Preventing endoplasmic reticulum calcium depletion mitigates platelet storage lesion

Background: Cold storage continuously decreases platelet count and function (platelet storage lesion, PSL). In resting platelets, calcium storage is maintained in endoplasmic reticulum due to the balance between inositol 1,4,5-trisphosphate (IP3)-mediated calcium release and sarcoendoplasmic reticulum calcium ATPase (SERCA)-mediated calcium uptake. However, there is evidence from other cell types that SERCA is more sensitive than IP3 synthesis to cold-induced inhibition. Therefore, we hypothesize that in cold stored platelets there is a slow and continuous depletion of ER calcium which contributes strongly to PSL. Methods: Healthy human platelets were stored at 4-6 °C 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: control (untreated), vehicle control (1% DMSO), U10 (10 mM U73122), U20 (20 mM U73122), and U50 (50 mM U73122), n = 5-8 for each group. The inhibitor or vehicle was applied after platelets rich plasma was mixed with PAS (day 1). Intracellular calcium levels, platelet counts, and platelet function were measured on days 1 (30 minutes after treatment), 3, 8, and 15. Intracellular calcium levels were measured by flow cytometry (Fluo-3-AM) before and after challenging with combined agonists thrombin (0.05 U) 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 cytosolic calcium levels prior to agonist continuously increased in control (from 16.7 ± 3.4 nM on day 1 to 38.2 ± 8.9 nM on day 15, P < 0.05) and vehicle groups (from 16.5 ± 2.7 nM on day 1 to 44.8 ± 9.5 nM on day 15, P < 0.05). With U73122 treatment, the increase in basal intracellular levels lasted for 3 days in U10 but was completely blocked in U20 and U50 from day 1. Agonists increased calcium release from ER in both control and vehicle groups on day 1 (106.3 ± 11.0 nM and 113.9 ± 10.7 nM, respectively), but calcium release became resistant to agonists during cold storage and was completely abolished on day 15 (not different from their baselines). Associated with the depleted calcium release, clot rate and clot strength continuously decreased in the control and vehicle groups. With U73122 treatment, agonist-induced calcium release, clot rate, and clot strength were inhibited at a dose dependent manner in U10, U20, and U50. Conclusion: The continuous increase in basal cytosolic calcium observed in cold-stored platelets was mediated by PLC-IP3 inhibition, which blocked agonist-induced ER calcium release and impaired coagulation function. These results suggest that cold-stored platelets undergo a slow but continuous ER calcium depletion that contributes to the PSL. 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. US Army Medical Research and Development Command This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.