Long‐term effects of thrombin require sustained activation of the functional thrombin receptor

Abstract

Thrombin is a potent activator of human glomerular epithelial cells (HGEC). Here we compare short-term and long-term effects of thrombin and thrombin receptor agonist peptide (TRAP) which selectively activates the functional thrombin receptor. TRAP, as thrombin, increases intracellular free Ca 2÷ concentration and acts synergistically with growth factors possessing tyrosine kinase receptors on DNA synthesis. Thrombin induces synthesis of proteins of the fibrinolytic system and cell proliferation if it is present for at least 8 h. TRAP alone does not stimulate protein synthesis and is not mitogenic. However, in the presence of the aminopeptidase inhibitor amastatin all long-term effects of thrombin can be fully mimicked by TRAR In conclusion, different effects of thrombin and TRAP may be related to the degradation of TRAP by cellular ectoenzymes. The recently cloned thrombin receptor accounts for early intracellular signals and long-term cellular effects that require sustained activation of this receptor.

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

“…Whether peptide degradation is mediated by proteases secreted into the medium, or via receptormediated endocytosis, as for EGF via its receptor, remains to be determined. Our results using amastatin were entirely in accord with the study of Zacharias and co-workers, who found that amastatin was required to observe a mitogenic action of the TRAP, SFLLRNP in cultured human glomerular epithelial cells (Zacharias et al, 1993).…”

Evidence for the presence of a protease-activated receptor distinct from the thrombin receptor in human keratinocytes.

“…As shown in Fig. 6, fibroblasts express a 3.4-kb thrombin receptor transcript, in agreement with an earlier report (21). Our analysis indicated that keratinocytes contain comparable levels of the thrombin receptor transcript.…”

“…1 C and D). Thrombin and SFLLRN induced inositol phosphate formation and calcium mobilization to similar extents, although the doses of SFLLRN required for receptor activation were greater than those for thrombin, as observed in other cell systems (4,20,21 1 and Fig. 2).…”

Thrombin-receptor agonist peptides, in contrast to thrombin itself, are not full agonists for activation and signal transduction in human platelets in the absence of platelet-derived secondary mediators

“…However, it is now evident that the transient nature of the responses to TRPs are due in some cases to the metabolism of the peptides by cellular aminopeptidase M, an enzyme present in plasma, leucocytes, smooth muscle, vascular endothelium and other cell types, which removes the terminal Ser to inactivate the TRPs, but which apparently does not attack the N-terminal Ser of the TL in the large native receptor molecule (Coller et al, 1992). Amastatin, a very potent inhibitor of the peptidase, potentiated aggregation of platelets in plasma by TRPs (Coller et al, 1992) and enabled a long-term mitogenic activity of TRPs in kidney epithelial cells (Zacharias et al, 1993). However, the differences between TRPs and thrombin in our experiments on washed human platelets were clearly not attributable to the metabolic destruction of the TRPs, because the incubation of TRPs with stimulated platelets and their secretory products did not diminish their agonist activity, and responses to TRPs were not potentiated or prolonged by amastatin.…”