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.     

Alterations in normal canine platelets during storage in EDTA anticoagulated blood

Abstract: Flow cytometric detection of platelet surface-associated IgG (PSAIgG) can be used to determine whether immunologic factors are contributing to thrombocytopenia in dogs. In vitro alterations in platelet activation and morphology, however, could impact the results of this test. The purpose of this study was to determine whether the PSAIgG test for immune-mediated thrombocytopenia was valid on whole blood in EDTA anticoagulant after 24–72 hours of storage, and to characterize other alterations in canine platelets that could impact immunologic testing. Platelets were harvested and analyzed immediately after blood collection and after 24, 48, and 72 hours of storage at 4°C. Spontaneous and thrombin-induced changes in the following platelet parameters were evaluated using flow cytometric techniques: PSAIgG, platelet microparticle formation, membrane expression of P-selectin and glycoprotein CD61, exogenous IgG binding, surface-exposed phosphatidylserine, and fibrinogen binding. The amount of PSAIgG increased 6-to 9-fold in stored samples compared with fresh samples. Platelet microparticle formation was spontaneous in stored samples and increased significantly over time. Membrane phosphatidylserine, P-selectin, and fibrinogen binding were not altered by storage, indicating that platelet activation was minimal in stored samples. Although storage decreased the percentage of platelets positive for CD61 by 8-to 10-fold compared with fresh samples, activation by high-dose thrombin partially restored the percentage of CD61-positive platelets in 24-hour-old samples. In conclusion, even though platelets stored in EDTA for up to 72 hours remain in a resting state, aged platelets have an increased tendency to form microparticles and have increased surface IgG and decreased surface CD61, which may contribute to false-positive results for tests of immune-mediated thrombocytopenia.     

Common variable immunodeficiency in three horses with presumptive bacterial meningitis

Three adult horses were evaluated for signs of musculoskeletal pain, dullness, ataxia, and seizures. A diagnosis of bacterial meningitis was made on the basis of results of CSF analysis. Because primary bacterial meningitis is so rare in adult horses without any history of generalized sepsis or trauma, immune function testing was pursued. Flow cytometric phenotyping of peripheral blood lymphocytes was performed, and proliferation of peripheral blood lymphocytes in response to concanavalin A, phytohemagglutinin, pokeweed mitogen, and lipopolysaccharide was determined. Serum IgA, IgM, and IgG concentrations were measured by means of radial immunodiffusion, and serum concentrations of IgG isotypes were assessed with a capture antibody ELISA. Serum tetanus antibody concentrations were measured before and 1 month after tetanus toxoid administration. Phagocytosis and oxidative burst activity of isolated peripheral blood phagocytes were evaluated by means of simultaneous flow cytometric analysis. Persistent B-cell lymphopenia, hypogammaglobulinemia, and abnormal in vitro responses to mitogens were detected in all 3 horses, and a diagnosis of common variable immunodeficiency was made.     

Isotype-specific antibodies in horses and dogs with immune-mediated hemolytic anemia

Classes of antibody bound to erythrocytes were determined using direct immunofluorescence (DIF) flow cytometry in 3 horses and 12 dogs with immune-mediated hemolytic anemia (IMHA). Background levels of antibody binding were determined in samples from 12 horses and 12 dogs that were free of clinical disease. The range of nonspecific binding of a fluorescein isothiocyanate (FITC)-conjugated goat anti-equine immunoglobulin G (IgG) was 19.9–36.7%, but was eliminated by the use of the F(ab)₂ fragment of FITC-conjugated goat anti-equine IgG. Background binding by other class-specific antibodies to equine and canine erythrocytes was negligible. The DIF results were compared to the direct antiglobulin (Coombs) test in 5 horses and 20 dogs with anemia. The former assay was more sensitive in dogs with IMHA than was the Coombs' test (100% versus 58%). In contrast, the Coombs' test had better specificity than the DIF assay (100% versus 87.5%, respectively). Using clinical parameters or response to therapy as the comparison, the positive and negative predictive values for the DIF test were 92% and 100% compared to the values of the Coombs' test of 100% and 62%. The DIF assay detected low levels of cells bound with antibody (<30%) in 5 dogs that were Coombs' test-negative. For both species, performance of the DIF test was independent of the prozone effect. Five dogs with IMHA had IgG and IgM on erythrocytes, 5 had IgG, and 2 had IgM. Three horses had surface-bound IgG, including a horse with suspected penicillin-induced IMHA, a foal with neonatal isoerythrolysis, and a foal with clostridial septicemia. The DIF method was valuable in monitoring the response to therapy in the foal with neonatal isoerythrolysis.     

