Platelet aggregation, storage pool deficiency, and protein phosphorylation in mice with Chediak-Higashi syndrome.

The beige (bgJ/bgJ) mouse is a well-described murine model of Chediak-Higashi syndrome. Platelet function was examined in normal and beige mice to better characterize the defective aggregation response in platelets from mice with Chediak-Higashi syndrome. Platelet aggregation after collagen, thrombin, and phorbol-12-myristate 13-acetate stimulation was significantly (P less than 0.025) decreased in platelets from beige mice, relative to platelets from normal mice. Compared with beige and normal mice, those heterozygous for the bg trait had intermediate responses to collagen and thrombin, but not phorbol-12-myristate 13-acetate. The defect(s) in aggregation of platelets from beige mice was associated with a dense granule storage pool deficiency and decreased stores of serotonin and adenine nucleotides in platelets. Mice heterozygous for the bg trait had normal platelet serotonin and adenine nucleotide concentrations. Platelets from beige mice were approximately 10 times more sensitive to prostacyclin inhibition of collagen-induced aggregation than were platelets from control mice. However, a significant difference in platelet cyclic AMP concentration was not apparent between beige and normal mice after prostacyclin stimulation. Platelet endoperoxide synthesis measured by quantification of thromboxane B2, was normal in beige mice. Protein phosphorylation patterns in mouse platelets were similar to those seen in human platelets. Thrombin and collagen-induced [32P] phosphorylation of 40- and 20-kD proteins in platelets from normal and beige mice was similar. Results indicate that the biochemical defect(s) in platelet function in beige mice is partially attributable to storage pool deficiency and does not result in an absolute defect in phosphorylation of 40- and 20-kD proteins.     

Comparative hematology: studies on goats.

Results of blood coagulation and hematologic studies on 6 goats, each tested 3 times, were compared to the values seen in persons. Special blood platelet studies were done on an additional goat. Blood coagulation values in the goats and in persons were similar, with these exceptions: In the goat, activated partial thromboplastin time was shorter and thrombin time was longer; one-stage assays of factors V, VIII, and IX were very high, and platelets aggregated poorly epinephrine and ristocetin. Both platelets and erythrocytes were small. On scanning electron microscopy, the erythrocytes appeared as flat disks or triangles, occasionally having a dimpled center, compared to the deeply dimpled doughnut shape of the larger human erythrocytes. Osmotic fragility of these small erythrocytes was greater than that of their human counterparts. By transmission electron microscopy of ultrathin sections of goat buffy coat, platelets had fine structures similar to those of human platelets. Unlike in human platelets, most of the dense bodies in goat platelets were surrounded by clear vacuoles. Biochemical studies showed higher than human levels of phosphorus, chloride, sodium, alkaline phosphatase, and serum glutamic oxaloacetic transaminase.     

A case of nontraumatic gas gangrene in a common marmoset (Callithrix jacchus)

The common marmoset is widely used in neuroscience and regenerative medicine research. However, information concerning common marmoset disorders, particularly infectious diseases, is scarce. Here, we report a case of a female common marmoset that died suddenly due to gas gangrene. The animal presented with gaseous abdominal distention at postmortem, and Clostridium perfringens type A was isolated from several tissues. Vacuoles, a Gram-positive bacteremia and intravascular hemolysis were observed microscopically in the muscles, liver and lungs. On the basis of these findings, we diagnosed nontraumatic gas gangrene caused by Clostridium perfringens type A in this common marmoset.     

