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

This study investigates the proteins released from activated platelets in horses, particularly focusing on the identification of thrombospondin, with a long term goal of developing a test to detect platelet activation in living horses.

Study Methods

• The study was conducted on 5 healthy, mature horses.
• Platelet-rich plasma (PRP) and platelet-poor plasma (PPP) were prepared from anticoagulated blood.
• Platelets were isolated from the plasma proteins using a process called gel filtration and then activated using a compound called platelet-activating factor.
• Protease inhibitors were used to prevent the degradation of proteins, and the proteins released from the platelets were then collected for examination.
• Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a method used to separate proteins based on their size, was performed on the collected proteins and compared to those in PPP.
• The proteins were then subjected to an immunoblot analysis using an antibody specific for human thrombospondin. Human platelet-derived proteins served as a control for the experiment.

Results

• In the presence of a compound called beta-mercaptoethanol, several proteins were identified in the activated platelets that were not found in the plasma, with sizes ranging from 95 to 194 kilodaltons (kd).
• Immunoblot results revealed a significant 180-kd protein in the activated platelets recognized by the human thrombospondin antibody. This protein was thrombospondin.
• Upon prolonged color development, two additional proteins sizes were observed (166 and 155 kd) – these were less prominent.

Conclusions

• The study concluded that there are several proteins that are only released during platelet activation in horses that are not present in normal equine plasma.
• One of the prominent high molecular weight proteins identified from activated equine platelets was thrombospondin.
• Finally, the study suggests that these findings could form the basis for developing a future assay to detect thrombospondin and potentially indicate platelet activation in live horses.