Neonatal Alloimmune Thrombocytopenia in a Quarter Horse Foal

Neonatal alloimmune thrombocytopenia is recognized as a spontaneous disease of human infants, piglets, and possibly mules, but it has not been previously reported in horses. A 1–day-old Quarter Horse foal presented to Michigan State University Large Animal Clinic with severe thrombocytopenia of unknown origin. Immunoglobulins that bound to the foal's platelets were identified in the mare's plasma, serum, and milk by indirect assays. The immunoglobulins were further shown to recognize platelets from the foal's full brother, born 1 year earlier. These findings, coupled with the clinical course of the foal during its period of hospitalization, strongly suggest that neonatal alloimmune thrombocytopenia can spontaneously occur in neonatal horses. This diagnosis should be considered for foals with severe thrombocytopenia when other causes can be excluded, and platelet antibody assays should be used to support this diagnosis.     

Peripheral Blood Lymphocyte Subpopulations and Immunoglobulin Concentrations in Healthy Foals and Foals with Rhodococcus equi Pneumonia

Infectious diseases are common in foals aged 1-5 months. The objectives of this investigation were to evaluate immunologic parameters in foals from birth to weaning to establish reference values for the proportion of circulating lymphocytes that were helper (CD4+) or cytotoxic (CD8+) T cells, or B cells; to measure serum immunoglobulin (IgM and IgG) concentrations; and to compare these immunologic parameters to values in foals with naturally occurring Rhodococcus equi pneumonia and in adult horses. Peripheral blood lymphocyte subpopulations were determined by flow cytometric analysis, and serum IgG and IgM concentrations were determined by radial immunodiffusion. Flow cytometric analysis of lymphocyte subpopulations suggested age-related changes in the cell-mediated immune system in horses. Absolute circulating CD4+ and CD8+ T lymphocytes and B cells increased linearly up to 3 months of age. Circulating B cell concentrations from birth to 6 months of age were greater than values in adult horses and the lymphocyte differences among the age groups are mainly due to variation in B lymphocytes. Both absolute and proportional B cell concentrations were greater in foals with R equi pneumonia than in healthy foals at the same age. The increase in absolute cell counts of each subpopulation was dependent on the increase of absolute peripheral blood lymphocyte count. Serum IgG concentration increased linearly from 1 to 3 months of age, and serum IgM concentrations increased from 1 to 6 months of age. These data suggest age-dependent cell-mediated and humoral development in young foals.     

Application of vincristine-loaded platelet therapy in three dogs with refractory immune-mediated thrombocytopenia

Three dogs presented with refractory immune-mediated thrombocytopenia (IMT). All patients failed to respond to prednisone, which is considered a mainstay of immunosuppressive therapy. Vincristine-loaded platelets (VLPs), which act selectively on mononuclear phagocytes,were introduced. After the VLPs were transfused, two dogs responded quickly withimproved clinical signs while the third dogwith recurrent IMT was euthanized due to its deteriorating condition. This case report describesthe efficacy of VLP therapy in refractory IMT patients.     

Thrombocytopenia associated with administration of trimethoprim/sulfadiazine in a dog.

