Effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation

OBJECTIVE: To investigate the effects of formaldehyde fixation on equine platelets using flow cytometric methods to evaluate markers of platelet activation. SAMPLE POPULATION: Blood samples from 6 Thoroughbreds. PROCEDURE: The degree of fluorescence associated with binding of fluorescein isothiocyanate (FITC)-conjugated anti-human fibrinogen antibody and FITC-annexin V in unactivated and adenosine diphosphate (ADP)-, platelet activating factor (PAF)-, and A23187-activated platelet samples in unfixed and 0.5, 1.0, and 2.0% formaldehyde-fixed samples was assessed by use of flow cytometry. RESULTS: In samples incubated with FITC-anti-human fibrinogen antibody prior to fixation, addition of 2.0% formaldehyde resulted in a 30% increase in total fluorescence in ADP- and PAF-activated samples and a 60% increase in A23187-activated samples. Fixation for 24 hours prior to addition of antibody resulted in reduced fluorescence of samples containing antihuman fibrinogen antibody for all 3 concentrations of formaldehyde in PAF-activated samples. The addition of all 3 concentrations of formaldehyde after incubation with FITC-annexin V resulted in significant increases in fluorescence in unactivated and activated platelet samples. As length of fixation time increased, there was a gradual increase in fluorescence that was significant at 24 hours. CONCLUSIONS: Because fixation with 2.0% formaldehyde results in significant changes in fluorescence in activated platelet samples containing anti-fibrinogen antibody, lower concentrations of formaldehyde should be used to fix equine platelet samples. Formaldehyde-fixed platelet samples should be analyzed within 12 hours of fixation to avoid artifactual increases in fluorescence. Fixation of samples containing FITC-annexin V should be avoided because of significant increases in fluorescence that may interfere with interpretation of results.     

Effects of sodium citrate, low molecular weight heparin, and prostaglandin E1 on aggregation, fibrinogen binding, and enumeration of equine platelets

OBJECTIVE: To investigate the effects of sodium citrate, low molecular weight heparin (LMWH), and prostaglandin E1 (PGE1) on aggregation, fibrinogen binding, and enumeration of equine platelets. SAMPLE POPULATION: Blood samples obtained from 4 Thoroughbreds. PROCEDURE: Blood was collected into syringes in the ratio of 9 parts blood:1 part anticoagulant. Anticoagulants used were sodium citrate, LMWH, sodium citrate and LMWH, or 300 nM PGE1/ml of anticoagulant. Platelet aggregation in response to ADP, collagen, and PGE1 was assessed, using optical aggregometry. Platelet activation was evaluated, using flow cytometry, to detect binding of fluorescein-conjugated anti-human fibrinogen antibody. Plasma concentration of ionized calcium was measured, using an ion-selective electrode. RESULTS: Number of platelets (mean +/- SEM) in samples containing LMWH (109.5+/-11.3 x 10(3) cells/microl) was significantly less than the number in samples containing sodium citrate (187.3+/-30.3 x 10(3) cells/microl). Increasing concentrations of sodium citrate resulted in reductions in platelet aggregation and plasma concentration of ionized calcium. Addition of PGE1 prior to addition of an agonist inhibited platelet aggregation in a concentration-dependent manner, whereas addition of PGE1 4 minutes after addition of ADP resulted in partial reversal of aggregation and fibrinogen binding. CONCLUSIONS AND CLINICAL RELEVANCE: A high concentration of sodium citrate in blood samples decreases plasma concentration of ionized calcium, resulting in reduced platelet aggregation and fibrinogen binding. Platelets tend to clump in samples collected into LMWH, precluding its use as an anticoagulant. Platelet aggregation and fibrinogen binding can be reversed by PGE1, which may result in underestimation of platelet activation.     

