-year-old dog was presented for evaluation of recurrent epistaxis. Platelet counts, biochemical tests, and coagulation tests were within the normal range, but a mucosal bleeding time was prolonged; there was hyperproteinemia and a monoclonal gammopathy. Heterogeneity of light chains appeared in urine, however, thus suggesting that the gammopathy was polyclonal. Platelet aggregation tests showed decreased responsiveness to collagen. An Ehrlichia canis indirect fluorescent-antibody titer was positive (1:40). Treatment with tetracycline, melphalan, and prednisone resulted in a rapid clinical improvement that persisted for at least 3 years.

Platelets from bleeding Simmental cattle have a long delay in both ADP-activated expression of GpIIB-IIIA receptors and fibrinogen-dependent platelet aggregation.

We have previously reported that platelets from bleeding Simmental cattle do not aggregate in vitro in response to ADP, collagen and calcium ionophore A23187, though calcium mobilization and myosin light chain phosphorylation do occur. The aggregation abnormality, measured by aggregometry, was ascribed to abnormal cytoskeletal expression, with the maximal numbers of activated GpIIb-IIIa receptors per platelet no different from that seen in normal bovine platelets activated with ADP. We have therefore sought to compare the kinetics of micro-aggregation with the rate of expression of GpIIb-IIIa receptors required for mediating fibrinogen (Fg)-dependent platelet aggregation, to provide a more direct molecular explanation for the aggregation abnormality. We compared aggregation kinetics of ADP-activated platelets using both aggregometry and particle counting to monitor micro-aggregation. Fibrinogen receptor expression was monitored with FITC-labelled human Fg and with the reporting antibody for activated GpIIb-IIIa, FITC-PAC1, using flow cytometry. The affected platelets show a marked delay in onset of microaggregation for ADP-activated platelets stirred with human Fg, paralleded by an unusual delay in activated GpIIb-IIIa receptor expression (DARE) for otherwise competent Fg binding. The on-rates for Fg binding to platelets maximally pre-activated with PMA are identical for normal and affected platelets, whether comparing the binding of human or bovine Fg. The unique DARE syndrome explains the observed delay in aggregation of platelets from affected Simmental cattle and predicts the bleeding problems due to delayed binding of adhesive proteins.

Use of blood and blood components in canine and feline patients with hemostatic disorders.

Therapy with blood and blood products related to hemostatic disorders in small animal practice is reviewed. Administration of platelet rich plasma and platelet concentrates in thrombocytopenia or thrombopathia is discussed. Vascular purpuras, vasculitis, and vascular inherited defects are also considered. Inherited coagulation disorders are summarized and the therapeutic choices in treating these disorders are also proposed. In addition, acquired coagulation disorders are briefly reviewed.

Platelet function defect in a 5-day-old Simmental calf.

A 5-day-old Simmental Heifer was evaluated for excessive bleeding from the skin following horse fly bites. A coagulation profile and platelet numbers were normal. In vitro platelet function, measured by whole blood aggregometry, was found to be abnormal when compared with age-matched controls. Therefore, this defect in hemostasis was attributed to a qualitative defect in platelet function.