HUMAN UMBILICAL VEIN SMOOTH-MUSCLE CELLS AS A MODEL TO STUDY THROMBINGENERATION AND FUNCTION - EFFECT OF THROMBIN INHIBITORS

The aim of the present work was to study how human umbilical vein smoo th muscle cells (HWSMC) can initiate the coagulation process and to in vestigate the responses of these cells to thrombin. Exposure of HUVSMC to recalcified human plasma led to a time-dependent production of thr ombin, measured both as amidolytic activity and as release of fibrinop eptide A. Thrombin activity was dose-dependently reduced by an anti-hu man tissue factor antibody (76 +/- 3% at 10 mu g/ml) and by inhibitors like heparin, rec-hirudin, hirulog-1, Napap and hirunorm, a novel hir udin-like thrombin inhibitor (IC50 2 +/- 0.4, 8 +/- 1, 130 +/- 22, 199 +/- 29 and 68 +/- 8 nM, respectively). The release of fibrinopeptide A was similarly prevented (IC50 = 14 +/- 1, 132 +/- 25 and 50 +/- 8 nM for rec-hirudin, Napap and hirunorm, respectively). Exogenously added thrombin increased thymidine incorporation into HUVSMC to 240 +/- 30% of basal (EC(50) = 0.49 +/- 0.09 nM) and thrombin inhibitors blocked this effect (IC50 = 10 +/- 3, 37 +/- 17, 343 +/- 165 and 1402 +/- 758 nM for rec-hirudin, hirunorm, Napap and hirulog-1, respectively). Also recalcified human plasma was mitogenic for HWSMC and its effect was m ainly due to endogenously generated thrombin, as shown by the use of t hrombin inhibitors. In conclusion, HWSMC are capable of initiating the extrinsic coagulation cascade, leading to the formation of thrombin w hich promotes clotting and stimulates DNA synthesis. Thrombin inhibito rs prevent both coagulative and cellular effects of thrombin.