Search

The fate of the circumferential bundle of microtubules in activated platelets has been a subject of disagreement. Thin sections of stimulated platelets fixed at multiple intervals following exposure to aggregating agents have revealed that the circumferential band is constricted into a tight ring around centrally concentrated organelles. However, studies of detergent-resistant platelet cytoskeletons fixed and either negatively stained or critical point dried after activation on polylysine-coated grids have revealed that microtubule rings disappear, leaving only fragments in the peripheral cytoplasm of spread cells. The present study has employed immunofluorescence on glass slides and the whole mount technique with detergent extraction and either negative staining or critical point drying to evaluate the fate of microtubules in surface-activated platelets treated with or without the microtubule stabilizing agent, taxol. Significant numbers of microtubule coils were visible in control and taxol-treated platelets stained indirectly with a fluorescein-coupled antibody to tubulin 30 to 60 minutes after surface activation on glass. Coils of microtubules were also visible in dendritic forms and in significant numbers of spread platelets on negatively stained or critical point dried whole mounts in the electron microscope. The findings support the concept that microtubule disassembly is not an integral step in early phases of platelet activation.

It is generally accepted that a circumferential microtubule supports the discoid shape of resting platelets. The fate of the many-coiled polymer following platelet activation, however, has been a subject of considerable debate. Morphological investigations have suggested that the circumferential coils are constricted into tight rings around centrally concentrated organelles during platelet shape change. Biochemical studies employing colchicine-binding assays, on the other hand, have indicated that the bundle of microtubules dissolves almost completely within seconds after activation and reassembles in a new location one to four minutes later. The present study has accepted the latter hypothesis in order to examine the second part of the disassembly-reassembly theory proposed in biochemical studies. Platelets exposed to low temperatures sufficient to remove all microtubules were placed on glass slides and microscope grids to cause surface activation during rewarming. The combined stimuli of rewarming and surface activation might have been expected to cause more rapid assembly than warming alone or activation alone. This was not the case. Reassembly of microtubules during rewarming and simultaneous surface activation was not accelerated. In contrast to the constriction of microtubule rings observed during activation in control platelets, the diameters of coils that developed in chilled platelets one to two hours after rewarming and surface activation were twice those of control cells.