“Platelet Fatigue” during Cold Storage

Introduction: The key steps for platelet activation are: 1) inositol trisphosphate (IP3)-mediated calcium release from endoplasmic reticulum (ER) in response to agonists, and 2) following store-operated calcium entry. At rest condition, ER calcium levels are well maintained by the balance between IP3-mediated calcium release and sarcoendoplasmic reticulum calcium ATPase (SERCA)-mediated calcium uptake. Notably, SERCA is very sensitive to cold-induced inhibition, while the IP3 synthesis and activity are relatively tolerant to cold temperature. Therefore, cold temperature may break the balance and cause a net loss of ER calcium. However, such mechanism has not been determined in cold stored platelets. We hypothesize that the impaired function in cold stored platelets is associated with ER calcium depletion and diminished agonist-induced calcium entry. Methods: Healthy human platelets were stored at 4-6 oC in platelet additive solution (PAS)-diluted plasma (PAS/plasma = 65/35 in volume) for 15 days without or with a phospholipase C (PLC) inhibitor U73122 to suppress IP3 synthesis. Five groups were included in the study: blank control (no treatment), vehicle control (1% DMSO), U10 (10 mM U73122), U20 (20 mM U73122), and U50 (50 mM U73122), n = 5-8 for each group. Intracellular calcium levels, platelet counts, and platelet function were measured on day 1, day 3, day 8, and day 15. Intracellular calcium levels were measured by flow cytometry (Fluo-3-AM) before and after challenging with combined agonists thrombin (0.05U) and convulxin (50 ng/mL), both are strong activators for PLC-IP3 mediated calcium entry. Coagulation function was evaluated by thromboelastography, and platelet counts were measured using a Beckman DH520 hematology analyzer. Results: The control group exhibited an earlier and faster decrease in platelet count compared to other groups during the 15 days of cold storage. Basal intracellular calcium levels prior to agonist were continuously increased in control (from 16.7 ± 3.4 on day 1 to 38.2 ± 8.9 nM on day 15, P < 0.05) and vehicle groups (from 16.5 ± 2.7 on day 1 to 44.8 ± 9.5 nM on day 15, P < 0.05). Such increase in basal intracellular levels only lasted for 3 days in U10 from and was completely blocked in U20 and U50 starting on Day 1 after U73122 treatment. Agonists increased intracellular calcium in both control and vehicle groups on day 1 (106.3 ± 11.0 nM and 113.9 ± 10.7 nM, respectively), but the calcium entry was continuous reduced during cold storage and became completely abolished on day 15 (not statistically different from their baselines). Correlated with the agonists-induced calcium entry, the alpha angle and maximum amplitude (MA) was progressively impaired in control and vehicle groups. U73122 directly and rapidly (within several hours) inhibited the agonists induced calcium entry, alpha angle, and MA in U20 and U50. Such inhibitory effects were not observed until 3 days after treatment in U10. Conclusion: Cold storage causes continuous increase in basal intracellular calcium mediated by PLC-IP3 and concomitant losses of agonists-induced calcium entry and coagulating function. These results suggest that cold-stored platelets undergo a slow but continuous ER calcium depletion –a status we termed as “platelet fatigue”. Therefore, preserving ER calcium could be a potential strategy to protect platelet function during cold storage. DoD disclaimer: The views expressed in this abstract are those of the author(s) and do not reflect the official policy or position of the U.S. Army Medical Department, Department of the Army, DoD, or the U.S. Government.