Binding of 125I-labeled endotoxin to bovine, canine, and equine platelets and endotoxin-induced agglutination of canine platelets

Endotoxin from Escherichia coli O127:B8, Salmonella abortus-equi and S minnesota induced clumping of some canine platelets (PLT) at a final endotoxin concentration of 1 microgram/ml. Endotoxin-induced clumping of canine PLT was independent of PLT energy-requiring processes, because clumping was observed with canine PLT incubated with 2-deoxy-D-glucose and antimycin A. The PLT responded to adenosine diphosphate before, but not after, incubation with the metabolic inhibitors. Endotoxin induced a slight and inconsistant clumping of bovine and equine PLT at high (mg/ml) endotoxin concentration. High-affinity binding sites could not be demonstrated on canine, bovine, and equine PLT, using 125I-labeled E coli O127:B8 endotoxin. Nonspecific binding was observed and appeared to be due primarily to an extraneous coat on the PLT surface that was removed by gel filtration. The endotoxin that was bound to PLT did not appear to modify PLT function. An attempt to identify plasma proteins that bound physiologically relevant amounts of endotoxin was not successful. The significance of the endotoxin-induced clumping or lack of it on the pathophysiology of endotoxemia is discussed.     

Lysis of bovine platelets by Pasteurella haemolytica leukotoxin

Pasteurella haemolytica A1 culture supernatants caused rapid cytolysis (less than 5 minutes) of isolated bovine platelets as measured by leakage of the cytoplasmic enzyme lactate dehydrogenase (LD). The platelet lytic factor had several features similar to P haemolytica leukotoxin. Like P haemolytica leukotoxin, the platelet lytic factor was produced by P haemolytica during logarithmic growth phase, was heat-labile, and was active against target cells (platelets) from ruminant species (cattle and sheep), but not from non-ruminant species (horses, pigs, and human beings). Additionally, the platelet lytic factor was neutralized with antileukotoxin rabbit serum. The amount of LD leaked by a fixed concentration of bovine platelets was proportional to the amount of toxin added at low toxic doses and became maximal at 88 +/- 11% of the total platelet LD activity for high doses of toxin. When a fixed dose of toxin was used and the platelet concentration was varied, LD leakage was initially proportional to the platelet concentration, but plateaued at higher platelet concentrations. The platelet lytic factor required Ca2+ and was inhibited by addition of the Ca2+ chelator ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetic acid. Toxin-mediated platelet damage may be important in thrombi formation and fibrin exudation typically associated with P haemolytica pleuropneumonia of cattle.     

Stimulus-dependent release of tissue-regenerating factors by equine platelets

Reasons for performing the study: Platelet‐rich plasma (PRP) is increasingly used for treatment of orthopaedic injuries. However, the effects of different stimuli on the release pattern of regenerative and proinflammatory factors from equine platelets are largely unknown and an optimal treatment protocol remains to be established. Objectives: The aim of this study was to identify a stimulus that enhanced release of histopromotive factors (platelet‐derived growth factor BB [PDGF] and transforming growth factor 1β[TGF]) without causing concurrent release of a proinflammatory mediator (CCL5). Methods: Washed platelets were prepared from 6 healthy ponies and release of growth factors and CCL5 measured using commercially available ELISAs for human proteins following incubation with or without thrombin, chitosan or equine recombinant tumour necrosis factor (erTNF) over 24 h and subsequently over 96 h. Additionally, noncoagulated samples were analysed. Results: Regardless of whether a stimulus was present or what stimulus was used, PDGF and TGF release was maximal by 0.5–1 h when clot formation took place and very little release was observed after 24 h. Growth factor release was minimal in noncoagulated samples. In contrast, CCL5 release was not associated with coagulation and appeared to persist for much longer. High concentrations of erTNF caused significantly greater release of CCL5 at 6 h than any other stimulus tested. Conclusions: Growth factor release from equine platelets is dependent on coagulation but independent of the initiating stimulus, and is accompanied by more sustained release of proinflammatory mediators. Potential relevance: Supernatants collected from coagulated platelets could be an alternative treatment to PRP.     

