Segregation of VWF from tubular α-granules. Platelets were fixed for immunoelectron microscopy as described in “Blood collection and platelet preparation.” Immunogold double-labeling was performed on approximately 60-nm-thick cryosections using 10-nm and 15-nm protein-A gold and analyzed on a JEOL 1200CX electron microscope. (A-E) Immunogold double labeling of VWF and fibrinogen in tubular and spherical granules. Labeling as indicated on the figures. White arrows indicate fibrinogen; and black arrows, VWF. (A) Long tubular profile showing distinct fibrinogen labeling at one end. VWF is absent from the tubules. Clathrin lattices are indicated by the arrowheads. (B-E) Fibrinogen is distributed over both subtypes. VWF and fibrinogen colocalize in spherical (C,E, ☆) and multivesicular subtypes (D). Spheres exclusively containing fibrinogen are also prominent (B,E). Tubular profiles containing fibrinogen but no VWF are indicated by arrowheads in panels B and E. Note that the sequence of immunogold labeling in panels C and E is reversed. (F-G) Quantitative evaluation of the VWF and Fg distribution and colocalization over spherical and tubular subtypes (n = 1000 granules). Bars represent 200 nm.