Thrombin active site regions required for fibroblast receptor binding and initiation of cell division.

Human a-thrombin was modified by four different procedures to identify specific active site regions required for receptor binding and stimulation of cell division. Conjugation of a-thrombin with diisopropylphosphofluoridate (DIP-F) or methylsulfonyl fluoride (MS-F) yielded catalytically inactivated preparations. Nitration or limited proteolysis of a-thrombin led to nitro-a-thrombin or y-thrombin preparations, respectively. Both possessed very little clotting activity but retained significant esterase activity; they were modified at regions necessary for the binding recognition of fibrinogen. Measurements of specific binding of these modified thrombins to cultured mouse, hamster, chicken, and human fibroblasts revealed no significant binding of nitro-a-thrombin or y-thrombin. Thus, binding of athrombin to each of the cell types examined involved regions of a-thrombin distinct from the catalytic apparatus that are related, if not identical, to regions required for fibrinogen recognition. Binding experiments with catalytic site-conjugated DIPor MS-athrombins revealed significant differences in the athrombin receptor among the four cell types; these thrombin forms bound as effectively as a-thrombin to mouse and hamster cells, but did not bind significantly to chick or human cells. Thus, thrombin binding to chick and human cells required the thrombin catalytic apparatus or adjacent active site regions, whereas binding to mouse or hamster cells did not. The four cell types examined all responded to the mitogenic action of a-thrombin. However, the derivative thrombin forms did not stimulate division of any of the cells significantly, with the exception of nitro-athrombin which possessed some residual activity for mouse cells. Since enzymatically inactive DIPand MSa-thrombins bound to mouse and hamster cells as effectively as active a-thrombin, the mitogenic activity of a-thrombin on these cells requires the intact catalytic apparatus of the enzyme to interact with and presumably cleave a specific protein component.