Equine platelets contain an anisotropic array of microtubules which reorganise upon activation

Electron microscopic and immunofluorescence studies of equine platelets reveal a distinctive distribution of microtubules. Resting cells have a crosslinked microtubule coil and an additional array of anisotropic microtubules. Thrombin activation of equine platelets results in platelet shape change accompanied by reorganisation of the microtubule arrays. The microtubule coil remains intact, while short linear microtubules are present within the pseudopodia. To examine the stability of these complex microtubule arrays, platelets were incubated with 2 μm-nocodazole for 2 h at room temperature. Immunofluoresence analysis of the distribution of tubulin showed that only the anisotropic array was depolymerised by nocodazole treatment. Western blot analysis demonstrates that in resting cells tubulin is only found in the detergent insoluble cytoskeleton, whereas in nocodazole-treated cells tubulin is present in the detergent-insoluble cytoskeleton as well as in the detergent-soluble supernatant. To determine whether post-translational modifications were associated with this differential stability, equine platelets were stained with antibodies to detyrosinated and acetylated tubulin. All parts of the complex microtubule array were stained by the polyclonal antibody to detyrosinated tubulin, and the monoclonal antibody to acetylated tubulin. Western blot analysis of resting and nocodazole-treated cells demonstrated that resting detergent-insoluble cytoskeletons contained both detyrosinated and acetylated tubulin. However, in nocodazole-treated cells detryosinated tubulin was also present in the detergent-soluble supernatant. Acetylated tubulin appears to be associated only with the stable microtubule fraction, the microtubule coil.