Factor Va binding sites and prothrombinase assembly localize to convex membrane ridges. Lipid bilayers containing 4% PS, 20% PE, and 76% PC were prepared on substrates with 10-nm radii ridges. Factor Va-Alexa 488, 10 nM (green) and 10 nM factor Xa-Alexa 647 (factor Xa-Glu-Gly-Arg-biotin/streptavidin-Alexa 647; blue) were incubated in the buffer overlaying the membrane ridges. (A) Differential interference contrast imaging mode localizes ridges (white) and troughs (dark) of the silica substrate. (B-C) Fluorescence images of bound factor Va–Alexa 488 and factor Xa–Alexa 647, respectively. (D) Overlay of images in panels A to C. (E) Fluorescence intensity line scans of the line in panel D shows that factors Va and Xa exhibited coincident and preferential binding to the convex ridges. See scheme in panel F.