Identification of thrombospondin as a high molecular mass protein released from activated equine platelets

Desireé L. Lipscomb From the Departments of Pathobiology (Lipscomb, Boudreaux, Spano, Welles), Physiology and Pharmacology (Paxton), and Large Animal Surgery and Medicine (Schumacher), College of Veterinary Medicine, Auburn University, AL 36849.

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Mary K. Boudreaux From the Departments of Pathobiology (Lipscomb, Boudreaux, Spano, Welles), Physiology and Pharmacology (Paxton), and Large Animal Surgery and Medicine (Schumacher), College of Veterinary Medicine, Auburn University, AL 36849.

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Ralph Paxton From the Departments of Pathobiology (Lipscomb, Boudreaux, Spano, Welles), Physiology and Pharmacology (Paxton), and Large Animal Surgery and Medicine (Schumacher), College of Veterinary Medicine, Auburn University, AL 36849.

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Joseph Spano From the Departments of Pathobiology (Lipscomb, Boudreaux, Spano, Welles), Physiology and Pharmacology (Paxton), and Large Animal Surgery and Medicine (Schumacher), College of Veterinary Medicine, Auburn University, AL 36849.

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Elizabeth G. Welles From the Departments of Pathobiology (Lipscomb, Boudreaux, Spano, Welles), Physiology and Pharmacology (Paxton), and Large Animal Surgery and Medicine (Schumacher), College of Veterinary Medicine, Auburn University, AL 36849.

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John Schumacher From the Departments of Pathobiology (Lipscomb, Boudreaux, Spano, Welles), Physiology and Pharmacology (Paxton), and Large Animal Surgery and Medicine (Schumacher), College of Veterinary Medicine, Auburn University, AL 36849.

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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)

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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