Microparticles from Aged Stored Platelets Impair Microvascular Pulmonary Endothelial Cell Barrier Function

Abstract

BACKGROUNDTransfusion‐related acute lung injury (TRALI) has a mortality of 5–15% and an incidence ranging from 0.02–1%. TRALI causes chest infiltrates and hypoxia in part due to lung endothelial cell (EC) barrier failure and edema formation. Aged platelets which have a high risk of causing TRALI, accumulate microparticles (PMPs) throughout storage. PMPs have been shown to mediate pro‐inflammatory processes such as neutrophil priming.RESEARCH QUESTIONConsidering the role of ECs in TRALI, we set out to determine if aged platelet derived PMPs could mediate EC barrier disruption?HYPOTHESISSpecifically, we hypothesized that PMPs from aged but not fresh platelets will lead to EC monolayer disruption.METHODSC57BL/6 mouse platelets were stored for 1 (fresh) or 5 (aged) days duration prior to PMP isolation (centrifugation/filtration) and enumeration (flow cytometry). Human microvascular pulmonary ECs (passage 6–8) were cultured with PBS (sham) or 0.1 mg/ml LPS two hours prior to addition of PBS, 5×105 of fresh or aged PMPs, or PMP depleted supernatants from stored platelet pools. Transendothelial electrical resistance (TEER) readings were obtained every 30 minutes for 8 hours in triplicate. Alternatively aged 10 ml/kg PMPs (≈ 5×106) or saline (sham) was transfused into primed (2 mg/kg LPS intraperitoneal) BALB/c mice for assessment of lung tissue wet‐to‐dry (W/D) ratios 6 hours post transfusion.RESULTSPMPs were found to be increased more than 2 fold in aged as compared to fresh platelet storage pools. ECs exposed to LPS alone did not reveal decreases in TEER relative to PBS sham. When aged PMPs were added 2 hours after PBS there was a marked drop in TEER within 2 hours which became maximal (nearly 50% decrease relative to PBS alone) by 5 hours. Addition of LPS instead of PBS prior to aged PMPs led to similar decreases in TEER readings but with faster onset. Addition of platelet and PMP depleted supernatants (fresh or aged) or fresh PMPs with LPS did not show signs of decreased TEER readings relative to PBS sham. Beyond being more numerous, aged PMPs appear more injurious than fresh PMPs when delivered in our EC model at identical doses. Aged PMPs are able to reduce EC monolayer integrity within 2 hours and sustain this effect for up to 6 hours +/− LPS. LPS primed mice transfused aged PMPs had higher W/D ratios than LPS primed mice transfused saline (p<0.05).CONCLUSIONSPMPs may in part contribute to TRALI when aged platelets which are a rich source of PMPs are transfused. In our model aged PMPs were injurious, impairing EC barrier integrity in vitro and leading to increased W/D ratios in vivo. Targeting aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationCanadian Institutes of Health Research (CIHR)Canadian Blood Services (CBS)