Mouse fibroblasts defective in thrombin mitogenesis possess functional proteolytically activated receptor for thrombin: requirement for a second signaling pathway.

Thrombin mitogenesis in fibroblasts requires two distinguishable subsets of signals; one generated by proteolytic cleavage, the other by high-affinity cell surface binding. Characterizing two closely related mouse embryo (ME) cell lines with high numbers of thrombin binding sites, we found that one line, B11-A, responds mitogenically to thrombin, epidermal growth factor (EGF), and serum, whereas the B11-B cell line is responsive to EGF and serum, but not to thrombin. The B11-B defect responsible for loss of thrombin responsiveness is not due to differences in the number of high-affinity binding sites, the affinity of thrombin binding to these sites, or to differences in cell surface expression of proteolytically activated receptors for thrombin (PART). The defect is also not associated with an inability of thrombin to activate PART since thrombin stimulates the cleavage-dependent induction of the proto-oncogene c-fos in both B11-A and B11-B cells. Various combinations of thrombin, synthetic thrombin receptor peptide, TRP-14 (SFFLRNPGENTFEL), platelet-derived growth factor (PDGF), and phorbol 12-myristate 13-acetate (PMA) were used to better define the defect in thrombin-mediated mitogenesis in B11-B cells. Direct activation of protein kinase C with PMA in combination with thrombin did not overcome B11-B nonresponsiveness. However, mitogenic responsiveness was regained in B11-B cells by simultaneous addition of PDGF and either thrombin or TRP-14. Therefore, the B11-B defect may involve a set of signals initiated by nonproteolytic thrombin interactions distinct from those initiated by PART, but related to the downstream signals initiated by the tyrosine kinase-associated growth factors, EGF and PDGF.