Calcium chelation: a novel approach to reduce cryopreservation-induced damage to frozen platelets

BACKGROUND Cryopreserved platelets are phenotypically and functionally different to conventionally stored platelets. Calcium may be released from internal stores during the freeze-thaw process, initiating signaling events which lead to these alterations. It was hypothesized that the addition of a calcium chelator prior to cryopreservation may mitigate some of these changes. METHODS Buffy coat-derived platelets that had been pooled and split were tested fresh and following cryopreservation (n = 8 per group). Platelets were cryopreserved using 5%-6% dimethylsulfoxide (DMSO) or were supplemented with increasing concentrations of the internal calcium chelator, BAPTA-AM (100 mu M, 200 mu M, or 400 mu M), prior to storage at -80 degrees C. RESULTS Supplementation of platelets with BAPTA-AM prior to freezing improved platelet recovery in a dose response manner (400 mu M: 84 +/- 2%) compared to standard DMSO cryopreserved platelets (70 +/- 4%). There was a loss of GPIb alpha, GPVI, and GPIIb/IIIa receptors on platelets following cryopreservation, which was rescued when platelets were supplemented with BAPTA-AM (400 mu M: p < 0.0001 for all). Platelet activation markers, such as phosphatidylserine and P-selectin, were externalized on platelets following cryopreservation. However, the addition of BAPTA-AM significantly reduced the increase of these activation markers on cryopreserved platelets (400 mu M: p < 0.0001 for both). Both cryopreserved platelet groups exhibited similar functionality as assessed by thromboelastography, forming clots at a faster rate than fresh platelets. CONCLUSIONS This study demonstrates that calcium plays a crucial role in mediating cryopreservation-induced damage to frozen platelets. The addition of the calcium chelator, BAPTA-AM, prior to cryopreservation reduces this damage.