Detection of activated platelets in canine blood by use of flow cytometry

OBJECTIVE: To evaluate whether markers of platelet activation, including P-selectin expression, phosphatidylserine exposure, platelet-leukocyte aggregates, and microparticle formation, could be measured in nonstimulated and stimulated canine blood samples and develop a standardized protocol for detection of activated platelet markers in canine blood. SAMPLE POPULATION: Blood samples from 10 dogs. PROCEDURE: Platelet activation was determined by flow cytometric measurement of platelets with P-selectin expression, platelet-leukocyte aggregates, platelet microparticles, and platelets with phosphatidylserine exposure. Changes in specific markers of platelet activation in nonstimulated versus stimulated samples were assessed by use of varying concentrations of 2 platelet agonists, platelet-activating factor (PAF) and adenosine diphosphate. Flow cytometry was used to detect platelet CD61 (glycoprotein IIIa), CD62P (P-selectin), and the leukocyte marker CD45. Annexin V was used to identify exposed phosphatidylserine. RESULTS: A significant difference was detected in the percentages of platelets with P-selectin, plateletleukocyte aggregates, microparticles, and platelets with annexin V exposure (phosphatidylserine) in samples stimulated with 10nM PAF versus the nonstimulated samples, with platelet-leukocyte aggregates having the greatest difference. CONCLUSIONS AND CLINICAL RELEVANCE: Platelet activation is essential for thrombus formation and hemostasis and may be potentially useful for evaluation of dogs with suspected thromboembolic disease. Prior to development of a thrombotic state, a prothrombotic state may exist in which only a small number of platelets is activated. Identification of a prothrombotic state by use of activated platelets may help direct medical intervention to prevent a thromboembolic episode.     

Assessment of platelet function in horses: ultrastructure, flow cytometry, and perfusion techniques

We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.     

Differential effects of phosphodiesterase inhibitors on platelet activating factor (PAF)- and adenosine diphosphate (ADP)-induced equine platelet aggregation

Compounds that activate adenylate cyclase, increasing intracellular cyclic adenosine monophosphate (cAMP), inhibit equine platelet aggregation. Cyclic AMP is broken down by phosphodiesterase (PDE) and, in the present study, the effects of theophylline, a nonselective PDE inhibitor, and selective inhibitors of PDE isoenzymes PDE3, PDE4 and PDE5, on equine platelet aggregation in response to platelet activating factor (PAF) and adenosine diphosphate (ADP) have been examined. Theophylline and the PDE3 inhibitors, trequinsin and quazinone, inhibited both PAF and ADP-induced aggregation in a concentration dependent manner. The inhibition of PAF-induced aggregation was, however, significantly greater than that of the response to ADP. The inhibitory effects of theophylline and the PDE3 inhibitors on ADP- but not PAF-, induced aggregation were reversed by addition of the calcium ionophore, A23187. Rolipram and zaprinast, inhibitors of PDE4 and PDE5, respectively, had no effect on either PAF- or ADP-induced aggregation. These results demonstrate that inhibition of aggregation caused by PAF or ADP can be achieved by selective inhibition of PDE3 but suggest that there may be agonist-specific differences in the intracellular signalling pathways that regulate equine platelet aggregation.     

Effects of etamsylate on equine platelets: in vitro and in vivo studies

The aim of this study was to investigate whether etamsylate produces equine platelet activation. In vitro and in vivo studies were designed in which seven and eight adult healthy horses were included, respectively. In the in vitro study, citrated blood was incubated with different concentrations of etamsylate, and P-selectin expression and annexin V binding were determined by flow cytometry. In the in vivo study, blood was collected before and 1 and 2h after IV administration of etamsylate, and P-selectin expression was evaluated. In the in vitro study, a significant increase in P-selectin expression, leukocyte-platelet aggregate formation and annexin V binding were observed. In the in vivo study, a marked increase in P-selectin expression and heterotypic aggregate formation was seen in two and five horses, respectively, although no significant differences were detected when analyzing results from all the animals together. The results of the in vitro study indicate that etamsylate produces a pre-activation state in equine platelets, but this fact could be confirmed by the in vivo study.     

