Focal Phosphatidylserine Exposure on Stressed Endothelial Cells In Relation to Membrane Topology: A Potential Basis for Compartmentalization of Procoagulant and Anticoagulant Activities

Abstract 4321

Exposure of endothelial cells to toxins or deprivation of nutrients leads to cell stress and, subsequently, to apoptosis. Apoptosis is associated with gross translocation of phosphatidylserine (PS) to the external membrane leaflet and exposed PS is required for coagulation enzyme complexes as well as some anticoagulant activities. However, in vivo studies of mouse thrombosis do not show apoptotic cells or gross PS exposure associated with clot formation, thus suggesting that PS supporting thrombosis is primarily derived from viable cells with limited PS exposure rather than apoptotic cells. In preliminary studies, we have found that cell stress, on multiple cell types, leads to limited, focal PS exposure before the onset of apoptosis. In this study, we have asked about the distribution of focal, PS-containing micro-domains on stressed endothelial cells. To induce stress we exposed Human Umbilical Vein Endothelial Cells (HUVEC) to 0.5 μM staurosporine for 15 minutes. PS exposure was detected by simultaneous staining using 10 nM lactadherin–Alexa 488 and annexin V–Cy 3.18, both exhibiting high affinity for PS. Stressed cells partially withdrew from their prior borders, leaving residual fibrils connected to original attachment points. In addition, they extended new thin fibrils that were up to several cell diameters in length. Confocal microscopy demonstrated focal staining, with little overlap between regions stained by lactadherin and regions stained by annexin V. Lactadherin stained the new thin fibrils, stretches of the cell margin, and patches near the nucleus. In contrast, annexin V stained in a reticular pattern in the perinuclear region. Based on the previously demonstrated finding, that lactadherin has a preference for highly curved vesicles and annexin V for flat or concave surfaces, we asked whether the selective binding might be determined by the membrane topology. To mimic the curvature of a cell membrane we prepared nano-fabricated silica substrates with ridges of 10 nm and 30 nm radii of curvature. The AFM topographic and fluorescent images of synthetic membrane bilayers supported by the substrates showed that, over a PS content of 4–15%, lactadherin preferentially binds to the convex nano-ridges with a ridge: valley staining ratio of approx. 280:1, while annexin V selectively binds the concave areas of the nano-trenches with a ridge: valley staining ratio of approx. 1:110. Combined fluorescence/AFM imaging of stressed HUVEC, demonstrated that the new thin filaments staining with lactadherin had radii of curvature of approx.12 nm, which was similar to the ridges of our synthetic bilayers. The reticular staining of the perinuclear region by annexin V correlated to concavities between nodules on the cell, suggesting that the selective staining is due, in part, to membrane topology. We are currently assessing the focal binding of procoagulant and anticoagulant proteins to the stressed endothelial cells. Because, factor V and factor VIII prefer the convex surfaces recognized by lactadherin we predict that procoagulant enzyme complexes will localize to these topographic features. Annexin II, which serves as a receptor for tPA, plasmin, and factor X, is predicted to prefer concavities, similar to annexin V. This could provide a basis for separate compartments of procoagulant and anticoagulant or fibrinolytic activity on the same cell.