RanBP10 modulates the tubulin-microtubule equilibrium and is essential for the platelet shape change. (A) Platelet activation with 0.04μM U46619 (left panel) still caused a shape change in wild-type (black curve), but not in RanBP10^−/− platelets (gray curve). Intermediate concentrations (0.1μM, middle panel) induced shape change without aggregation in both strains, whereas high concentrations led to similar shape changes in platelets of wild-type and mutant platelets with full aggregation (right panel). (B) The amount of glutamylated (Glu-tub) or tyrosinated tubulin (Tyr-Tub) as well as EB1 proteins were unaffected in resting, ADP, or PAR4p-stimulated knockout platelets (−/−) compared with wild-type (WT) controls. (C) RanBP10 prevents premature polymerization of α/β1–tubulin dimers. Megakaryocytes lacking RanBP10 (−/−) show an increased fraction of β1 and α-tubulin in the pelleted fraction (P) representing polymerized microtubules compared with the soluble (S) fraction isolated from the supernatant. Other β-tubulins recognized by a commerical β-tubulin antibody remain unaffected. In the presence of taxol filaments polymerize independent of RanBP10. (D) Platelets were incubated with either 0.5mM taxol or PBS before aggregometry with 5 μg/mL collagen. When microtubules were stabilized with taxol, the shape change is delayed and aggregation diminished, mimicking the RanBP10-null phenotype.