Activation of Equine Platelet-Rich Plasma: Comparison of Methods and Characterization of Equine Autologous Thrombin

Objective To investigate and compare clinically relevant Platelet-rich plasma (PRP) activation methods. Study Design Experimental. Methods PRP was prepared from 6 equine subjects. Activation of the PRP was performed by 4 methods (autologous thrombin, bovine thrombin, calcium chloride (CaCl2), or freeze-thaw). The resultant PDGF-BB (where PDGF is platelet-derived growth factor) and TGF beta 1 (where TGF beta is transforming growth factor beta) levels in PRP releasates were quantified by Enzyme-linked immunosorbent assay (ELISA) and compared. Growth factor contents were also compared between platelet-rich clots produced by thrombin or CaCl2. The composition and function of equine autologous thrombin were characterized by Western blot analysis and platelet aggregometry. Results CaCl2 (23 mM) activation of PRP yielded significantly greater PDGF release than did any other method. TGF beta release was comparable after PRP activation by CaCl2, bovine thrombin, and freeze thaw. Autologous thrombin was significantly less effective than all other activation methods in eliciting platelet growth factor release and induced significantly less platelet aggregation than bovine thrombin at 5 U/mL. Clots retained substantial concentrations of growth factor, and the amount in the releasate versus the clot differed between activation methods. Conclusions PRP activation methods differ in terms of growth factor output as well as logistical considerations. Autologous thrombin is not recommended for PRP activation. CaCl2 (23 mM) is an effective and inexpensive method of PRP activation. The PRP releasate derived from CaCl2 activation contains 80% of the total PDGF content and is easily produced, making it a convenient product for clinical use.