THROMBIN INDUCES ENDOTHELIAL-CELL GROWTH VIA BOTH A PROTEOLYTIC AND ANONPROTEOLYTIC PATHWAY

Binding of I-125-thrombin to human umbilical vein endothelial cells (H UVECs) was specifically displaced by the synthetic tetradecapeptide SF LLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which ha s recently been described as a peptide mimicking the new N-terminus cr eated by cleavage of the thrombin receptor, and F-14, a tetradecapepti de representing residues 365-378 of the human alpha-thrombin B chain. Binding of I-125-TRAP to HUVECs was time-dependent, reversible and sat urable, showing high affinity (K-D = 1.5 +/- 0.4 mu M) and high bindin g capacity (B-max. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabell ed thrombin and TRAP competitively and selectively inhibited the speci fic binding of I-125-TRAP With IC50 values of 5.8 +/- 0.7 nM and 2.8 /- 0.4 mu M respectively, whereas F-14 remained ineffective at displac ing I-125-TRAP from its binding sites, suggesting the presence of at l east two different types of thrombin-binding sites on HUVECs. TRAP was a potent mitogen for HUVECs in culture. Both TRAP and alpha-thrombin stimulated the proliferation of HUVECs with half-maximum mitogenic res ponses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitog enic effects of F-14 and TRAP were additive. N ha-(2-Naphthylsulphonyl glycyl)-DL-p-amidinophenyl- alanylpiperidine (NAPAP) and hirudin (two specific inhibitors of the enzymic activity of thrombin) specifically inhibited thrombin-induced HUVEC growth (IC50 values 400+/-60 and 52+/ -8 nM respectively) but remained without effect on the mitogenic effec t of TRAP or F-14. This demonstrated that the mitogenic effect of alph a-thrombin for HUVECs was intimately linked to its esterolytic activit y but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fa ct that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracel lular portion of the thrombin receptor, which both strongly reduced th rombin-induced proliferation of HUVECs. Thrombin-, TRAP- or F-14 induc ed HUVEC proliferation was strongly inhibited by a neutralizing monocl onal antibody directed against basic fibroblast growth factor (bFGF), suggesting that thrombin regulates the autocrine release of bFGF in HU VECs.