Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes

We produced recombinant human thrombin mutants to investigate the correlati on between the thrombin enzyme and mitogenic activity. Single amino acid su bstitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205 T), in the oxy-anion binding site (G203A) and in the anion binding exosite- l region (R73E). Proteins were produced as prethrombin-2 mutants secreted i n the culture medium of DXB11-derived cell lines. All mutants were activate d by ecarin to the corresponding thrombin mutants; the enzymatic activity w as assayed on a chromogenic substrate and on the procoagulant substrate fib rinogen. Mutations S205A and G203A completely abolished the enzyme activity . Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activi ty and the clotting activity of the protein. The ability of thrombin mutant s to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astr ocyte stellation was also studied, The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enz ymatic activity. Furthermore the receptor occupancy by the inactive S205A m utant prevented the thrombin effects providing strong evidence that a prote olytically activated receptor is involved in cellular responses to thrombin . (C) 1999 Elsevier Science B.V. All rights reserved.