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), and platelet activation (CD62 expression) in platelet-rich plasma from Cavalier King Charles Spaniels with various stages of MMVD and assess the influence of thrombocytopenia on those variables. Materials and Methods Animals The study was

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in American Journal of Veterinary Research

Lipopolysaccharide NADPH Reduced form of nicotinamide adenine dinucleotide phosphate PG Prostaglandin PMA Phorbol 12-myristate 13-acetate PRP Platelet-rich plasma ROS Reactive oxygen species TNF Tumor necrosis factor Footnotes a. IRAP II system

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in American Journal of Veterinary Research

SUMMARY

Ten adult dogs (5 Beagles and 5 mixed-breed dogs) were inoculated IV with canine platelets containing Ehrlichia platys. Inclusions and morulae of E platys developed in platelets of infected dogs at 10 to 14 days after inoculation, followed by marked thrombocytopenia at 14 to 21 days. Parasitemia and marked thrombocytopenia recurred at 24 to 28 days after inoculation. Increased numbers of megakaryocytes were observed in marrow aspirate smears from infected dogs, indicative of regenerative thrombocytopenia. Prior to infection, platelet-rich plasma from these dogs was determined to have similar aggregatory response to arachidonate. After infection with E platys, the aggregatory response of platelet-rich plasma to collagen or 3 dilutions of adenosine diphosphate was evaluated. A statistically significant (P < 0.05) inhibition of platelet aggregatory response to the lowest dilution of adenosine diphosphate was detected for mixed-breed dogs, whereas aggregation responses were unchanged in Beagles. Results indicate that platelet activation may occur in dogs with acute ehrlichial infection.

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in American Journal of Veterinary Research

Abstract

Objective

To evaluate a method for detecting thiazole orange-positive (TO+, reticulated) platelets in equine blood, using flow cytometry.

Animals

16 healthy, equine infectious anemia virus (EIAV)-negative horses and ponies; 9 thrombocytopenic, ElAV-positive horses and ponies; and 2 thrombocytopenic, ElAV-negative horses.

Procedure

Blood from healthy and thrombocytopenic horses was collected by jugular venipuncture. Appropriate sample requirement and incubation time for the assay were evaluated, using blood anticoagulated with EDTA or sodium citrate, or platelet-rich plasma in sodium citrate. The sample of blood or platelet-rich plasma was incubated with thiazole orange, and flow cytometric analysis was performed. Percentage of circulating TO+ platelets was determined from fluorescence (FL-1) logarithmic histograms.

Results

Healthy ponies (n = 9) had 1.28 to 2.83% (mean ± SD, 2.03 ± 0.50%) and horses (n = 7) had 0.9 to 3.44% (2.12 ± 1.14%) TO+ platelets in circulation. Thrombocytopenic ponies (n = 7) had 11.14 to 48.41 % (26.51 ± 11.99%) and thrombocytopenic horses (n = 4) had 2.33 to 8.52% (6.19 ± 2.68%) TO+ platelets in circulation. Mean platelet counts for the thrombocytopenic ponies and horses were 24,400 ± 20,500 and 39,300 ± 13,500 platelets/μl, respectively (reference range, 94,000 to 232,000 platelets/μl).

Conclusion

Thiazole orange-positive platelets can be detected in equine blood and percentages of TO+ platelets are increased in thrombocytopenic horses.

Clinical Relevance

Enumeration of TO+ platelets may prove to be a helpful noninvasive clinical measurement of bone marrow platelet production and aid in the assessment of platelet kinetics in thrombocytopenic horses. (Am J Vet Res 1997;58:1092–1096)

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in American Journal of Veterinary Research

Abstract

Objective

To establish the existence of platelet-derived proteins in equine plasma, with the future goal of developing an assay for the detection of in vivo platelet activation.

Animals

5 mature healthy horses.

Procedure

Platelet-rich plasma and platelet-poor plasma were prepared from anticoagulated blood. Platelets were separated from plasma proteins by gel filtration, then activated with 0.5 μM platelet-activating factor. Protease inhibitors were added, and the released platelet proteins were harvested. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on the released platelet proteins and platelet-poor plasma, and the resultant silver-stained bands were compared. Immunoblot analysis was performed on released platelet proteins, using an antibody to human thrombospondin; human platelet-derived proteins served as the positive control for the antibody.

