INTACT MICROTUBULES ARE NECESSARY FOR COMPLETE PROCESSING, STORAGE AND REGULATED SECRETION OF VON WILLEBRAND FACTOR BY ENDOTHELIAL CELLS

The importance of intact microtubules in the processing, storage and regulated secretion of von Willebrand factor (vWf) from Weibel-Palade bodies in endothelial cells was investigated. Human umbilical vein endothelial cells treated for one hour with colchicine (10^-6M) or nocodozole (10^-6M) lost their organized microtubular network. Stimulation of these cells with secretagogues (A23187, thrombin) produced only 30% release of vWf in comparison to control cells containing intact microtubules. The nocodazole treatment was reversible. One hour incubation in the absence of the drug was sufficient for microtubules to reform and to restore the full capacity of the cells to release vWf.

Long-term incubation (24 hours) of endothelial cells with microtubule destabilizing agents had a profound effect on vWf distribution. In control cells vWf was localized to organelles in the perinuclear region (i.e. endoplasmic reticulum and Golgi apparatus) and to Weibel-Palade bodies. In drug-treated cells vWf staining was dispersed throughout the cytoplasm and Weibel-Palade bodies were absent. The vWf synthesized in the absence of microtubules contained significantly less large multimers than that produced by control cells. This was not due to possible side effects of the drugs on the cells because the presence of lumicolchicine, an inactive analogue of colchicine, had no effect on vWf processing. Since Weibel-Palade bodies specifically contain the large multimers (Sporn et al, Cell 46:185-190, 1986), we hypothesize that the structural defect in vWf secreted by cells in the absence of microtubules is due to the lack of Weibel-Palade bodies in these cultures.

In summary, the intact microtubular cytoskeleton in the endothelial cells in culture, appeared to be crucial for normal release of Weibel-Palade bodies after stimulation with secretagogues, for reformation of new Weibel-Palade bodies and for the efficient intracellular multimerization of vWf dimeric molecules.