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)     

Distribution of protein gene product 9.5-immunopositive and NADPH-diaphorase-positive neurons in the common marmoset (Callithrix jacchus) accessory olfactory bulb

The principal center of the accessory olfactory system is the accessory olfactory bulb (AOB). In primates, simians are divided into two groups, New and Old World monkeys, and the AOB is present in only New World monkeys. The common marmoset (Callithrix jacchus) is a species of New World monkey. Although the morphology of the common marmoset AOB has been demonstrated, the distribution patterns of the mitral/tufted and granule cells of the AOB remain unclear. In the present study, therefore, the distribution of the mitral/tufted and granule cells in the common marmoset AOB was examined using two histochemical markers including immuno-staining for protein gene product (PGP) 9.5 and NADPH-diaphorase staining. The vomeronasal nerves, gomeruli and mitral/tufted cells showed PGP 9.5-immunoreactivity. The mitral/tufted cells were arranged in only one or two rows along the margin of the glomerular layer to form the mitral/tufted cell layer (MTL). Since the mitral/tufted cells occurred sparsely in the common marmoset, the MTL was illegible. NADPH-diaphorase reactivity was primarily detected in the rostral and caudal areas of the AOB. In these areas, granule cells showed NADPH-diaphorase reactivity. Since the granule cells were sparse, the common marmoset AOB displayed less-developed granule cell layer. Although the functional significance of the AOB remains to be solved in the common marmoset, small-sized and less-laminated AOB may show that sexual behavior of the common marmoset has lesser dependence on the accessory olfactory system.     

A case of intestinal mucormycosis in a common marmoset (Callithrix jacchus)

A 3-year-old female common marmoset was euthanatized because of wasting. Grossly, a perforating lesion was present in the jejunum and hemorrhagic lesions in the cecum and colon. Histopathologically, these gross lesions were a perforated jejunal ulcer and necrotizing colitis, respectively. Necrotizing colitis was characterized by extensive mucosal necrosis along with numerous ribbon-shaped aseptate hyphae. These aseptate hyaline hyphae were positively stained with PAS and GMS, and reacted immunohistochemically with the antibody against the family Mucoraceae. This case was diagnosed as intestinal mucormycosis. This is the first report on mucormycosis in a common marmoset.     

Phenylbutazone inhibition of equine platelet function.

Nonsteroidal anti-inflammatory drugs impair platelet aggregation and secretion in man, pigs, and rabbits and inhibit platelet thromboxane/prostaglandin synthesis. The present investigation studied the effects of phenylbutazone on platelet aggregation and bleeding times in the horse. Aggregation responses to adenosine diphosphate and collagen were markedly impaired 15 minutes and 2 hours after treatment, but 4 hours after treatment, platelet responses approximated those prior to treatment. The in vivo effect of phenylbutazone correlated with its plasma concentrations. Phenylbutazone, like aspirin, appeared to exert its effect by inhibiting thromboxane/prostaglandin synthesis, because thrombin-induced malondialdehyde formation was inhibited. However, unlike aspirin, free arachidonate-induced malondialdehyde synthesis was reduced but not eliminated, which suggested that phenylbutazone may have more than one site of action. Although collagen-induced platelet aggregation was impaired, a response was still present, and bleeding times were not altered by phenylbutazone treatment. To account for these findings, it is proposed that equine platelets can respond to collagen by thromboxane/prostaglandin independent pathways. The physiologic and pathophysiologic importance of these findings is discussed.     

Increased platelet aggregation response in Cavalier King Charles Spaniels with mitral valve prolapse

Mitral valve prolapse (MVP) is a fundamental feature of myxomatous mitral valve disease in the dog. In humans, primary MVP is associated with increased platelet reactivity. In Cavalier King Charles Spaniels (CKCS), a breed predisposed to myxomatous mitral valve disease, there is a high prevalence of hypomagnesemia and platelet anomalies, such as thrombocytopenia and macrothrombocytosis. The objective of this study was to evaluate platelet aggregation responses in CKCS and to determine the relationship between the platelet aggregation response and serum magnesium concentration, MVP, mitral regurgitation (MR), and platelet count. In 19 CKCS with MVP and 7 control dogs (not CKCS), the platelet aggregation response to 3 different agonists was compared. The CKCS with >100,000 platelets/microL (n = 10) had a significantly higher maximum aggregation response with regard to all tested agonists than the CKCS with <100,000 platelets/microL (n = 9) and control dogs (n = 7). The CKCS with <100,000 platelets/microL had a platelet aggregation response similar to the control dogs. There was no correlation between degree of MVP and platelet aggregation response. Platelet diameter increased (P = .006) and serum magnesium concentration decreased (P = .04) with lower platelet concentration. In conclusion, CKCS with MVP appeared to separate into 2 groups--1 group with <100,000 platelets/microL, normal platelet aggregation, low serum magnesium concentration, and enlarged platelets, and another group with >100,000 platelets/microL, increased platelet aggregation, and normal serum magnesium concentration and platelet size.     

