Ceramide Containing Microparticles from Aged Stored Platelets Recapitulate Aspects of Murine Transfusion Related Acute Lung Injury

Abstract

BACKGROUNDTransfusion‐related acute lung injury (TRALI) presents as chest infiltrates and hypoxia after 0.02–1% of transfusions with an associated mortality of 5–15%. Aged platelet products elicit TRALI via a mechanism invovling formation of the sphingolipid ceramide (CER) by acid sphingomyelinase (ASM), which causes lung injury in vivo and impairs pulmonary endothelial cell (EC) barrier integrity in vitro. Concomitantly, aged platelets form abundant microparticles (PMPs) which can shuttle nucleic acids, proteins, and ‐ importantly ‐ lipids, to target cells.OBJECTIVETo test the hypothesis that aged (5 days) but not fresh (1 day) platelets will generate PMP that mediate TRALI in a CER‐dependent manner.METHODSPMPs from male C57BL/6 wild type (WT) or Smpd1−/− (ASM knockout) platelets stored for 1 (fresh) or 5 (aged) days duration were isolated by differential centrifugation and filtration before enumeration (flow cytometry) and CER analysis (mass spectroscopy). Cultured human microvascular pulmonary ECs (passage 6–8) were incubated with PBS (sham) or 5×105 fresh or aged PMPs for 4 hours and transendothelial electrical resistance (TEER) was measured every 30 minutes. For TRALI induction, BALB/c male mice primed with 2 mg/kg intraperitoneal LPS two hours prior to intravenous (IV) infusion of 10 ml/kg PMPs (≈ 5×106) or saline (sham) and were assessed (6 hours later) by measuring lung tissue wet‐to‐dry (W/D) ratios, bronchoalveolar lavage fluid protein (BAL‐p) and lung tissue myeloperoxidase activity (MPO).RESULTSCompared with fresh WT PMP, aged WT PMPs were more numerous and CER‐enriched. In contrast, aged ASM KO PMPs were less numerous compared with fresh ASM KO PMP but contained similar CER content as WT aged PMPs. Aged (CER rich) but not fresh (CER poor) PMPs lead to reductions in TEER (< 2 hours) in vitro in keeping with EC loss of barrier integrity which persisted for > 4 hours. Reductions in TEER seen over 4 hours with aged PMPs was prevented with pre‐treatment of WT PMPs with ceramidase (0.75 μg/mL) prior to incubation with ECs. LPS primed mice receiving IV aged WT PMPs exhibited increased W/D (p < 0.05), MPO and BAL‐p (p<0.05) relative to mice receiving LPS priming in addition to saline, fresh PMPs or ASM KO aged PMPs.CONCLUSIONSCeramide formation in aged platelets contributes to PMP formation and impairs human EC barrier integrity resulting in TRALI. Removal or modification of aged PMPs may prove a therapeutic option to reduce the risk of aged cellular blood products from causing TRALI.Support or Funding InformationThis work was jointly funded by Canadian Blood Services and the Canadian Institute of Health Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.