Binding and internalization of thrombin by normal and transformed chick cells.

Abstract

Thrombin stimulates cell proliferation in cultures of normal chick embryo fibroblasts but not in cells transformed with Rous sarcoma virus. Analysis of medium conditioned by Rous-sarcoma-virus-transformed cultures demonstrates that these cells do not secrete molecules that can inhibit or inactivate thrombin. The interaction of thrombin with these cells was investigated with enzymatically active 125I-thrombin. The total amount of cell-associated 125I-thrombin was 3 times greater with normal cells than with transformed cells. In both types of cell, greater than 50% of the total cell-associated 125I-thrombin was found as a component that was not dissociated from the cells by trypsin treatment, an observation suggesting that a significant portion was not on the cell surface. The amount of the trypsin-insensitive fraction increases with time up to 12 h, whereas the trypsin-sensitive fraction is saturated after 1-4 h. Autoradiography of thin sections of 125I-thrombin-treated cells observed by EM reveals that after 10 h incubation greater than 70% of the label is localized in the cytoplasm of normal and transformed cells. Autoradiograms of sodium dodecyl sulfate/polyacrylamide slab gels demonstrate that 40% of the intracellular label is the size of native thrombin with the remainder in 2 large fragments of 22,000 and 19,500 daltons.