Platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor antagonists have been highly effective inhibitors of platelet aggregation in preclinical studies and in clinical trials. However, decreased platelet counts have been documented in preclinical studies and in some patients receiving GPIIb/IIIa antagonists. We evaluated changes in platelet kinetics and fate in dogs receiving the GPIIb/IIIa receptor antagonist RPR 109891 orally for 4 days. Dogs receiving RPR 109891 had a 22-52% decrease in platelet count with the nadirs at 3-5 days after initiation of treatment. Platelet survival time was reduced by 19%, and platelet half-life was reduced by 63%. Indium-111-labeled platelets were rapidly cleared from the blood within 1 hour after administration of RPR 109891 on treatment days 1 and 2. This clearing was associated with a sharp increase in radioactivity in spleen but not in liver or lung. Platelet clearance was markedly attenuated on treatment days 3 and 4. Platelet counts returned to baseline within 1 week after discontinuation of treatment. These data indicate that RPR 109891 causes rapid and selective sequestration of platelets in the spleen.

Three flow cytometric techniques were evaluated for determination of differential cell counts on canine clinical bone marrow specimens. Techniques included staining bone marrow specimens with 2'7'-dichlorofluorescein (DCF) or 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and evaluation of forward-angle light scatter vs. side-angle light scatter plots. Flow cytometric evaluation of bone marrow cells stained with DCF failed to separate bone marrow cells into distinct cell populations. Staining with DiOC6 resulted in separation of bone marrow cells into populations of mature and immature erythroid cells, mature and immature myeloid cells, and lymphocytes. The scatter plot method resulted in identification of mature and immature erythroid cells, immature myeloid cells, metamyelocytes, and bands and segmenters. Lymphocytes could not be differentiated from mature erythroid cells by the scatter plot method. When the results of the DiOC6 method and the scatter plot method were compared with manual bone marrow differential cell counts, the scatter plot method had more similar mean values and higher correlation coefficients. The scatter plot method has the potential of providing rapid semiquantitative assessment of bone marrow differential cell counts in dogs for specimens that contain low numbers of lymphocytes.

OBJECTIVE:To determine the capacity of inflammatory mediators tumor necrosis factor-alpha (TNF-alpha), interleukin-8 (IL-8), platelet-activating factor (PAF), lipopolysaccharide (LPS), and leukotoxin to prime, activate, or alter deformability of adult bovine neutrophils.SAMPLE POPULATION:Blood collected from 5 healthy adult Holstein cows.PROCEDURE:Isolated neutrophils or whole blood was incubated with TNF-alpha, IL-8, PAF, LPS, or leukotoxin, and neutrophil chemiluminescence, degranulation, deformability, shape change, CD11b expression, and size distribution was measured.RESULTS:Incubation with TNF-alpha, IL-8, PAF, and LPS primed neutrophils for oxygen radical release but caused minimal oxygen radical release by themselves. None of the inflammatory mediators induced degranulation. Incubation with TNF-alpha and PAF resulted in a decrease in neutrophil deformability and induced shape change in neutrophils. Incubation with PAF consistently resulted in an increase in neutrophil size as measured by use of flow cytometry. Only IL-8 caused an increase in expression of CD11b by neutrophils.CONCLUSIONS AND CLINICAL RELEVANCE:Inflammatory mediators tested had minimal effects on neutrophil oxygen radical production or degranulation but did prime neutrophils for oxygen radical production. Incubation with PAF and TNF-alpha caused a decrease in neutrophil deformability and altered neutrophil shape and size. Results of our study indicate that PAF- and TNF-alpha-induced changes in neutrophil deformability and size may cause integrin- and selectin-independent trapping of neutrophils in the lungs of cattle with pneumonic pasteurellosis.

The filterability of platelet-neutrophil aggregates through filters containing 5 μm pores was determined to evaluate the potential of plateletneutrophil aggregates to alter blood flow in the microvasculature. Incubation of platelet-neutrophil mixture with platelet-activating factor (PAF) and with 0.5 μM adenosine diphosphate (ADP) resulted in large platelet-neutrophil aggregates which rapidly plugged filter pores. Incubation of platelet-neutrophil mixtures with lower concentrations of ADP resulted in small platelet-neutrophil aggregates that contained only one neutrophil. Filtration of these samples resulted in a 1.3-, 3.2- and 3.7-fold increase in initial filtration pressure at ADP concentrations of 0.05, 0.15 and 0.25 μM, respectively. We conclude that platelet-neutrophil aggregates have reduced deformability and may alter blood flow in the microvasculature.