Detection of antiplatelet antibody: comparison of platelet immunofluorescence,agglutination, and immunoinjury tests using rabbit antiequine platelet serum

Immunofluorescence, tube agglutination, and platelet factor-3 immunoinjury tests for detecting antiplatelet antibody were compared using a heterologous system of equine platelets and rabbit antiequine platelet serum. Platelet immunofluorescence tests were performed using paratormaldehyde-fixed platelets in suspension as well as in air-dried smears on glass slides (solid phase). Bright homogeneous, membranous, specific fluorescence was seen in both assays with anti-immunoglobulin G (IgG) and protein G fluorescein isothiocynate conjugates (FITC-conjugates). Protein A conjugate gave nonspecific fluorescence irrespective of normal or antiserum treatment. Anti-IgG and protein G conjugates in suspension immunofluorescence tests with the same antiserum yielded antibody titers of 1:1024 and 1:128, respectively. Similarly, respective titers of 1:512 and 1:64 were obtained with solid phase immunoassay. Platelet suspension assay was slightly better than the solid phase assay. These observations indicated that anti-IgG was more sensitive than protein G in detecting antiplatelet antibody by fluorescence microscopy, while protein A was ineffective because of its nonspecificity. Chloroquine treatment of platelets failed to reduce the nonspecific fluorescence. Platelet agglutination and platelet factor-3 tests were relatively less sensitive to detect equine antiplatelet antibody.     

Actions and interactions of ADP, 5- HT, histamine and PAF on equine platelets

Platelets are thought to play a role in equine diseases such as acute laminitis and verminous arteritis and may be involved in allergic disease. Mediators implicated in the pathophysiology of these conditions activate platelets and responses may be enhanced by interactions between mediators. The present study compared platelet aggregation, thromboxane production and release of radiolabelled [(3)H]5- HT in response to 5- HT, histamine, ADP and PAF alone and in combination in vitro.PAF caused concentration-related aggregation, [(3)H]5- HT release and thromboxane production. In contrast, ADP caused aggregation and 5- HT induced the release of [(3)H]5- HT with little effect on other platelet functions. Histamine had little or no effect on equine platelets. Addition of 5- HT (10 microM) prior to ADP significantly displaced the aggregation response curve to the left.The profile of responses to PAF, ADP and 5- HT suggests differential activation of intracellular signalling pathways regulating these events. The enhanced response to ADP in the presence of 5- HT may have implications in thromboembolic disease in the horse.     

Effects of WEB 2086, an antagonist to the receptor for platelet-activating factor (PAF), on PAF-induced responses in the horse.

Platelet-activating factor (PAF) causes oedema and neutrophil accumulation when injected into the skin of normal horses. PAF is also known to induce aggregation of horse platelets in vitro. The selective PAF receptor antagonist WEB 2086 has now been used to determine whether these effects are mediated by PAF receptor activation. Addition of WEB 2086 to equine platelets in vitro inhibited PAF-induced aggregation in a competitive reversible manner (pA2 = 7.14). Inhibition of in vivo inflammatory responses to PAF occurred after local administration of WEB 2086: wheal formation induced by 0.1 micrograms PAF/site was reduced by 1-10 micrograms WEB 2086/site. PAF (1 micrograms/site)-induced neutrophil accumulation was also inhibited by co-administration of 10 micrograms WEB 2086/site. Systemic administration of WEB 2086 (3 mg/kg iv) to 4 normal ponies inhibited PAF-induced wheal formation and ex vivo platelet aggregation. At 30 min after drug administration the concentration of PAF required to produce a half maximal aggregation response was increased 496 +/- 137 fold. At 6 h the degree of inhibition was markedly reduced and responses had returned to pre-treatment values by 24 h. PAF-induced increases in cutaneous vascular permeability were also reduced by 80% as early as 30 min after iv administration of WEB 2086 in these animals, the inhibition persisting for at least 6 h. These results suggest that in vitro and in vivo responses to PAF in the horse are mediated via activation to PAF receptors. WEB 2086 will therefore be a useful agent for studying the role of PAF in the pathogenesis of equine inflammatory conditions.     

