Background/Case Studies: Red blood cells (RBCs) have limited repair capacity: mild stress prompts microvesicle (MV) shedding, whereas severe stress causes hemolysis. Both processes can harm transfusion recipients – MVs deliver pro-inflammatory, pro-coagulant signals, while hemolysis releases free hemoglobin and heme that drive harder-to-contain oxidative and vasoactive injury. Because hemolysis of transfused RBCs also eliminates viable oxygen carriers, steering stressed RBCs toward controlled vesiculation is desirable. Accordingly, this study investigates whether phospholipase A₂ (PLA₂), which generates curvature-inducing lysophospholipids under membrane stress, can shift the balance between vesiculation and hemolysis.
Study
Design/Methods: RBCs from CPD/SAGM leukoreduced units were pre-treated with bromoenol lactone (BEL) to inhibit PLA₂, then challenged with 5 µM calcium ionophore A23187 + 1 mM CaCl₂ for 60 min at 37 °C. Supernatants were collected by centrifugation at 200 g for 10 min, then cleared of residual debris by sequential spins (5,000 g and 10,000 g, 10 min each). MVs in the final supernatant were labeled with 0.5 µM fluorescein-DHPE and 1 µg/mL Annexin V, then enumerated on a CytoFlex S flow cytometer (Beckman Coulter, model C01159). EVs and phosphatidylserine-positive (PS⁺) subsets were gated using FlowJo v10.8.1, with thresholds set against buffer-only controls. Hemolysis was quantified by measuring total and supernatant hemoglobin via a conventional Drabkin’s assay.
Results/Findings: A clear drop in hemolysis was seen in every BEL-treated sample after the ionophore challenge (p = 0.038). A matching decline in MV counts did not, however, materialize. In fact, MV counts trended slightly higher in BEL-treated samples (1.09 – 10.22 % increase), though the difference between groups was insignificant (p = 0.3924). Interestingly, BEL markedly reduced both the total number and overall proportion of PS⁺ EVs generated in response to the ionophore (57.7–67.4 % reduction; p < 0.0001).
Conclusions: Inhibiting PLA₂ diverted calcium-stressed RBCs from hemolysis toward vesiculation and reduced PS-positive MV release, identifying PLA₂ as a possible tunable switch between premature rupture and controlled membrane shedding. These benefits require validation under the milder, multifactorial stresses encountered during storage or post-transfusion, using graded – rather than complete – PLA₂ inhibition to partially preserve its protective membrane-remodelling functions.