Objectives-To determining whether inhibition of platelet aggregation prevents development of carbohydrate overload-induced alimentary laminitis.Animals-22 healthy adult ponies.Procedures-Acute laminitis was induced by oral administration of corn starch/wood flour to 16 ponies, 8 of which were treated with a synthetic analogue of the platelet fibrinogen receptor antagonist peptide (RPR) RGDS (arginine-glycine-aspartic acid-serine) 110885; 6 ponies served as negative controls. Blood was collected before and at 4, 8, 12, 24, 28, and 32 hours after administration of carbohydrate overload, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, in vivo platelet activation, and platelet-neutrophil aggregates were evaluated.Results-Of 16 ponies given carbohydrate, 6 of 8 untreated ponies developed laminitis and 0 of 8 ponies treated with RPR 110885 developed laminitis. The RPR 110885 treatment attenuated the increase in platelet-neutrophil aggregates observed in untreated ponies.Conclusions-Platelets are involved in the pathogenesis of equine alimentary laminitis.Clinical Relevance-Platelet aggregation inhibitors may be useful for prevention or treatment of laminitis, or both.

Objectives-To determine whether platelets become activated and form platelet-platelet or platelet-neutrophil aggregates, or both, when subjected to shear.Sample Population-Blood obtained from 3 Thoroughbreds.Procedures-Blood, with PCV adjusted to 32 (low hematocrit) or 60 (high hematocrit)%, was subjected to shear rates of 11.25, 22.5, 45, 90, 225, and 750/s for 3 minutes by use of a cone-plate viscometer. Flow cytometric techniques were used to identify activated platelets, platelet-platelet aggregates, and platelet-neutrophil aggregates.Results-Shear resulted in decreased platelet count, increased mean platelet volume, platelet activation, and formation of platelet-platelet and platelet-neutrophil aggregates. These changes occurred at lower shear rates in blood with high hematocrit. Platelet-neutrophil aggregate formation was inhibited by blocking P-selectin, but not CD11/CD18 receptors.Conclusions-Shear-induced platelet activation and aggregate formation occur at physiologic shear rates.Clinical Relevance-Shear-induced platelet activation may explain the exercise-associated platelet-neutrophil aggregates observed in Thoroughbreds undergoing treadmill exercise.

OBJECTIVE To determine whether plasma protein concentrations were altered in ponies with alimentary laminitis. ANIMALS 12 adult ponies. PROCEDURE Acute laminitis was induced in 6 ponies by oral administration of carbohydrate (85% corn starch, 15% wood flour); the other 6 ponies were used as controls. A physical examination was performed and blood samples were collected immediately before and 4, 8, 12, 24, and 28 hours after administration of carbohydrate. Plasma protein concentrations were determined by means of sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS 19 plasma proteins ranging from a molecular weight of 24,000 to a molecular weight of 350,000 were identified in all 12 ponies. Plasma concentrations of proteins with molecular weights of 350,000 (fibrinogen), 130,000 (ceruloplasmin), 118,000 (c-reactive protein), 67,000 (alpha1-antitrypsin I), 65,000 (alpha1-antitrypsin II), 50,000 (haptoglobulin), and 45,000 (acid glycoprotein) were significantly increased in ponies with laminitis, compared with concentrations in control ponies. CONCLUSION Changes in plasma protein concentrations are detectable within 4 hours after the onset of alimentary laminitis in ponies. Clinical Relevance-Measurement of plasma protein concentrations may be useful in monitoring the progression of laminitis in ponies.

Objectives-To determine whether platelets are hyperaggregable or form platelet-neutrophil aggregates during the prodromal stages of acute laminitis of ponies.Animals-Healthy adult ponies: 8 experimental and 6 control,Procedures-Acute laminitis was induced by oral administration of corn starch and wood flour to 8 ponies, and indices of platelet activation were evaluated, Blood samples were collected before and at 4, 8, 12, 24, 28, and 32 hours after carbohydrate administration, and PCV, total plasma protein concentration, platelet count, activated clotting time, whole blood recalcification time, spontaneous platelet aggregation, ex vivo platelet aggregation responses, and platelet-neutrophil aggregates were determined. When lameness was first detected, ponies were euthanatized and arteriography and histologic examination of hooves were performed.Results-Carbohydrate overload was associated with hyperaggregability of platelets throughout the prodromal stages of laminitis and increased numbers of platelet-neutrophil aggregates. Reduction of blood supply to affected hooves was Variable, and blood clots were found in 6 of 11 laminitis-affected hooves.Conclusions-Platelets were hyperaggregable throughout the prodromal stages of carbohydrate-induced laminitis and formed platelet-neutrophil aggregates. Platelet-neutrophil aggregates may initiate or contribute to development of acute laminitis.Clinical Relevance-Anti-platelet therapy may be useful for treatment of acute alimentary laminitis in horses.