Synergistic actions of a thrombin‐derived synthetic peptide and a thrombin receptor‐activating peptide in stimulating fibroblast mitogenesis

We measured the ability of the thrombin receptor activating peptide, SFLLR‐NH 2 (P5A) to stimulate 3 H‐thymidine incorporation in hamster CCL‐39 fibroblasts either alone or in combination with the thrombin‐derived polypeptides, YPPWNKNFTENDLL (TDP‐1) and AGYKPDEGKRGDACEGDSGGPFV (TDP‐2). In the presence (but not absence) of the amino peptidase inhibitor amastatin (10 μM), P5A alone (7.5 to 100 μM) caused a 1.5‐ to 2‐fold stimulation of thymidine incorporation above basal, even though this inhibitor did not abrogate the degradation of P5A by other peptidases present in the assay medium. Neither TDP‐1 nor TDP‐2 alone had any effect on thymidine incorporation. However, TDP‐1 (30 to 90 μM) considerably augmented P5A‐mediated thymidine incorporation at low P5A concentrations (7.5 to 30 μM), shifting the P5A concentration‐effect curve to the left. TDP‐2 was inactive in this regard. The EC 50 for this potentiating action of TDP‐1 was approximately 40 μM. Further, thrombin, rendered proteolytically inactive by a low‐molecular‐weight bifunctional inhibitor, hirutonin‐6, also acted synergistically with P5A to stimulate CCL‐39 cell thymidine incorporation. We hypothesize that thrombin can cause its cellular effects, such as thymidine incorporation, not only via the proteolytic activation of its G‐protein‐coupled receptor, but also via the concurrent and synergistic interaction of its TDP‐1 peptide domain with a separate cell surface docking site. © 1996 Wiley‐Liss, Inc.