Regulation of neurite outgrowth from differentiated human neuroepithelial cells: A comparison of the activities of prothrombin and thrombin

The mechanism by which thrombin and prothrombin control neurite retraction was studied in Ad12E1HER10 human neuroepithelial cells. Morphological changes in differentiated cells were apparent within minutes of the addition of very low concentrations of thrombin (3 pM). Higher concentrations (2 nM) of prothrombin were required to elicit a similar response. Doses of thrombin and prothrombin sufficient to cause neurite retraction stimulated protein tyrosine kinase activity. Protein tyrosine kinase activation also correlated positively with thrombin- and prothrombin-induced phosphoinositide 3-kinase activation and InsP₆ dephosphorylation. However, thrombin-stimulated Ins(1,4,5)P₃ generation and intracellular Ca²⁺ mobilization only occurred at concentrations in excess of those needed to induce retraction. No fluctuations in Ins(1,4,5)P₃ were detected after stimulation with prothrombin, and no rapid synchronized release of Ca²⁺ was observed, even at very high concentrations. Prothrombin did, however, cause small oscillations in the intracellular Ca²⁺ concentration, similar to those produced by low concentrations of thrombin, after ~ 30 min. We conclude that prothrombin- and thrombin-induced neurite retractions are not dependent on PtdIns(4,5)P₃ and Ca²⁺ mobilization, but are more probably mediated through an effector mechanism involving protein tyrosine kinase activation. No intracellular Ca²⁺ mobilization, protein tyrosine kinase activity or neurite retraction was observed after treatment of cells with proteolytically inactive mutant thrombin (S205 → A). Prothrombin-mediated intracellular Ca²⁺ mobilization and neurite retraction were inhibited by hirudin, which was shown to interact with thrombin but not prothrombin. It is concluded that cleavage of prothrombin to thrombin is a necessary prerequisite for biological activity on differentiated Ad12E1HER10 cells and that differences in agonist concentration are capable of coupling the thrombin receptor to different pathways within the cell.