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-NH2 (P5A) to stimulate 3H-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 microM), P5A alone (7.5 to 100 microM) 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 microM) considerably augmented P5A-mediated thymidine incorporation at low P5A concentrations (7.5 to 30 microM), shifting the P5A concentration-effect curve to the left. TDP-2 was inactive in this regard. The EC50 for this potentiating action of TDP-1 was approximately 40 microM. 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.
Hollenberg
M D
MD
Department of Pharmacology and Therapeutics, University of Calgary, Faculty of Medicine, Canada.
Mokashi
S
S
Leblond
L
L
DiMaio
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004357:Drug Synergism; D005347:Fibroblasts / Q000166:cytology*; D008938:Mitosis / Q000187:drug effects*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000494:pharmacology*; D010455:Peptides / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D018179:Receptors, Thrombin / Q000502:physiology*; D015398:Signal Transduction; D013917:Thrombin / Q000737:chemistry* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; C090291:C186 65; D009842:Oligopeptides; D010455:Peptides; D011480:Protease Inhibitors; D018179:Receptors, Thrombin; C016977:amastatin; C047981:arginyl-glycyl-aspartic acid; D013917:Thrombin