Evaluation of glycoprotein Ib expression on feline platelets

OBJECTIVE: To determine whether platelets obtained from cats expressed glycoprotein Ib (GPIb). SAMPLE POPULATION: Platelets obtained from 11 specific-pathogen-free cats. PROCEDURE: Platelets were analyzed by use of immunofluorescence microscopy, flow cytometry, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and immunoprecipitation. RESULTS: Immunofluorescence microscopy and flow cytometry revealed the protein on the surface of feline platelets. Biochemical studies (western immunoblot analysis and immunoprecipitation) revealed a 140-kd membrane glycoprotein. Additional biochemical studies revealed that feline GPIb was sensitive to proteolysis, because platelet cytoskeletons prepared with low concentrations of a calpain inhibitor (ie, leupeptin; 100 microg/ml) had substantial proteolysis, and there was an association of protein fragments with the actin cytoskeleton. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these results indicate that feline platelets express a 140-kd membrane protein that is recognized by monoclonal antibodies developed against GPIb. Application of standardized ELISA to quantitate glycocalicin, the water-soluble fragment of GPIb, may provide important information on the production of microvesicles, increased platelet turnover, and abnormal proteolysis.     

Evaluation of techniques for counting bovine platelets.

Visual and electronic techniques for counting bovine platelets were investigated. The reference method used was hemacytometer counting of platelets in whole blood diluted with 0.85% NaCl solution. A whole blood platelet-rich plasma technique was imprecise and inaccurate. Isopycnic centrifugation of blood diluted in 8.01% NaCl solution (same density as platelets) was a precise technique, but the whole blood platelet count was underestimated. The most precise and accurate technique investigated was unit gravity sedimentation of a 1:100 dilution of blood with 10 ml of Isoton followed by electronic counting of platelets in the supernatant. This technique correlated very well with visual counting of bovine platelets (N = 77, y = 55 + 0.80x, r = 0.89, P less than 0.01).     

Aggregation of bovine platelets by Fusobacterium necrophorum.

Washed cell suspensions of biovar A strains of Fusobacterium necrophorum aggregated cattle platelets, but similar suspensions of biovar B strains did not. Platelets were also aggregated by heat-treated bacterial cells or the lipopolysaccharide of biovar A. No platelet aggregation occurred in the presence of the cell-free culture supernatant of biovar A and of all samples prepared from biovar B. Scanning electron microscopy revealed that aggregated platelets were not damaged. Platelet aggregation was inhibited by EDTA, aspirin and quinacrine, and lag time was retarded by these inhibitors, indicating the reaction was a Ca(2+)-dependent, cyclo-oxygenase sensitive event. Platelet aggregation may be a virulence marker, probably mediated by the lipopolysaccharide of F. necrophorum biovar A strains.     

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.     

Open canalicular system of platelets in porcine stress syndrome

A study was undertaken to test whether a previously reported alteration in platelet morphology could be of predictive value for the detection of stress-susceptibility in pigs. Platelets from 20 normal pigs, nine pigs classified as stress-susceptible on the basis of their response to halothane challenge, and 11 siblings of halothane reactors belonging to two different breeds were subjected to electron microscopic examination. A quantitative analysis of electron micrographs, based on the extent of dilatation of the open canalicular system in platelets and the percentage of affected platelets, revealed that halothane reactor pigs could be distinguished from normal animals on the basis of their open canalicular system score. The discrete nature of the score categories in siblings indicates that platelet alteration may be an inherent component of the porcine stress syndrome and suggests that some of the false negatives in the halothane test may be identified as stress-susceptible on this criterion. Further studies involving a larger number of halothane reactors and siblings are needed to ascertain the consistency of the open canalicular system features and eventually, to develop a simple test system based on platelet alterations for the detection of stress-susceptibility in pigs.     

