We have developed a unique in vitro model that promotes differentiation of megakaryocytes into platelets. When megakaryocytes isolated from guinea pig bone marrow were cultured on hydrated rat tail collagen gels, cells spontaneously formed elongated, beaded processes that fragmented to yield cytoplasmic pieces with the same size and internal composition as individual platelets. Addition of nocodazole at the initiation of cultures blocked process formation, while addition of nocodazole to cells with previously established processes resulted in their retraction. The addition of taxol to cultures resulted in abnormally thick processes that were tightly adherent to the underlying substratum, and did not bead or fragment. Cytochalasin D accelerated process formation and fragmentation of megakaryocytes cultured on collagen gels by twofold. On the basis of these results, we propose a model for platelet formation in culture that involves the following steps: adherence of megakaryocytes to the underlying extracellular matrix; dilation of the demarcation membrane system and breakdown of the actin-rich peripheral zone; microtubule-based extension of pseudopodia, which are no longer adherent to the substratum; and fragmentation into platelets by the coalescence and fusion of demarcation membrane vesicles with the plasma membrane. We feel that this distinctive culture system closely approximates thrombocytopoiesis in vivo, thus allowing detailed elucidation of this important process.
We have demonstrated the presence of a 210K (K = 10(3) Mr) microtubule-associated protein (MAP) in blood platelets and have studied its relationship to tubulin and to the cytoskeleton, using a well-characterized polyclonal antibody for the analysis. When platelet lysates were enriched for tubulin by an assembly cycle at 37 degrees C, the 210K MAP was also enriched, as detected by Western blotting, while the antigen was not detected in pellets from cold-treated samples that lacked stabilized tubulin. Immunofluorescence of resting platelets showed that the 210K antigen colocalized with the microtubule coil in ring-like structures. On the other hand, in preparations of platelet cytoskeletons, the 210K antigen was present in samples from platelets in which the coil was disassembled (cold-treated without taxol pretreatment) as well as from platelets in which the coil was preserved (at 37 degrees C without taxol, or 4 degrees C with taxol pretreatment). In chilled platelets with disassembled microtubule coils, indirect immunofluorescence using antibodies to 210K or tubulin gave a diffuse signal throughout the platelet cytoplasm. However, immunofluorescence of the 210K antigen in both resting and cold-treated platelets displayed discrete or patchy staining as compared to the continuous staining with antitubulin. We conclude that 210K MAP is present in platelets, that it copurifies with tubulin and that it is localized along the microtubule coil. Our results also suggest that the 210K MAP may interact with some other element(s) of the cytoskeleton, and hence that it might serve as a linking protein.