PKC isoenzymes in equine platelets and stimulus induced activation

Protein kinase C (PKC) is an important regulator of platelet activation and different isoenzymes can play positive and negative regulatory roles. The PKC isoenzymes expressed in equine platelets have not been documented but pharmacological inhibition has suggested a role for PKC delta (δ) in modulating responsiveness to platelet activating factor (PAF) (Brooks et al., 2009). Here the PKC isoenzyme profile in equine platelets has been characterised and PKCδ activation by PAF investigated. Platelet lysates were probed by Western blotting using a panel of antibodies against individual PKC isoenzymes. PKCδ and eight other isoenzymes were identified, namely classical PKCs alpha (α), beta (β), (both βI and βII) and gamma (γ), the novel PKCs epsilon (?), eta (η) and theta (θ) and atypical PKC zeta (ζ). Having shown PKCδ to be present, a method was developed to measure PAF-induced isoenzyme translocation by preparing cytosolic and membrane fractions from digitonin permeabilised platelets. Phorbol 12-myristate 13-acetate (PMA) was shown to cause translocation of PKCδ to the membrane within 5s. PAF also caused PKCδ translocation although the response occurred more slowly; a significant, 7.6 ± 1.2 fold, increase in band density compared to unstimulated platelets was observed at 15 min; p=0.036, n=3. These data support a role for PKCδ in regulating PAF-induced functional responses in equine platelets.     

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.     

Regulation of platelet activating factor-induced equine platelet activation by intracellular kinases

Lipopolysaccharide (LPS) can activate equine platelets directly or indirectly, via leukocyte-derived platelet activating factor (PAF). Thromboxane (Tx) production by LPS-stimulated equine platelets requires p38 MAPK and this kinase has been suggested as a therapeutic target in endotoxaemia. The present study has utilised selective inhibitors to investigate the role of p38 MAPK and two other kinases, phosphatidylinositol-3 kinase (PI3K) and protein kinase C (PKC), in regulating PAF-induced Tx production, aggregation and 5-HT release in equine platelets, and the modification of these responses by LPS. LPS enhanced PAF-induced 5-HT release, an effect that was reduced by the p38 MAPK inhibitor, SB203580 (60 ± 8% reduction; n  = 6). SB203580 did not affect responses to PAF alone; whereas inhibition of PKC reduced PAF-induced 5-HT release, Tx production and aggregation (maximal inhibition by the PKCδ inhibitor, rottlerin: 69 ± 13%, 63 ± 14% and 97 ± 1%, respectively; n  = 6). Wortmannin and LY249002, which inhibit PI3K, also caused significant inhibition of PAF-induced aggregation (maximal inhibition 78 ± 3% and 88 ± 2%, respectively; n  = 6). These data suggest that inhibition of platelet p38 MAPK may be of benefit in equine endotoxaemia by counteracting some of the effects of LPS. However, detrimental effects of platelet activation mediated by PAF and not enhanced by LPS are unlikely to be markedly affected.     

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.     

Flow cytometric detection of activated platelets in the dog

Background: Platelet activation appears to play a role in a variety of canine thrombotic disorders. At present, tests for the detection of activated platelets are not used routinely in veterinary clinical laboratories. Objective: The purpose of this study was to develop a clinically applicable method to detect activated canine platelets. Methods: A flow cytometric assay was developed to detect activated platelets, platelet aggregates, and platelet microparticles in the dog. Blood was collected from healthy dogs using EDTA or sodium citrate as the anticoagulant, and platelet‐rich plasma was prepared. Platelets were activated by adding phorbol myristate acetate. In some experiments, platelets were fixed by incubation with 0.5% paraformaldehyde. In other experiments, platelets were stored for 4 or 24 hours at 4°C before analysis. Activated platelets were detected by measuring surface expression of P‐selectin and by determining the percentages of platelet aggregates and microparticles using forward‐angle vs side‐angle light scatter plots. Results were analyzed by using 2‐way ANOVA and the SchefféF‐test. Results: Platelets collected in EDTA had minimal expression of P‐selectin, whereas platelets collected in sodium citrate had greater median fluorescence intensity. Fixation with 0.5% paraformaldehyde before labeling platelets with anti‐P‐selectin did not affect antibody binding or the percentages of platelet aggregates and microparticles. Storage of platelet‐rich plasma at 4°C for 4 hours did not affect antibody binding or the percentages of platelet aggregates or microparticles. Activation of platelets ex vivo by addition of 10 ng/mL phorbol myristate acetate resulted in a large increase in expression of P‐selectin but only slight increases in platelet aggregates and microparticles. Conclusion: Determination of platelet P‐selectin expression and percentages of platelet aggregates and platelet microparticles may provide a clinically applicable means for detection of activated platelets in dogs. The capacity to use EDTA‐anticoagulated blood samples and to fix platelets for evaluation at a later time makes the test attractive as a routine diagnostic tool.     

