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

Binding of I-125-thrombin to human umbilical vein endothelial cells (HUVECs) was specifically displaced by the synthetic tetradecapeptide SFLLRNPNDKYEPF, named thrombin receptor agonist peptide (TRAP), which has recently been described as a peptide mimicking the new N-terminus created by cleavage of the thrombin receptor, and F-14, a tetradecapeptide representing residues 365-378 of the human alpha-thrombin B chain. Binding of I-125-TRAP to HUVECs was time-dependent, reversible and saturable, showing high affinity (K-D = 1.5 +/- 0.4 mu M) and high binding capacity (B-max. = 7.1 +/- 0.6 x 10(6) sites/cell) (n = 3). Unlabelled thrombin and TRAP competitively and selectively inhibited the specific 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 displacing I-125-TRAP from its binding sites, suggesting the presence of at least 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 responses between 1 and 10 nM. F-14 also promoted HUVEC growth. The mitogenic effects of F-14 and TRAP were additive. N alpha-(2-Naphthylsulphonylglycyl)-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 effect of TRAP or F-14. This demonstrated that the mitogenic effect of alpha-thrombin for HUVECs was intimately linked to its esterolytic activity but also showed that thrombin can stimulate HUVEC growth via another non-enzymic pathway. This hypothesis was further reinforced by the fact that F-14-induced proliferation of HUVECs remained unaltered by two antibodies directed against TRAP or the cleavage site on the extracellular portion of the thrombin receptor, which both strongly reduced thrombin-induced proliferation of HUVECs. Thrombin-, TRAP- or F-14 induced HUVEC proliferation was strongly inhibited by a neutralizing monoclonal antibody directed against basic fibroblast growth factor (bFGF), suggesting that thrombin regulates the autocrine release of bFGF in HUVECs.