Cell surface action of thrombin is sufficient to initiate division of chick cells

Thrombin covalently linked to carboxylate-modified polystyrene beads initiated division of quiescent chick embryo (CE) cells either in medium containing low levels of serum or in serum-free medium. Release of thrombin was monitored by measuring acid-precipitable radioactivity released from 125I-thrombin beads into the medium during incubation with cells. Even if all of the acid-precipitable material released from the beads were active thrombin, it was not sufficient to account for any of the observed cell division, and was 10-30 fold less than the amount necessary to produce the increase in cell number caused by the thrombin beads. Two other kinds of experiments also showed that material released into the medium did not account for the observed initiation of cell division. First, medium taken from cultures incubated with thrombin beads did not initiate cell division when added to new quiescent cultures. Second, in coverslip experiments where populations of cells with an without thrombin feads shared the same medium, only bead-contacted cells divided. Several results suggested that the material which was released from the thrombin beads resulted from cell-associated proteolysis rather than from "leakage" of intact thrombin from the beads. For example, after incubating 125I-thrombin beads with or without CE cells, we were unable to detect any intact thrombin released into the medium. In addition, most of the material released from the beads was acid-soluble and was only released in the presence of CE cells. A few thrombin beads were endocytosed by CE cells, but they were surrounded by an intact plasma membrane. Thus they did not directly interact with the cytoplasm. The close association of many of the beads with the cell surface and the presence of a few beads in endocytic vesicles made it important to consider the possibility that thrombin might be released from the beads directly into the cells. This possibility was explored using ultrastructural (EM) autoradiography. With this technique (where one grain represented 700--900 thrombin molecules), we found that beads inside the cells had approximately the same number of grains as beads not in contact with cells. This suggested that little, if any, additional radioactive material had been released from the beads which were in contact with the cells. In addition, we were unable to detect any grains in the cytoplasm which could be attributed to released thrombin, even using an amount of 125I-thrombin beads which was 8 fold greater than the amount which produced maximal cell division. Taken together, these results provide direct evidence that thrombin action at the cell surface is sufficient to initiate division of CE cells.