Charge Screening by Extracellular Ca2+ Shields Endothelial Cells from Histone Injury and Permits Membrane Repair

**Background:** Extracellular histones released after trauma and in sepsis injure the vascular endothelium and contribute to coagulopathy and organ failure. Hypocalcemia frequently accompanies resuscitation with citrated blood products and predicts poor outcome, an association usually attributed to coagulation-factor dysfunction. Whether extracellular Ca2+ directly governs endothelial vulnerability to circulating cytotoxic histones, and whether the resulting membrane injury is reparable, are unknown. **Methods:** We combined live-cell spinning-disk confocal imaging of plasma membrane dynamics (FM1-43), Ca2+ imaging, flow cytometry, electric cell-substrate impedance sensing, and protein-lipid binding assays in cultured human endothelial cells, ORAI1/2/3 triple-knockout HEK-293 cells and surgically opened en face murine mesenteric arteries. Membrane repair was assessed with a sequential two-dye live-cell viability assay. **Results:** Histones evoked rapid, irregular endothelial Ca2+ elevations that were unaffected by deletion of all three ORAI homologs and were blocked by Gd3+ only at concentrations two orders of magnitude above those required to inhibit store-operated Ca2+ entry, excluding this pathway. Histones instead bound anionic membrane phospholipids, deforming the plasma membrane: lamellipodial ruffling, retraction, blebbing, extracellular vesicle release, phosphatidylserine externalization, and permeabilization in a heterogeneous subset