Platelet morphology and the TUBB1 R318W mutation. (A) Peripheral blood smears were stained with May-Grünwald Giemsa for a normal control (TUBB1 43QQ/307RR) and the patient (original magnification ×1000). The patient showed giant platelets with morphologically normal leukocytes. The number in each panel shows the mean platelet size in microns (n = 200). (B) Ultrastructure of platelets. Platelet-rich plasma prepared from acid-citrate-dextrose citrated whole blood was fixed in 2% glutaraldehyde and postfixed in 1% osmium tetroxide. The platelet samples were embedded in epoxy resin (Quetol 651; Nissin EM, Tokyo, Japan). Ultra-thin sections were cut, stained with uranyl acetate and lead citrate, and observed under a transmission electron microscope (JEM 1011, JEOL, Tokyo, Japan) Original magnification ×8000. (C) The entire coding regions of the patient's TUBB1 gene were amplified from genomic DNA by polymerase chain reaction (PCR) and amplified DNA fragments were subjected to direct cycle sequence analysis. A C-to-T transition at nucleotide 952 (952C>T), changing Arg318 to Trp (R318W), was detected. The arrow shows the position of the substitution. (D) β-Tubulin sequence alignment. Amino acid sequence alignment is shown for the 7 human β-tubulin isoforms and the known β1-tubulin from other species. R318 is conserved in other species and other human β-tubulin isoforms. The substituted amino acid is indicated in bold. (E) Structural analysis of R318 using a tubulin 3D model. (i) Position of R318 (red) in the whole tubulin molecule. The residue is almost buried beneath the taxol pocket of β-tubulin but exposed slightly. Blue indicates β-tubulin; gray, α-tubulin. (ii) R318 (orange) is located near the α and β intradimer interface. D249 (yellow) is located at the interface. GTP (magenta) bound on the N-site of α-tubulin is also shown. (iii), Residues surrounding the side chain of R318. All the figures are depicted from 1JFF with the aid of MacPyMol (DeLano Scientific, Palo Alto, CA; http://www.delanoscientific.com/).