We are evaluating whether the generation of microvesicles (MV) known to occur during storage of platelet concentrates involves calpain-induced proteolysis of cytoskeletal proteins, including 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 blood bank storage of platelet concentrates (PC), platelet actin is hydrolyzed into two fragments (SP-10, 29kd and SP-2, 27kD) and this cleavage may also be due to calpain activation. We analyzed cytoskeletal proteolysis in MV obtained from units of PC stored under blood bank conditions for up to 10 days. PC were incubated with 0.9% NaC1 (control); calcium ionophore A23187 (calpain agonist); and leupeptin and E64d (calpain inhibitors). MV were prepared from PC by high speed centrifugation. SDS-PAGE and immunoblotting was performed with probes for actin binding protein(ABP), talin, vinculin and glycoprotein Iib. In addition, the MV pellet was solubilized in urea for 2D-IEF/SDS-PAGE (2D-PAGE). Results showed that during blood bank preparation and storage, MV actin, ABP, talin and vinculin were degraded with concomitant generation of specific fragments over time. Glycoprotein Iib levels increased in MV during the storage period which indicated that MV generation increased with time. Degradation of MV actin was enhanced by exposure of PC to A23187, and inhibited by incubation of PC with E64d. These results imply that calpain activation occurs in PC during storage and that activation increases over time. Preliminary results suggest that shear stress may contribute to MV generation. Cytoskeleton disruption by cytochalasin D or vinblastine was not sufficient to cause MV release or calpain activation. Initial results of this study were presented at the annual meeting of the American Society for Hematology, December, 1991. A manuscript is in preparation.