Natural occurrence of amyloid-Abeta deposits in the brain of young common marmosets (Callithrix jacchus): a morphological and immunohistochemical evaluation

The spontaneous incidence and distribution of beta-amyloid (Abeta) peptide and argyrophilic deposits were extensively investigated in the common marmoset (Callithrix jacchus). In 12 young marmosets, up to 67 coronal step sections were evaluated in the brain. One brain hemisphere was stained by a silver method and also immunohistochemically with a primary antibody directed against Abeta. Argyrophilic deposits were observed in 4 (33%) of 12 animals with silver stain, and Abeta was seen in 4 (33%) of 12 animals with anti-4G8 antibody. The overall incidence was 6 (50%) of 12 affected animals. These results show that the young marmoset brain displays Abeta deposits at a low incidence and that immunohistochemistry is more suitable than silver staining to reveal rare amyloid plaques. These findings also provide evidence for the early onset of amyloid deposits in marmoset brain.     

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.     

Estimation of platelet counts on feline blood smears

Blood samples were obtained from 50 cats admitted for hematologic evaluation at the Royal (Dick) School of Veterinary Studies. Manual platelet counts were done using a hemacytometer, and the average number of platelets per oil immersion field (1,000X magnification) was determined on stained blood smears. A hemacytometer count was not obtained for one sample because of a failure in erythrocyte lysing. In nine samples, obvious platelet clumps in the blood smear prevented accurate determination of the number of platelets per oil immersion field. Hemacytometer counts on these nine samples ranged from 260-587 X 10 (3) platelets/microliter, suggesting that platelet clumps on a blood smear were usually associated with adequate platelet numbers. Simple regression analysis of hemacytometer counts and the average umber of platelets per oil immersion field for the remaining 40 samples yielded correlation coefficients (r) of 0.776 on untransformed data, and 0.892 on log10-transformed data. Each platelet per oil immersion field represented a circulating platelet count of approximately 20 X 10(3)/microliter, similar to conversion factors reported for dogs and human beings. It was concluded that estimation of platelet number on stained blood smears is a simple and quick method that appears to be reliable over a wide range of platelet counts in cats.     

Alteration of release and role of adenosine diphosphate and thromboxane A2 during collagen-induced aggregation of platelets from cattle with Chediak-Higashi syndrome

OBJECTIVE: To compare the interaction of endogenous ADP with collagen and thromboxane A(2) (TXA(2)) during collagen-induced platelet aggregation between platelets from healthy cattle and those with Chediak-Higashi syndrome (CHS). POPULATION SAMPLE: Platelets harvested from blood samples from healthy Japanese Black cattle and those with CHS. PROCEDURES: Aggregation of gel-filtered platelets; release of ATP-ADP; and generation of thromboxane B(2) (TXB(2)), a metabolite of TXA(2), were measured. RESULTS: The potency of collagen to induce aggregation in platelets of cattle with CHS (ie, CHS platelets) was less than a tenth of that in platelets of healthy cattle (ie, control platelets). Platelet aggregation induced by collagen at an intermediate concentration depended on the coexistence of ADP and TXA(2), suggesting that released ADP cannot cause platelet aggregation by itself. Collagen-induced ADP release was markedly decreased, whereas TXB(2) production was slightly low in CHS platelets, compared with that in control platelets. A combination of subthreshold amounts of ADP and 9,11-dideoxy-9alpha, 11alpha-methano-epoxy-prostaglandin F(2) (U46619), a TXA(2) analogue, caused platelet aggregation. Similarly, a combination of subthreshold amounts of collagen and ADP caused platelet aggregation, whereas collagen and U46619 were not synergistic. CONCLUSIONS AND CLINICAL RELEVANCE: Deficient ADP release ensuing from the delta-storage pool deficiency in platelets from cattle with CHS resulted in reduction of collagen-induced platelet aggregation, through attenuation of synergism between TXA(2) and ADP and between ADP and collagen. Furthermore, results of the study reported here indicated that TXA(2) was important for aggregation of bovine platelets.     