A diagnosis of trimethoprim/sulfadiazine-induced, immune-mediated thrombocytopenia in a dog was made, using a novel in vitro assay for thrombolytic activity. The assay quantifies thrombolytic activity by measuring the amount of platelet fragments in normal canine platelets before and after incubation with plasma from the thrombocytopenic dog. This report confirms previous reports of the development of thrombocytopenia after administration of trimethoprim/sulfadiazine, and describes a new assay that, after further validation, may be useful in the diagnosis of immune-mediated thrombocytopenia when an adequate sample of platelets cannot be obtained for quantification of platelet-associated IgG.     

Development of a sensitive immunoradiometric assay for detection of platelet surface-associated immunoglobulins in thrombocytopenic dogs

OBJECTIVE: To develop a direct assay to measure platelet surface-associated immunoglobulins (PSAIg) in dogs and to determine whether the assay is useful in the diagnosis of immune-mediated thrombocytopenia (IMT). ANIMALS: 20 healthy dogs were used to develop reference intervals, and 23 dogs with IMT and 17 with non-IMT were used to evaluate the clinical use of this assay. PROCEDURE: After optimization of platelet collection and assay conditions, concentrations of PSAIg were measured, using radiolabeled staphylococcal protein A (SpA) and polyclonal antibodies against canine IgG (anti-gamma) and IgM (anti-micro). Concentrations of PSAIg were expressed as the percentage of radiolabeled immunoglobulin detector bound. RESULTS: Cut-off values (mean + 3 SD) were as follows: SpA, 1.1%; anti-gamma, 1.3%; and anti-micro, 3.5%. Values greater than these cut-off values were considered positive. Values determined by use of radiolabeled SpA for all dogs with IMT were greater than the cut-off value; values were considered high positives (> 5 times cut-off value) for 22 of these 23 dogs. Although 9 of 17 dogs with non-IMT also had PSAIg concentrations greater than the cut-off value, values were considered high positives for only 3 of these 9 dogs. CONCLUSIONS AND CLINICAL RELEVANCE: The immunoradiometric assay developed is a reliable and sensitive method to detect PSAIg in dogs. However, to obtain accurate results, optimum temperature, time, and storage conditions must be used. Detection of increased concentrations of PSAIg in dogs presumed to have non-IMT should alert clinicians to reconsider an immune-mediated basis for the thrombocytopenia.     

Sarcocystis neurona-specific immunoglobulin G in the serum and cerebrospinal fluid of horses administered S neurona vaccine

A vaccine against Sarcocystis neurona, which induces equine protozoal myeloencephalitis (EPM), has received conditional licensure in the United States. A major concern is whether the immunoglobulin G (IgG) response elicited by the vaccine will compromise the use of Western blotting (WB) as a diagnostic tool in vaccinated horses with neurologic disease. Our goals were to determine if vaccination (1) causes seroconversion: (2) causes at least a transient increase in S neurona-specific IgG in the cerebrospinal fluid (CSF); and (3) induces an IgG response that can be differentiated from that induced by natural exposure. Horses included in the study (n = 29) were older than 6 months with no evidence of neurologic disease. The presence or absence of anti-S neurona antibodies in the serum of each horse was determined by WB analysis. Seropositive horses had CSF collected and submitted for cytology, CSF index, and WB analysis. The vaccine was administered to all the horses and boostered 3-4 weeks later. On day 14 after the 2nd administration, serum and CSF were collected and analyzed. Eighty-nine percent (8 of 9) of the initial seronegative horses seroconverted after vaccination, of which 57% (4 of 7) had anti-S neurona IgG in their CSE Eighty percent (16 of 20) of the seropositive horses had an increase in serum S neurona IgG after vaccination. Of the 6 of 20 horses that were initially seropositive/CSF negative, 2 were borderline positive for anti-S neurona IgG in the CSF, 2 tested positive, and 2 were excluded because the CSF sample had been contaminated by blood. There were no WB banding patterns that distinguished samples from horses that seroconverted due to vaccination versus natural exposure. Caution must be used in interpreting WB analysis from neurologic horses that have been recently vaccinated for EPM.     

