Spatial uncoupling of endothelial calcium signaling from phosphatidylserine externalization in vascular injury

Ca ²⁺ is both the signal that spreads injury information through the endothelium and the trigger for phospholipid scrambling that makes a membrane procoagulant. How a blood vessel can disseminate the first without broadcasting the second is unknown. Here, we combine single-cell laser injury in endothelial monolayers with imaging of intact mesenteric vessels in vitro and intravital Ca ²⁺ imaging in vivo. Injury generated a coherent Ca ²⁺ wavefront that spread rapidly before progressively slowing and terminating at a reproducible boundary 197 ± 7 µm from the wound, engaging 50 ± 8 cells. The first ring of neighboring cells exhibited a distinct response that was prevented by carbenoxolone, whereas propagation beyond this ring required extracellular ATP, purinergic receptor activation, and IP₃-dependent Ca ²⁺ release. Wavefront kinematics were consistent with ATP diffusion. In vivo, injury elicited vasoconstriction and bidirectional Ca ²⁺ waves propagating 205 ± 28 µm before terminating. Remarkably, despite widespread Ca ²⁺ elevations, phosphatidylserine (PS) externalization remained confined to the wounded cell in monolayers and intact vessels in vitro. In contrast, ionomycin-induced Ca ²⁺ overload exposed phosphatidylserine in nearly all endothelial cells. These findings reveal an unexpected spatial uncoupling between endothelial Ca ²⁺ signaling and phospholipid scrambling, demonstrating that Ca ²⁺ -dependent scramblase activation is subject to additional spatial regulation. The endothelium can therefore propagate injury signals across a broad field of cells while maintaining a highly localized procoagulant membrane response.