Studies On Process Of Human Platelets Loaded With Trehalose Before Lyophilization And Characteristics Of Rehydrated-Lyophilized Human Platelets

Objective The aim was to study the technology of loading trehalose into cytoplasm of platelets before lyophilization, to optimize conditions for loading trehalose, to inhibit reversibly platelets activation, and to study the characteristics of rehydrated and lyophilized human platelets. Methods The sulfuric-anthrone reaction was used to determine the intracellular trehalose concentration loaded by human platelets.The figures of loading efficiency or intracellular trehalose concentration versus incubation temperature, time, and external trehalose concentration were protracted respectively to optimize loading conditions. The responses of platelets to agonists were assayed by APACT-2 aggregometer before and after platelets incubation. The expressions of CD62_P and PAC-1 on platelets membranes were measured by flow cytometry (FCMs) before and after incubation of platelets in plasma or loading buffer at 37℃ for 4h_s, in the presence and absence of 2% DMSO in loading buffer, respectively. The sulfuric-anthrone reaction was used to measure the intracellular trehalose concentrations after incubation of platelets at 37℃ for 4h_s in loading buffer, in the presence and absence of 2% DMSO, respectively. The intracellular LYCH concentration and distribution versus incubation temperature, and time, were visualized respectively by fluorescence microscopy in the presence and absence of 2% DMSO. The expressions of CD_62P and PAC-1 on platelets membranes, and intracellular trehalose concentration were measured by FCMs and sulfuric-anthrone reaction respectively, in the presence and absence of reversible platelets activation inhibitors-Adenosine and Prostaglandin E₁, before and after incubation of platelets at 37℃ for 4h_s. The effects of external trehalose or human serum albumin concentration added to drying buffer, and storage time at room temperature on numerical recoveries of rehydrated-lyophilized platelets were analyzedrespectively by counting platelets with a Blood Cell counter MEK-6108K. The dose-responses of rehydrated and fresh platelets to agonists and the effects of intracellular or external trehalose concentration on aggregation were measured respectively by APACT-2 aggregometers. The effects of intracellular or external trehalose concentration and 2% DMSO on activation of (p)rehydrated platelets were analyzed respectively by FCMs. The Hemostasis Analyzer was used to measure the activities of unstable coagulation factorâ…§ and factor V in plasma before and after platelet-rich plasma were lyophilized and rehydrated. Results (1) The method for intracellular trehalose concentration was established. Incubation temperature of 37 degrees C, incubation time of 4 h_s, and external trehalose concentration < 50mmol/L in loading buffer were the optimal conditions for platelets loaded with trehalose before lyophilization. (2) Compared to untreated groups(before incubation in plasma), the responses of platelets to agonists in treated groups (after incubation in plasma) showed no significant difference, P>0.05;The expression of CD_62P was higher after incubation of platelets in loading buffer for 4 h_s, P<0.01, and decreased again with the reversible platelets activation inhibitors-Adenosine ( 5mmol/L ) and Prostaglandin E₁ (PGE₁,10 μ g/ml) added to loading buffer, P>0.05;The expressions of PAC-1 kept relatively stable. Compared to untreated groups(before incubation in plasma), the expressions of CD_62P and PAC-1 showed no significant difference after incubation of platelets in plasma at 37℃ for 4h_s, P>0.05. (3) Compared to controlled groups (loading in buffer), the expressions of CD62_P and PAC-1 in treated groups (loading in plasma) decreased significantly, (8.29 + 8.69) % versus (20.67 + 7.557) %, respectively, P<0.01. (4) Compared to controlled groups (loading in buffer in the absence of 2% DMSO), the expressions of CD_62P and the intracellular trehalose concentration in treated groups (loading in buffer in the presence of 2% DMSO) decreased significantly, P<0.01;The intracellular LYCH concentration was higher, and homogeneously distributed in the cytoplasm of platelets. (5) Reversible platelets activation inhibitors-Adenosine (5 mmol/L) and PGE₁ (10 μ g/ml) significantly inhibited the expressions of glycoprotein CD_62P and PAC-1, and significantly increased the loading efficiency of trehalose. (6) The numerical recoveries of lyophilized and rehydrated platelets loaded with trehalosevaried with external trehalose or human albumin concentration in freeze-drying buffer, and were much more stable after rehydration of lyophilized platelets at room temperature for 8 h_s. (7) The dose-response curves of rehydrated platelets to agonists were similar to that of fresh platelets. Compared to untreated groups (without loading trehalose before lyophilization, only in presence of external trehalose in drying buffer), the values of aggregation of rehydrated platelets to ADP, and the expressions of glycoprotein CD_62p in the treated groups (loading trehalose before lyophilization, in the presence of external trehalose in the freeze-drying buffer) showed significant difference, respectively, P<0.01. The expressions of CD_62P decreased significantly with 2% DMSO, Adenosine or/and PGE₁ added to drying buffer, P<0.01, respectively. (8) The activities of unstable coagulation factor â…§ and V before and after lyophilization showed no significant differences in the presence of trehalose in drying buffer, P>0.05. Conclusions Incubation temperature of 37 degrees C, incubation time of 4 h_s, and external trehalose concentration <50mmol/L in loading buffer were the optimal conditions for platelets loading trehalose. Loading trehalose in plasma more effectively inhibited platelets activation than in loading buffer. Reversible platelets activation inhibitors-adenosine (5mmol/L) and PGE₁ (10μg/ml) significantly inhibited platelets activation. DMS0(2%) significantly accelerated the intake of trehalose and made trehalose homogeneously distributed in cytoplasm of platelets, and effectively inhibited platelets activation in all processing of platelets lyophilization and rehydration. The numerical recoveries of lyophilized and rehydrated platelets were higher and much more stable at room temperature. The rehydrated-lyophilized platelets had good responses to agonists. The activities of unstable coagulation factor â…§ and V in PRP kept stable before and after lyophilization in the presence of trehalose.