Spatial uncoupling of endothelial calcium signaling from phospha-tidylserine externalization in vascular injury

Abstract

Ca2+ 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 Ca2+ imaging in vivo. Injury generated a coherent Ca2+ wavefront that spread rapidly before progressively slowing and terminating at a reproducible boundary 197 {+/-} 7 m 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 Ca2+ release. Wavefront kinematics were consistent with ATP diffusion. In vivo, injury elicited vasoconstriction and bidirectional Ca2+ waves propagating 205 {+/-} 28 m before terminating. Remarkably, despite widespread Ca2+ elevations, phosphatidylserine (PS) externalization remained confined to the wounded cell in monolayers and intact vessels in vitro. In contrast, ionomycin-induced Ca2+ overload exposed phosphatidylserine in nearly all endothelial cells. These findings reveal an unexpected spatial uncoupling between endothelial Ca2+ signaling and phospholipid scrambling, demonstrating that Ca2+ 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.