Our studies on the mechanism by which thrombin initiates division of cultured fibroblasts have shown that action at the cell surface is sufficient for cell activation. This prompted studies on the cell surface for interactions and events necessary for the stimulation of cell division. These studies led to the identification of protease nexin, a cell-secreted protein which mediates much of the specific binding of thrombin to the cell surface. Protease nexin is released by cells into the culture medium where it forms a covalent linkage with thrombin. The thrombin-protease nexin complexes then specifically bind to cells and are internalized and degraded. Our studies have shown that linkage of thrombin to protease nexin inactivates the thrombin. Moreover, protease nexin inhibits the stimulation by thrombin, and thus it represents a mechanism by which cells modulate their mitogenic response to thrombin. We have also demonstrated a cell surface binding site for unlinked thrombin and have shown that binding of thrombin to this site is not necessary for the stimulation of cell division. Past studies have shown that the proteolytic activity of thrombin is necessary for cell activation. In fact, all of our results point to a requirement for cleavage of one or more cell surface proteins by thrombin for the stimulation. At this point we have shown that several cell surface proteins are cleaved by thrombin. One of these has been identified as fibronectin. In addition, cell surface proteins of about 140 kilodaltons and 55 kilodaltons were thrombin-sensitive, but they have not yet been identified. Studies are underway to better resolve membrane proteins so we will be able to identify additional ones that might be thrombin-sensitive. We will study which of these cleavages are necessary for cell activation by determining whether they occur in a large series of cloned cell populations that are either responsive or unresponsive to the mitogenic action of thrombin. (A)
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