Binding and internalization of thrombin by normal and transformed chick cells

Area of Science:

• Cell Biology
• Biochemistry
• Virology

Background:

• Thrombin is a key enzyme in coagulation and has known roles in cell signaling.
• Rous sarcoma virus (RSV) transformation alters cellular properties, including growth regulation.

Purpose of the Study:

• To investigate the differential interaction of thrombin with normal chick embryo fibroblasts and RSV-transformed cells.
• To elucidate the mechanism behind thrombin's effect on cell proliferation.

Main Methods:

• Enzymatically active 125I-thrombin was used to study cell association.
• Cell-associated thrombin was analyzed for trypsin sensitivity and localization via electron microscopy and autoradiography.
• Intracellular thrombin and its fragments were identified using SDS-PAGE.

Main Results:

• RSV-transformed cells exhibited significantly lower association with 125I-thrombin compared to normal cells.
• A substantial portion of cell-associated thrombin was trypsin-insensitive, indicating intracellular localization.
• Autoradiography confirmed thrombin's presence within the cytoplasm of both cell types after 10 hours.
• Intracellular thrombin was found as native enzyme and two large fragments (22,000 and 19,500 daltons).

Conclusions:

• RSV transformation impairs thrombin binding and cellular uptake.
• Thrombin and its fragments are internalized and localized within the cytoplasm.
• The reduced interaction with thrombin in transformed cells correlates with their altered proliferation characteristics.