Disseminated Intravascular Coagulation Associated with Colic in 23 Horses (1984–1989)

Disseminated intravascular coagulation (DIC) secondary to colic was diagnosed in 23 horses. Each horse was categorized retrospectively as to the cause of the colic based on surgical and/or necropsy findings: group 1 consisted of 14 horses with compromised intestine that required resection and anastomosis; group 2 consisted of 3 horses with nonstrangulating intestinal displacement and/or impactions; and group 3 consisted of 6 horses with colic associated with enteritis and/or colitis. Horses were considered to be affected with DIC if at least three of five hemostatic parameters were significantly abnormal: decreased antithrombin III (AT III) values, increased level of fibrin degradation products (FDP), thrombocytopenia, prolonged activated partial thromboplastin time, and prolonged prothrombin time. The most consistent hemostatic abnormalities were decreased AT III activity, increased FDP titers, and thrombocytopenia. Clotting times were more variable and did not always correlate with the presence of excessive hemorrhage. Excessive hemorrhage was present during surgery in seven horses and occurred within 1 to 12 hours after surgery in nine other horses. In addition to treatment of the primary disease, 19 horses received treatment for DIC consisting of heparin and/or plasma or fresh whole blood transfusions. Heparin alone was used in 12 horses. Heparin, in addition to fresh whole blood transfusions or fresh plasma, was administered to four horses. Three horses were treated with plasma alone. Four other horses were not treated specifically for the DIC. Eight horses (34%) survived the acute coagulopathy. Although a greater proportion of the surviving horses received heparin therapy (87.5%; 7/8) than did those that died (60%; 9/15), the difference was not statistically significant (P = 0.345).(ABSTRACT TRUNCATED AT 250 WORDS)     

Thrombocytopenia and hemorrhages in veal calves infected with bovine viral diarrhea virus

The relationship between bovine viral diarrhea virus (BVDV) infection and thrombocytopenia was studied in 18 veal calves experimentally infected with BVDV. All calves were free of BVDV, and 13 calves were free of serum neutralizing antibodies to BVDV before virus inoculation. Calves were inoculated at approximately 10 days of age, and platelet counts were monitored over a period of several weeks. Ten additional calves housed in close proximity were kept as uninoculated controls. A profound decrease in platelet counts by 3 to 11 days after inoculation was seen in all calves that had neutralizing antibody titers less than 1:32 before infection. Severe thrombocytopenia (less than 5,000 platelets/microliter) was seen in 12 calves, 11 of which also developed hemorrhages. Necropsy findings in 3 severely thrombocytopenic calves that died included multiple hemorrhages throughout the body. Calves that recovered had increased platelet counts, and in most instances, a corresponding increase in neutralizing antibody titers to BVDV. At 11 days after inoculation, BVDV was detected on platelets by use of immunofluorescence, but evidence of surface-bound immunoglobulin was not found. The results suggest that a nonimmunoglobulin-mediated method of platelet destruction or sequestration develops as a sequela to BVDV infection.     

Detection of antiplatelet immunoglobulin in thrombocytopenic dogs

Indirect enzyme-linked immunosorbent assays (ELISA) were used to detect platelet-associated immunoglobulin in sera from dogs with immune-mediated thrombocytopenia (IMT) and/or other autoimmune syndromes. One ELISA, utilizing whole platelets as the antigen substrate, readily detected antibody associated with platelets, either as specific, antiplatelet antibody or as immune complexes. This assay apparently lacked specificity because of the position reactions with sera from dogs with miscellaneous autoimmune disorders and no concurrent thrombocytopenia. Although the second ELISA, utilizing immunoaffinity purified platelet antigens was not influenced as much by immune complexes, absorbance values apparently were slightly increased. However, a small number of dogs with non-thrombocytopenic autoimmune disease tested positive. Immunoadsorption and Western immuno-blotting techniques demonstrated a complex pattern of specificities for antiplatelet antibodies. Clinical significance of these findings is discussed.     