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.     

Evaluation of a citrate-based anticoagulant with platelet inhibitory activity for feline blood cell Counts

Abstract: Aggregation of feline platelets in vitro results in difficulty assessing platelet number. A citrate-based anticoagulant containing the platelet inhibitors theophylline, adenosine, and dipyridamole (CTAD; Diatube-H, Becton Dickinson, Oxford, UK) has been developed for use in human platelet studies and heparin assays. To evaluate the efficacy of CTAD in reducing platelet aggregation in feline blood samples, aliquots of blood from 51 cats were anticoagulated with EDTA, CTAD, and for 12 samples, citrate solution. Samples preserved in CTAD had significantly higher (P ≤ .001) platelet counts, as determined by an impedance counter, hemacy-tometer, and smear estimation, than samples preserved in EDTA. In addition, subjective assessment of blood smears showed significantly fewer platelet aggregates (P<.001) in CTAD-treated samples compared with EDTA samples. Although values were similar, automated platelet counts and smear estimates of platelet number were significantly higher (P < .05) and platelet aggregation was significantly less (P < .05) in CTAD samples than in citrate samples. These results suggest that the platelet inhibitory activity of CTAD reduced feline platelet aggregation. Automated total WBC counts in CTAD samples were significantly lower (P<.001) than automated counts in EDTA samples but were similar to manual WBC counts in EDTA samples. Differences in both platelet and WBC counts between CTAD and EDTA or citrate samples were clinically relevant. Mean platelet volume and MCV were significantly lower (P< .05) in CTAD samples than in EDTA samples. No effect was seen on cell morphology or staining characteristics. The anticoagulant CTAD offers an advantage over both EDTA and citrate for feline hematologic analysis, by decreasing pseudothrombocytopenia and pseudoleukocytosis.     

Characterization of the equine platelet aggregation response.

Equine platelet aggregation responses to bovine collagen, adenosine diphosphate (ADP), serotonin, epinephrine, and arachidonate in a platelet aggregometer were recorded. Equine platelets exhibited irreversible aggregation when incubated with ADP at a final concentration of 10 microM and bovine collagen. A secondary aggregation wave was recorded from platelets from certain horses at final ADP concentrations of 1 to 5 microM. Serotonin and arachidonate induced a weak reversible aggregation response, but a response was not observed following epinephrine addition. Equine platelet aggregation was influenced by concentration of anticoagulant (sodium citrate). Platelet aggregation responses at 37 C were indistinguishable from those recorded at 39 C. Platelet aggregation responses also were altered if the aggregation tests were not performed within 4 hours of blood sample acquisition. An assessment of platelet aggregation from multiple blood samples from the same horse indicated that the procedures described provide a reliable method to assess equine platelet aggregation in vitro.     

Evaluation of single and double centrifugation tube methods for concentrating equine platelets

The aim of this study was to evaluate single and double centrifugation tube methods for concentrating equine platelets. Whole blood samples were collected from clinically normal horses and processed by use of single and double centrifugation tube methods to obtain four platelet concentrates (PCs): PC-A, PC-B, PC-C, and PC-D, which were analyzed using a flow cytometry hematology system for hemogram and additional platelet parameters (mean platelet volume, platelet distribution width, mean platelet component concentration, mean platelet component distribution width). Concentrations of transforming growth factor beta 1 (TGF-beta(1)) were determined in all the samples. Platelet concentrations for PC-A, PC-B, PC-C, and PC-D were 45%, 44%, 71%, and 21% higher, respectively, compared to the same values for citrated whole blood samples. TGF-beta(1) concentrations for PC-A, PC-B, PC-C, and PC-D were 38%, 44%, 44%, and 37% higher, respectively, compared to citrated whole blood sample values. In conclusion, the single and double centrifugation tube methods are reliable methods for concentrating equine platelets and for obtaining potentially therapeutic TGF-beta(1) levels.     