Results

Released platelet proteins in the presence of β-mercaptoethanol (reduced samples) contained several proteins that were not observed in plasma including (mean ± SEM) 194 ± 2, 159 ± 2, 151 ±2, 104 ± 2, and 95 ± 1 kd. Immunoblots of released platelet proteins had a prominent 180 ± 2-kd protein in reduced samples that was recognized by an antibody to human thrombospondin, and with prolonged color development, 2 additional less prominent proteins (166 ± 1 and 155 ± 1 kd) were observed.

Conclusions

Several proteins are released from activated equine platelets that are not detectable in normal equine plasma. Thrombospondin is one of the high molecular mass proteins released by activated equine platelets.

Clinical Relevance

An assay can be developed for detection of thrombospondin in equine plasma and may be useful for detection of in vivo platelet activation in horses. (Am J Vet Res 1997;58:954–960)

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in American Journal of Veterinary Research

Summary

A protocol was developed for preparation of platelet concentrates (pc) to support thrombocytopenic dogs. Four clinically normal dogs with platelet counts that ranged from 200 to 330 × 109 platelets/L were used as donors. One unit (450 ml) of blood was collected by venipuncture into a double blood bag. Whole blood (wb) was centrifuged for 4 minutes at 1,000 × g (braking time = 2 minutes, 30 seconds) to prepare platelet-rich plasma (prp). The prp was expressed into the satellite bag and was centrifuged for 10 minutes at 2,000 × g (braking time = 2 minutes, 36 seconds). The platelet-poor plasma was expressed, leaving 40 to 70 ml of plasma and the pelleted platelets in the satellite bag. The resulting pc was left undisturbed for 60 minutes to promote disaggregation, and the platelets were then resuspended by gentle manual agitation.

Forty-eight pc were prepared. Mean (± sd) platelet yield from wb to prp was 78 (±13)% (range, 35 to 97%); yield from prp to pc was 94 (± 6)% (range, 75 to 100%); and overall yield (pc from wb) was 74(± 13)% (range, 36 to 91%). Mean pc platelet count was 8.0 (± 3.0) × 1010 platelets/pc (range, 2.3 to 13.4 × 1010 platelets/pc). The wbc content was 0.1 to 2.3 × 109 platelets/pc, representing 3 to 74% of wbc in the wb. Hematocrit was 0.1 to 26.2%. Results of bacterial and fungal culturing were negative.

The pc were irradiated (18 Gy) and transfused to 5 cross-matched dogs undergoing bone marrow transplantation that developed profound thrombocytopenia of up to 8 weeks’ duration. Transfusions were given over 5 to 15 minutes, using a latex-free transfusion set. Platelet count in recipients was monitored to confirm reduction in the degree of thrombocytopenia. Mean actual 24-hour post-pc transfusion platelet count was significantly (P < 0.0001) higher than mean expected 24-hour platelet count without pc transfusion. Recipients were closely observed for hemorrhage, which did not occur. Prevalence of transfusion reactions was 17%; signs were fever (5/46), urticaria (1/46), vomiting (1/46), and anaphylaxis (1/46).

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in American Journal of Veterinary Research

Questions statistical power of study on release of growth factors from platelet-rich plasma We would like to raise several concerns regarding the article “Effects of preparation method, shear force, and exposure to collagen on release of

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in American Journal of Veterinary Research

. Platelet-rich plasma is becoming popular as a treatment for various orthopedic injuries of horses. Platelet-rich plasma may promote healing of tissues via release of growth factors from the concentrated platelets. Results of in vitro studies 3–5 indicate

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in Journal of the American Veterinary Medical Association

, Minneapolis, Minn. o. Equine IL-1Ra duo set, R&D Systems Inc, Minneapolis, Minn. p. SAS, version 9.2, SAS Institute Inc, Cary, NC. q. PROC MIXED, SAS, version 9.2, SAS Institute Inc, Cary, NC. References 1. Textor J . Platelet-rich plasma

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in American Journal of Veterinary Research

growth factors involved in tendon healing are contained within the α-granules of platelets. 10 Several experimental and preclinical studies 11–19 have provided promising results following the use of PRP to enhance tendon healing. Platelet-rich plasma

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in American Journal of Veterinary Research