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.     

von Willebrand factor in lysates of washed canine platelets

Canine and human platelets (washed 4 times in a solution containing EDTA, prostaglandin E1, and theophylline to prevent release of alpha-granule constituents) were lysed by being frozen and thawed in the presence of detergent. Radioelectroimmunoassay for von Willebrand factor (vWf) in 5 human platelet lysates produced precipitin rockets, shaped like those produced from vWf in plasma from healthy human beings, and indicated that the mean von Willebrand factor antigen (vWf:Ag) content in platelets from healthy human being was 526 +/- 87 human U/10(12) platelets. Radioelectroimmunoassay for vWf in platelet lysates from 17 healthy dogs with normal plasma. vWf:Ag concentration produced precipitin rockets that looked different from those produced from canine plasma and indicated vWf:Ag content of 59 +/- 35 canine U/10(12) platelets. Inclusion of protease inhibitors in the lysing solution did not normalize the appearance of the precipitin rockets or substantially alter the measured platelet content of vWf:Ag. The array of vWf multimers revealed by sodium dodecyl sulfate-agarose gel electrophoresis of canine platelet lysates had a distinct appearance that differed from that of vWf in canine or human plasma and platelets; the intensity of the canine platelet vWf multimer bands was skewed, with relatively greater density in the lower molecular weight region and faint or undetectable multimer bands in the higher molecular weight region. Electrophoretograms with visible multimers in the high molecular weight region had vWf components that had higher molecular weight than did any vWf components in canine plasma. Radioelectroimmunoassay for fibronectin in these same canine platelet lysates indicated that the fibronectin content in platelets was 2.89 +/- 1.10 mg/10(12) platelets.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum thromboxane generation by platelets in several domestic animal species

Blood collected from calves, sheep, goats, pigs, dogs, horses, ponies and donkeys, was allowed to clot under standard conditions. Thromboxane B2 generated during the clotting process was measured by radioimmunoassay in serum harvested from each sample. Highly significant differences were found between species and also between genera within a species. Highest concentrations of thromboxane B2 were detected in the dog samples (887.7 +/- 123.7 ng/ml) and lowest concentrations in samples from sheep (2.7 +/- 0.2 ng/ml). The amount of thromboxane produced per unit number of circulating platelets or per unit volume of platelets in each species was not the same and it would appear that platelets from each species have different inherent ability to produce thromboxane under the stimulus applied, or that some species generate thromboxane from other sources during the clotting process.     

Immunocytochemical detection of Ehrlichia platys antigens in canine blood platelets

An avidin-biotin immunoperoxidase complex (ABC) immunocytochemical (ICC) stain procedure was optimized for detection of Ehrlichia platys antigens. Positive immunoreactivity was detected with dilutions of canine immune serum on acetone-fixed smears of platelet-rich plasma from E. platys-infected dogs. No E. platys antigens were detected when this ICC stain was applied to frozen or paraffin-embedded formalin- or acetone-fixed tissue sections from dogs with acute E. platys infection. Acetone fixation and freezing preserved ICC staining of ehrlichial antigens in infected blood platelets, whereas formalin treatment of similarly preserved E. platys-infected platelets nullified positive immunoreactivity. Significant E. platys infection of cells and tissues other than platelets may not occur.     

Effects of a P2Y(12) receptor antagonist on the response of equine platelets to ADP. Comparison with human platelets

Horses show susceptibility to platelet-related disorders. Equine platelets differ from human platelets in some of their responses, so information available about human platelets must be validated in the horse. Aggregation of platelets by ADP involves both P2Y(1) and P2Y(12) receptors on the platelet surface. We have compared the effect of the P2Y(12) antagonist, AR-C67085, on equine and human platelets in vitro using turbidimetric aggregometry to measure the rate and final extent of aggregation. Aggregation profiles, concentration-response curves and pA(2) values show that the rate of aggregation of equine platelets is much more susceptible to inhibition by AR-C67085 than that of human platelets. This species difference may reflect differences in the relative numbers of P2Y(1) and P2Y(12) receptors, or in intracellular signalling pathways, but will need to be considered by equine clinicians before using P2Y(12) antagonists in the treatment of thrombotic conditions.