Phosphodiesterase isoenzymes in equine platelets and their influence on platelet adhesion

OBJECTIVE: To determine the phosphodiesterase (PDE) isoenzymes in equine platelets and evaluate their influence on platelet adhesion. SAMPLE POPULATION: Platelets obtained from healthy New Forest Pony geldings that ranged from 12 to 20 years of age (mean +/- SEM, 17.3 +/- 1.1 years). PROCEDURES: PDE isoenzyme activity in equine platelets was determined by use of a 2-step radioactive assay. Functional importance of PDE isoenzymes was established by use of selective inhibitors in a colorimetric adhesion assay. RESULTS: PDE1, PDE2, PDE3, and PDE5 and small amounts of PDE4 were found in equine platelets. Inhibition of PDE3 abolished platelet adhesion almost completely, whereas inhibition of PDE4 and PDE5 had little effect. CONCLUSIONS AND CLINICAL RELEVANCE: Function of equine platelets can be influenced by inhibition of PDE3. Selective PDE3 inhibitors may be clinically useful to regulate platelet function. They offer the advantage of increased potency with fewer adverse effects, compared with those for nonselective PDE inhibitors.     

Hemostatic studies in racing standardbred horses with exercise-induced pulmonary hemorrhage. Hemostatic parameters at rest and after moderate exercise.

The purpose of this study was to determine whether a defect in hemostasis might be a factor in the etiology of exercise-induced pulmonary hemorrhage (EIPH). Hemostatic parameters were evaluated in 22 EIPH-positive and ten EIPH-negative racing horses while in a rested state. Nineteen EIPH-positive and ten EIPH-negative horses were further evaluated just before and immediately after a 15 min exercise period on a 260 m oval track. When EIPH-positive and EIPH-negative horses were compared at rest, there was no significant difference in any of the coagulation and fibrinolytic parameters studied. There was however, a significant difference in platelet function as assessed by aggregometry. The platelets from affected horses were significantly less responsive than those from nonaffected horses when exposed in vitro to the platelet agonists adenosine diphosphate, collagen and platelet activating factor. Exercise tended to increase the packed cell volume and factor VIII/von Willebrand factor and to decrease platelet aggregation responses to low concentrations of adenosine diphosphate. These effects of exercise however were quantitatively similar in both EIPH-positive and EIPH-negative horses. Reduced platelet function may therefore be a contributing factor in the bleeding characteristic of horses with EIPH.     

Measurement of the activation of equine platelets by use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody

OBJECTIVE: To investigate the potential use of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-human thrombospondin antibody for detection of the activation of equine platelets by use of flow cytometry. SAMPLE POPULATION: Platelets obtained from 6 Thoroughbreds. PROCEDURE: Flow cytometry was used to assess platelet activation as indicated by detection of binding of fluorescent-labeled annexin V, anti-human fibrinogen antibody, and anti-thrombospondin antibody to unactivated and ADP-, collagen-, platelet activating factor (PAF)-, and A23187-activated equine platelets. Human platelets were used as control samples. Determination of 14C-serotonin uptake and release was used to assess the extent of platelet secretion. RESULTS: Anti-human thrombospondin antibody failed to bind to equine platelets. Annexin V bound to platelets activated with PAF or A23187 when platelets had undergone secretion. Anti-human fibrinogen antibody bound to ADP-, PAF-, and A23817-activated platelets, but binding was not dependent on platelet secretion. The extent of binding of anti-fibrinogen antibody was less in equine platelets, compared with that for human platelets, despite maximal stimulation. CONCLUSIONS AND CLINICAL RELEVANCE: Activation of equine platelets can be detected by use of fluorescent-labeled annexin V and anti-human fibrinogen antibody but not by use of anti-human thrombospondin antibody. These flow cytometric techniques have the potential for detection of in vivo platelet activation in horses at risk of developing thrombotic disorders.