We are evaluating whether the generation of microvesicles (MV) known to occur during storage of platelet concentrates involves calpain-induced proteolysis of cytoskeletal actin. MV, which are shed when calpain is activated by physiological agonists, may be the result, in part, of proteolytic cleavage of actin-binding protein (ABP) by calpain at the actin-membrane interface. During routine blood bank storage of platelet concentrates, platelet actin is hydrolyzed into two fragments (SP-1, 29 kD; SP-2, 27 kD) and this cleavage may also be due to calpain activation. In the present study, platelets concentrates were stored under routine blood bank conditions for up to 6 days. Concentrates were incubated with 0.9% NaCl/0.1% DMSO (Control); calcium ionophore A23187 (10uM, a calpain activator); or leupeptin or E64d (1mM and 200 ug/ml, calpain inhibitors). SDS-PAGE and immunoblotting with rabbit anti-ABP and anti talin were used to analyze changes in ABP and talin as evidence of calpain activation during storage. MV were prepared from platelet concentrates by high speed centrifugation and the MV pellet was solubilized in urea for two-dimensional IEF/SDS-PAGE (2D-PAGE). Results showed that over 6 days of blood bank storage, ABP and talin in concentrates were proteolyzed as seen on immunoblots by A23187 and increased storage time, but were inhibited by leupeptin and E 64d. 2D- PAGE showed formation of SP-1 and SP-2 during storage and their incorporation into MV. These data suggest that calpain activation occurs in platelet concentrates during storage and that activation increases over time. Calpain generated during storage may promote MV formation due to alteration of the actin-membrane interface possibly involving cleavage of actin, as well as ABP and talin. Results of this study were submitted as an abstract to the American Society for Hematology, August, 1991.