Endotoxin and dietary amines may increase plasma 5-hydroxytryptamine in the horse

Uptake of 5-hydroxytryptamine (5-HT) into platelets is an important mechanism by which low plasma concentrations are maintained, and platelet activation may therefore result in significant release of this vasoconstrictor. The present study examined the kinetics of active uptake of radiolabelled [3H]5-HT by washed equine platelets in vitro, and investigated the effects on this process of 4 other naturally occurring monoamines which may be released from the caecum in conditions of carbohydrate overload. The release of [3H]5-HT by platelets was also studied, since platelet accumulation and activation has been associated with acute laminitis. Release of [3H]5-HT was measured in response to platelet activating factor (PAF), unlabelled 5-HT and the indirect activation of platelets by endotoxin in the presence of blood leucocytes. Km value for the uptake of 5-HT by equine platelets was 2.4 +/- 0.6 micromol/l and the Vmax was 8.3 +/- 0.6 pmol [3H]5-HT/10(7) platelets/min. The rate of uptake of 5 micromol/l [3H]5-HT was significantly decreased by the uptake inhibitors fluvoxamine and clomipramine. The 4 other monoamines examined all inhibited the uptake of [3H]5-HT in a noncompetitive manner, decreasing Vmax by between 17 and 82%. Incubation of platelets with LPS (0.1 mg/ml) in the absence of leucocytes did not result in significant release of [3H]5-HT; however, in the presence of leucocytes 3.8 +/- 1.7 pmol [3H]5-HT/10(7) platelets (mean +/- s.e.) were released. This release was significantly inhibited by parthenolide and WEB2086, but not by aspirin. This suggests that PAF from activated leucocytes was responsible for the 5-HT release. These data show that 5-HT uptake by equine platelets is a saturable process operating most efficiently at substrate concentrations in the low micromolar range. The noncompetitive inhibition of 5-HT uptake by other naturally occurring monoamines may result in increased plasma concentrations of 5-HT, as would its release by endotoxin. Such a rise in plasma 5-HT concentrations may contribute to selective vasoconstriction in the equine digital circulation.     

Detection of equine antiplatelet and antineutrophil antibodies by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (elisa) was standardised and applied for the detection of anti-platelet and antineutrophil antibodies using a heterologous system consisting of equine platelets or neutrophils and antisera raised in rabbits. The standardised technique consisted of using Immulon type 3 plate, I per cent gelatine as a blocking solution, poly-L-lysine buffer as a coating solution, unfixed antigen, 90 μl test serum, horseradish peroxidase conjugated antibody and o-phenylenediamine dihydrochloride as a substrate. The number of unfixed platelets or neutrophils required for optimum detection of antibodies was 250,000 per well. Unfixed cellular antigens were as good as their extracts and superior to paraformaldehyde-fixed antigens in detecting specific antibodies. Microtitre plates coated with platelet or neutrophil antigens could be stored at 4° and −70°C for four to five weeks without significant loss of antigenicity. The ELISA was very sensitive in that antiplatelet antibody was detected up to a titre of 1:204,800 and antineutrophil antibody to a titre of 1:51,200. Some cross-reactivity (1:1600) was detected in antiplatelet and antineutrophil sera for neutrophil and platelet antigens, respectively. Platelet-associated antibody was also detected in extracts from platelets pretreated with 1:2 and 1:8 dilutions of antiplatelet serum. Standardised elisa detected antiplatelet antibodies in nine and antineutrophil antibodies in three of 100 isologous equine blood typing sera.     

