[Process of human platelets loaded with rehalose before lyophilization].
The aim of this research was to study the technology and methods of loading lyoprotectant-trehalose into cytoplasm of human platelets before lyophilization, to optimize experimental conditions of loading trehalose, to investigate the changes of platelets response to agonists and activation after incubation of platelets for 4 hours at 37 degrees C in the presence of lyoprotectant-trehalose, to protract the figures of loading efficiency and intracellular trehalose concentration versus incubation time, temperature and external trehalose concentration, to optimize loading parameters. The response of platelets to different agonists--thrombin, ADP, collagen and ristocetin were measured respectively by APACT2 aggregometer before and after loading trehalose into platelets; the expressions of CD62p and PAC-1 on platelet membranes in the presence and absence of reversible platelets activation inhibitors were measured by flow cytometry respectively before and after loading trehalose into cytoplasm of platelets. The results showed that the loading efficiency was linear to incubation time (2 hours later) and incubation temperature (rang from 30 degrees C to 40 degrees C), respectively. The loading efficiency almost reached 60% when the platelets were incubated at 37 degrees C for 4 hours. The intracellular trehalose concentration was higher with the increase of the extracellular trehalose concentration (< 50 mmol/L). Compared to untreated groups, the values of MPV and aggregation to different agonists in treated groups showed no significant difference, respectively (P > 0.01). After incubation of platelets for 4 hours, the expression of CD62p increased to some extent, however, the expression of CD62p decreased again when the reversible platelets activation inhibitor PGE-1 and adenosine were added to the incubation buffer. It is concluded that 37 degrees C, 4 hours and the extracellular trehalose concentration < 50 mmol/L are the optimal conditions for loading with trehalose. The processing of loading with trehalose before platelet lyophilization has no significant effects on response of platelets to agonists and activation.
Department of Blood Transfusion, General Hospital of PLA, Center for Clinical Blood Transfusion of PLA, Beijing 100853, China.
Fa-Qiang
FQ
Lu
Jing-Han
JH
Liu
Xi-Lin
XL
Ouyang
Xi-Jin
XJ
Li
Jun
J
Zhou
Yuan
Y
Zhuang
D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D001793:Blood Preservation / Q000379:methods*; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods; D005612:Freeze Drying; D006801:Humans; D014199:Trehalose / Q000097:blood / Q000494:pharmacology*
D014199:Trehalose