Hemangiosarcoma in 11 young horses

Hemangiosarcoma is a rare neoplasm of horses and hemangiosarcoma in young horses might behave differently than in mature horses. The purpose of this study was to identify the characteristics of hemangiosarcoma occurring in horses < or = 3 years of age. Medical records from 1982 to 2004 were searched for horses < or = 3 years of age with a histopathologic diagnosis of hemangiosarcoma. Eleven records were identified. Thoroughbred and Thoroughbred crosses predominated. Age ranged from 9 days to 3 years. All horses presented with cutaneous or leg swellings or joint effusion. Physical examination findings included tachycardia, fever, and depression. Laboratory abnormalities included anemia (5/11), hyperfibrinogenemia (4/11), hypofibrinogenemia (3/11), thrombocytopenia (2/11), and neutrophilic leukocytosis (1/11). Ultrasonographic and radiographic evaluation was not diagnostic in any case. Antemortem histopathologic diagnosis was obtained in 10 cases. Six of 11 horses were euthanized. Surgical resection was performed in 5 horses, 2 of which were later euthanized. Diagnosis was confirmed histologically at postmortem examination in all euthanized horses. Two cases resolved spontaneously. Early histopathologic diagnosis may allow cure if the mass is localized and amenable to surgical resection. In cases where the horse is medically stable, and masses are not interfering with quality of life, a period of observation may be warranted.     

Platelet-bound antibodies detected by a flow cytometric assay in cats with thrombocytopenia

In cats, primary or secondary immune-mediated thrombocytopenia have rarely been described or characterised. The objective of this study was to determine platelet-bound antibodies (PBA) by a flow cytometric assay in both healthy and thrombocytopenic cats. Direct PBA testing was performed in 42 thrombocytopenic cats (platelet counts 6-179 x 10(9)/l, median 56 x 10(9)/l). Of these 42 cats, 19 had positive PBA test results, 17 of which were considered to have secondary immune-mediated thrombocytopenia (sITP). Underlying diseases included fat necroses (four cases), feline infectious peritonitis (three), feline leukaemia virus (two) or feline immunodeficiency virus (two) infections, lymphoma (two), leukaemia (one), hepatitis (one), pyelonephritis (one), or hyperthyroidism (one). In two cats, no underlying disease was found suggesting a primary immune-mediated thrombocytopenia (pITP). The PBA test was negative in 23 cats diagnosed with varying underlying diseases and in 47 healthy control cats with platelet values within the reference range. Only seven of the 42 cats with thrombocytopenia (platelet count 10-57 x 10(9)/l, median 34 x 10(9)/l) had spontaneous bleeding. This study suggests that immune-mediated destruction of platelets might be an important pathological mechanism for feline thrombocytopenia caused by various underlying diseases. In cats, pITP appears to be rarely diagnosed.     

Disseminated hemangiosarcoma in the horse: 35 cases

Thirty-five cases of disseminated hemangiosarcoma (21 clinical cases and 14 previously reported cases) were reviewed to describe the disease in horses. Hemangiosarcoma occurred in mature, particularly middle-aged horses, with no apparent sex predilection. Thoroughbreds seemed to be overrepresented (13 cases) but a true breed predilection could not be established. The respiratory and musculoskeletal systems were most commonly affected and presenting complaints included dyspnea (26%), subcutaneous or muscular swelling (24%), epistaxis (17%), and lameness (12%). Heart and respiratory rates were usually increased and mucous membrane color was frequently pale or icteric. Capillary refill time and rectal temperature were often normal. Anemia (88%), neutrophilic leukocytosis (62%), and thrombocytopenia (48%) were common. Examination of tissue samples collected by fine-needle aspirate or biopsy established an antemortem diagnosis in 4 horses. The diagnosis was made during postmortem examination in the remaining 31 horses. The lung and pleura (77%), skeletal muscle (46%), and spleen (43%) were most commonly affected. A primary site of tumor involvement could be identified in 22 horses. Hemangiosarcoma should be included as a differential diagnosis for horses with evidence of hemorrhage into body cavities, skeletal muscle, or subcutaneous locations.     

