Microtubule coils versus the surface membrane cytoskeleton in maintenance and restoration of platelet discoid shape

The discoid form of blood platelets is important to their function in hemostasis. Recent studies have suggested that the spectrin-rich surface membrane cytoskeleton and the cytoplasmic, actin-rich cytoskeleton are responsible for discoid shape, shape change, and recovery after activation or chilling. Earlier studies had suggested that circumferential coils of microtubules supported the disc shape of resting platelets and that their repositioning or reassembly restored disc shape after exposure to low temperature. The present study has used the chilling-rewarming model, together with microtubule stabilizing (taxol) and disassembling (vincristine) agents to retest the relative importance of the surface membrane cytoskeleton and circumferential microtubules in platelet discoid shape and its restoration. Washed platelet samples were rested at 37 degrees C and chilled to 4 degrees C; chilled and rewarmed to 37 degrees C for 60 minutes; or chilled, rewarmed, and exposed to the same cycle in the presence or absence of vincristine or taxol and fixed for study by disseminated interference phase contrast microscopy and electron microscopy. Rhodamine-phalloidin and flow cytometry were used to measure changes in actin filament assembly. Chilling caused loss of disc shape, pseudopod extension, disassembly of microtubule coils, and assembly of new actin filaments. Rewarming resulted in restoration of disc shape, pseudopod retraction, disassembly of new actin filaments, and reassembly of circumferential microtubule coils. Vincristine converted discoid platelets to rounded cells that extended pseudopods when chilled and retracted them when rewarmed, leaving spheres that could undergo the same sequence of changes when chilled and rewarmed again. Taxol prevented cold-induced disassembly of microtubules and limited pseudopod formation. Rewarming caused retraction of pseudopods on taxol-treated, discoid cells. Cytochalasin B, an agent that blocks new actin filament assembly, alone or in combination with taxol, inhibited the cold-induced shape change but not dilation of the open canalicular system. Rewarming eliminated open canalicular system dilation and restored lentiform appearance. The results indicate that microtubule coils are the major structural elements responsible for disc shape and its restoration after submaximal stimulation or rewarming of chilled platelets.