Platelet activation in ponies with airway inflammation

REASON FOR PERFORMING STUDY: Platelet activation occurs in human obstructive airway diseases and in laboratory animal models. However, there is limited evidence that platelets may be involved in equine recurrent airway obstruction (RAO) and other inflammatory diseases. This study investigated whether platelet activation also occurred in RAO. HYPOTHESIS: Platelet function is altered in ponies with active RAO. This alteration can be detected ex vivo by measuring platelet adhesion. METHODS: An in vitro platelet adhesion assay measuring acid phosphatase (AcP) activity colorimetrically was adapted for use with equine platelets and responses to selected agonists were established. Platelet adhesion and aggregation was evaluated in vitro on platelets isolated from 6 ponies with RAO before, during and after a 7 h natural antigen challenge. Three ponies with no history of airway disease were also studied. RESULTS: Adhesion of equine platelets to serum coated plastic was detected at concentrations of 10-100 radicaló 10(9)/l. Adhesion increased in response to stimulation with platelet activating factor and thrombin, but not equine interleukin 8. Prior to the antigen challenge, adhesion of nonstimulated platelets was low and increased significantly (P<0.05) 24 h after initiation of the challenge in RAOs, but not in the normal animals. No changes in platelet aggregation were noted in either group. CONCLUSIONS: The described assay offers an alternative method to evaluate platelet function in healthy and diseased horses and can detect changes not observed using a classic aggregation assay. Circulating platelets are activated 24 h after antigen challenge of ponies with RAO and may play a role in pulmonary inflammation and/or the pathophysiology of RAO. POTENTIAL RELEVANCE: Investigating platelet function in RAO and airway inflammation may reveal new aspects of the pathogenesis of inflammatory lung disease in the horse.     

Alterations in bovine platelet function and acute phase proteins induced by Pasteurella haemolytica A1.

Platelet function was assessed by aggregometry in 10 Holstein calves before and after exposure to Pasteurella haemolytica (biotype A, serotype 1) by intrabronchial challenge. At 24 h after exposure the platelets had become more reactive to stimulation with known platelet agonists such as adenosine diphosphate (ADP) and platelet-activating factor (PAF) and the platelet aggregates that formed were more resistant to disaggregation. The activation of platelets was an early response in the challenged calves as platelet function had returned to pretreatment levels 72 h after exposure to the bacteria while the acute phase reactant proteins, haptoglobin and fibrinogen, were approaching their peak values and alpha 2-macroglobulin levels had also risen significantly (P < 0.05) at this time. The plasma levels of these proteins were still elevated and albumin levels were depressed 6 d post-treatment. At post-mortem all calves exhibited pneumonic tissue damage. When P. haemolytica leukotoxin was added directly to bovine platelet suspensions both spontaneous aggregation and an increase in the aggregation response to ADP and PAF stimulation were observed. The morphological appearance of the platelet aggregates exhibited the typical pattern for bovine platelets with 2 distinct zones of cells being visible within each aggregate. One zone contained platelets in which the cytoplasmic granules were still evident and the other zone contained irregularly shaped platelets devoid of granular content. In the latter zone, discrete gaps, or pores, were evident in the plasma membrane of numerous platelets. This pore formation is characteristic of leukotoxin action and is not observed in ADP or PAF induced aggregates.     

Functional and biochemical characterization of immunologically derived equine platelet-activating factor

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets pre-incubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after base-catalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.     

Effects of physiologic agonists on canine whole blood flow cytometry assays of leukocyte-platelet aggregation and platelet activation

Platelets play a role in both the innate and adaptive immune systems. Methods for detecting activated platelets and leukocyte-platelet aggregates (LPAs) are useful for basic and applied research concerning the role of platelets in inflammation and immune disorders. The aim of the study was to develop flow cytometric assays for detection of platelets binding to monocytes and neutrophils and for activated platelets in canine whole blood and to investigate the effect of physiologic agonists. Citrate anticoagulated whole blood was incubated with monoclonal antibodies against CD14 and CD61 for detection of LPAs, and the effect of various agonists was investigated. For detection of activated platelets, whole blood was incubated with monoclonal antibodies against CD62P and against a receptor-induced binding site on fibrinogen (CAP1) with CD61 as a platelet identifier. Isotype controls were prepared in parallel. The individual physiologic agonists ADP, collagen and epinephrine increased LPAs, CD62P and CAP1 binding only modestly. However, combinations of agonists gave more substantial increases. A dose-response relationship was seen using alpha- and gamma-thrombin, and ADP as agonists. In conclusion, we have developed flow cytometry assays to measure LPAs and platelet activation in canine whole blood, and have explored the effect of various physiologic agonists at different concentrations.