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 correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-...

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Published in:	Biochimica et biophysica acta Vol. 1451; no. 1; pp. 173 - 186
Main Authors:	Arcone, Rosaria, Pagliuca, M.Gabriella, Chinali, Alberto, Grimaldi, Maurizio, Schettini, Gennaro, Gast, Alain, Pietropaolo, Concetta
Format:	Journal Article
Language:	English
Published:	Netherlands Elsevier B.V 12-08-1999
Subjects:	3T3 Cells Animals Astrocyte stellation Astrocytes - drug effects Astrocytes - metabolism Astrocytes - physiology Blood Coagulation - drug effects Bucladesine - pharmacology Cell Differentiation Cell proliferation Cells, Cultured Chromogenic Compounds - metabolism Dipeptides - metabolism DNA, Complementary - genetics Endopeptidases - pharmacology Enzyme Activation - drug effects Enzyme Precursors - biosynthesis Enzyme Precursors - genetics Enzyme Precursors - pharmacology Methotrexate resistant cell Mice Microscopy, Phase-Contrast Mitogens - genetics Mitogens - metabolism Mitogens - pharmacology Point Mutation Protease-activated receptor expression Prothrombin - biosynthesis Prothrombin - genetics Prothrombin - pharmacology Rats Rats, Wistar Receptors, Thrombin - drug effects Reverse Transcriptase Polymerase Chain Reaction Thrombin - genetics Thrombin - metabolism Thrombin - pharmacology Thrombin mutant Thrombin receptor Cell proliferation Methotrexate resistant cell Astrocyte stellation Thrombin receptor Protease-activated receptor expression Thrombin mutant
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Summary:	We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. 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 activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.
ISSN:	0167-4889 0006-3002 1879-2596
DOI:	10.1016/S0167-4889(99)00086-5