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 (CaCl(2) ), or freeze-thaw). The resultant PDGF-BB (where PDGF is platelet-derived growth factor) and TGFß1 (where TGFß 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 CaCl(2) . The composition and function of equine autologous thrombin were characterized by Western blot analysis and platelet aggregometry. RESULTS: CaCl(2) (23 mM) activation of PRP yielded significantly greater PDGF release than did any other method. TGFß release was comparable after PRP activation by CaCl(2) , 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. CaCl(2) (23 mM) is an effective and inexpensive method of PRP activation. The PRP releasate derived from CaCl(2) activation contains 80% of the total PDGF content and is easily produced, making it a convenient product for clinical use.

Macroscopic domain formation during cooling in the platelet plasma membrane: an issue of low cholesterol content.

There has been ample debate on whether cell membranes can present macroscopic lipid domains as predicted by three-component phase diagrams obtained by fluorescence microscopy. Several groups have argued that membrane proteins and interactions with the cytoskeleton inhibit the formation of large domains. In contrast, some polarizable cells do show large regions with qualitative differences in lipid fluidity. It is important to ask more precisely, based on the current phase diagrams, under what conditions would large domains be expected to form in cells. In this work we study the thermotropic phase behavior of the platelet plasma membrane by FTIR, and compare it to a POPC/Sphingomyelin/Cholesterol model representing the outer leaflet composition. We find that this model closely reflects the platelet phase behavior. Previous work has shown that the platelet plasma membrane presents inhomogeneous distribution of DiI18:0 at 24 degrees C, but not at 37 degrees C, which suggests the formation of macroscopic lipid domains at low temperatures. We show by fluorescence microscopy, and by comparison with published phase diagrams, that the outer leaflet model system enters the macroscopic domain region only at the lower temperature. In addition, the low cholesterol content in platelets ( approximately 15 mol%), appears to be crucial for the formation of large domains during cooling.

Comparison of 4 blood storage methods in a protocol for equine pre-operative autologous donation.

OBJECTIVE: To compare viability of equine whole blood stored by 4 different methods, and to establish optimal storage protocols for an equine autologous blood donation program. STUDY DESIGN: In vitro study of stored equine whole blood. Animals- Six healthy adult horses. METHODS: Blood from each horse was collected into 4 different containers: glass bottles containing acid-citrate-dextrose solution (ACD), plastic bags containing ACD, citrate-phosphate-dextrose (CPD), and CPD with supplemental adenine (CPDA-1). Blood was stored for 5 weeks and sampled at 2-day intervals. Standard hematologic and biochemical variables were evaluated, and adenosine-5-triphosphate (ATP) and 2,3-diphosphoglycerate (2,3-DPG) concentrations were measured and normalized to total hemoglobin content. RESULTS: Plasma hemoglobin, % hemolysis, lactate, potassium, ammonia, and lactate dehydrogenase (LDH) increased, whereas glucose concentration and pH decreased in all stored blood over 5 weeks. There was a temporal increase in hemolysis with all storage methods, but the increase was greatest in glass bottles. Lactate and ammonia were highest in CPD and CPDA-1 samples, indicating more active red blood cell (RBC) metabolism. 2,3-DPG concentrations decreased during storage, but were optimally preserved with CPDA-1. ATP concentrations were significantly higher for blood stored in CPDA-1, and were lowest in glass bottles. CONCLUSIONS: Hematologic and biochemical values measured for blood stored in CPDA-1 are suggestive of improved RBC viability compared with other storage methods. With the exception of ATP, results from stored equine blood were similar to those reported for other species. CLINICAL RELEVANCE: Commercial CPDA-1 bags appear to be the optimal storage method for equine whole blood.