Scanning EM and whole-mount immuno-EM analysis of tethers and microparticles. (A-E) Citrated whole blood (platelet count, 250 000 platelets/μL) perfused over dVWFA1 at γw of 30 000 s–1. (A) Scanning electron micrograph of a perfusion-fixed activated platelet, shown for comparison. The activated platelet shows multiple filopodia protruding from the surface in all directions. (B) A nonactivated platelet adhering to VWF through GPIbα. The membrane surface is smooth, and the long passively pulled tether has less than half the diameter of filopodia. Two short protruding membrane areas (arrowheads) are indicative of nascent tethers. (C) Enlargement of a detail highlighted in panel B, showing thin and short tethers connecting the platelet body to the adhesive substrate. (D-E) Isolated microparticles and detached tethers. Scale bars = 1 μm (A-D) or 0.5 μm (E). See Movie S7. (F-K) Citrated whole blood was perfused at γw of 30 000s–1 over dVWFA1-coated EM grids, fixed under flow, and immunolabeled with monoclonal anti-GPIbα antibody followed by protein A coupled to 10-nm gold particles (GPIb10 ). (F-G) Severed tethers exhibiting tubular and globular domains, the latter (highlighted) corresponding to DAPs. (H-I) GPIbα in the globular tether end (DAP) is predominantly peripheral and excluded from the central zone. (J) Bifurcating tether showing peripheral GPIbα distribution in the DAP-like bifurcation zone (arrow). (K) Severed tubular tether exhibiting subdomains with variable GPIbα content. Scale bar in panel A = 1 μm; bar in panel B = 500 nm; bars in panels C to F = 200 nm.