Thrombocytopenia in Horses: 35 Cases (1989–1994)

The records of 3,952 equine patients presenting to the Veterinary Teaching Hospital at North Carolina State University College of Veterinary Medicine were evaluated to determine risk factors associated with thrombocytopenia. Of 2,346 horses from which a CBC was obtained, 35 (1.49%) were thrombocytopenic (platelet count < 75,000/μL). A reference population of 189 horses with normal platelet counts (75,000 to 300,000/μL) was also studied. Standardbred horses were at increased risk for thrombocytopenia. but age and gender were not identified as significant risk factors. Horses with infectious or inflammatory diseases were at increased risk for thrombocytopenia. The potential association of clinical and clinicopathologic factors with thrombocytopenia were assessed by reviewing a series of multiple logistic regression models. Clinical and clinicopathologic variables significantly associated with thrombocytopenia in the final model included increased PCV, increased band neutrophil count, increased total WBC, and decreased plasma protein concentration. Increased mature neutrophil count was associated with normal platelet counts. Thrombocytopenic horses were significantly more likely to die or be euthanized than were horses with normal platelet counts. J Vet Intern Med 1996;10:127–132. Copyright © 1996 by the American College of Veterinary Internal Medicine .     

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.     

Diagnosis of microthrombocytosis and immune-mediated thrombocytopenia in dogs with thrombocytopenia: 68 cases (1987-1989).

The mean platelet volume (MPV) was evaluated in 68 dogs with thrombocytopenia attributable to various causes. Platelet size was high or low in some dogs. The most clinically useful observation was that low MPV (microthrombocytosis) was a specific indicator of immune-mediated thrombocytopenia (IMT) in these thrombocytopenic dogs. All but one case of microthrombocytosis (MPV less than 5.4 fl) was found in dogs with IMT. Microthrombocytosis was detected in 17 of 31 dogs with IMT and appeared at the onset of the disease. Macrothrombocytosis (MPV greater than 9.4 fl) indicated active thrombopoiesis, but was not unique to any disease category. Macrothrombocytosis was detected in 18 of 31 dogs with IMT, 3 of 17 dogs with disseminated intravascular coagulation, and 3 of 9 dogs with primary bone-marrow disease.     

Investigation of a novel, heritable bleeding diathesis of Thoroughbred horses and development of a screening assay

BACKGROUND: Bleeding in racing horses associated with exercise appears to be multifactorial, and clinical investigation into severe cases rarely occurs. Previously, we reported a severe bleeding diathesis in a Thoroughbred mare. Herein, we describe the cellular physiology of this defect, provide a diagnostic tool for identifying it, and demonstrate that the dysfunction is heritable. HYPOTHESIS: The subject has a heritable defect in platelet secretion that reduces thrombin generation in the absence of additional plasma factors and delays the onset of thrombin production even in the presence of these factors. ANIMALS: The study included 3 clinically normal Thoroughbred horses: the subject and her offspring. METHODS: Washed platelets were examined for their ability to (1) translocate phosphatidylserine to the outer leaflet of the platelet membrane as determined by annexin-V binding, (2) generate thrombin as assessed by the activity of the prothrombinase enzyme complex, and (3) bind fibrinogen and form aggregates as determined by flow cytometry. RESULTS: Subject and offspring platelets created procoagulant surfaces by translocating phosphatidylserine. The subject's platelets demonstrated reduced prothrombinase activity, resulting in decreased production of thrombin relative to control platelets. Subject and offspring platelets bound less fibrinogen than control platelets when stimulated with thrombin. CONCLUSIONS AND CLINICAL IMPORTANCE: The subject mare has a transmissible defect that involves reduced generation of thrombin by activated platelets, resulting in decreased aggregation and ineffective clotting. A flow cytometric assay of fibrinogen binding to washed platelets discriminates individuals with this platelet dysfunction and may be useful for discerning subclinical congenital or acquired platelet dysfunctions.