Unraveling the mysteries of platelet fatigue in cold storage: Calcium release dynamics and energy metabolism as therapeutic targets.

Abstract Background: Cold storage improves platelet shelf-life due in part to suppressed metabolism, but platelet function still rapidly declines during storage. Calcium mobilization in platelets is critical for coagulation but remains vulnerable to cold stress; therefore, we hypothesized that the loss of platelet function during cold storage is due to impaired endoplasmic reticulum (ER) calcium release capacity (CRC) and/or sensitivity (CRS). Study Design and Methods: Platelet rich plasma (PRP) was stored at 4oC in platelet additive solution-diluted plasma (PRP/PAS = 65% / 35%) for 15 days in five groups (n=5-8): control, vehicle (1% DMSO), U10, U20, and U50 (10, 20, or 50 mM of U73122 on day 1 to inhibit 1,4,5-trisphosphate signaling). Platelet counts, thromboelastography, glucose, lactate, and the ER CRC following high concentrations of thrombin-convulxin were measured on Days 1 (30 minutes posttreatment), 3, 8, and 15. In another experiment, platelets were stored in PRP at both room (RT) and cold temperature (CT, n = 6 for each) for a week to compare platelet function, metabolism, and CRS determined by challenging platelets with the minimal effective dose of thrombin on Days 1, 3, and 8. Results: Resting cytosolic calcium levels continuously increasedduring cold storage, doubling by Day 15 (from 16.7 ± 3.4nM on Day 1 to 38.2 ± 8.9nM on Day 15, P < 0.01). CRC was impaired over storage time and abated by Day 15 (from 89.6 ± 10.5nM on Day 1 to 13.4 ± 3.9nM on Day 15, P < 0.01), correlating with diminished clot rate/strength. U73122 inhibited calcium release, clot formation, glycolysis, and lactate accumulation in a dose-dependent manner. CRS was unaltered in RT-stored platelets (from 65.7 ± 22.1nM on Day 1 to 46.4 ± 6.4nM on Day 7, P = 0.66) but was impaired over time and completely diminished on Day 7 in cold-stored platelets (from 52.1 ± 12.7nM on Day 1 to 1.0± 1.0nM on Day 7, P < 0.01). In addition, compared to RT, cold-stored platelets had a lower platelet count (decreased by 21% on Day 3 and 31% on Day 7 at RT; 54% on Day 3 and 55% on Day 7 at CT, P < 0.01) and an earlier decrease in maximal amplitude (from 73.6 ± 0.7mm on Day 1 to 73.5 ± 1.1mm on Day 3 at RT; from 74.6 ± 1.6mm on Day 1 to 60.7 ± 4.9mm on Day 3 at CT, P < 0.01), despite the reduced glycolysis (glucose 18 ± 1.7 mg/dL on Day 7 at RT, 71.6 ± 6.0 mg/dL on Day 7 at CT, P < 0.01), oxygen consumption, and lactate accumulation (10.0 ± 0.6 on day 7 at RT, 3.4 ± 0.3 on Day 7 at CT, P < 0.01) during 1 week of storage. Conclusion: Cold storage slows metabolism but triggers platelet fatigue characterized by progressive reduction in the CRC and CRS, and these altered calcium activities are correlated with cytosol glycolysis and suppressed coagulation function. Thus, alleviating platelet fatigue by maintaining the balances of calcium and energy homeostasis provides therapeutic targets for further improvement of cold-stored platelet quality. Additionally, these results potentiate a novel strategy for platelet storage through enabling a hibernation-like state with fewer toxic waste products via ER calcium preservation.