Thrombin is a potent mitogenic agent. Clot‐associated thrombin retains its amidolytic and pro‐aggregant activity. We therefore studied the ability of fibrin clots to induce proliferation in CCL39 cells (Chinese hamster lung fibroblasts), in the absence and presence of the thrombin inhibitors PPACK, recombinant hirudin (rHV2 Lys47) and heparin:antithrombin III. Fibrin clots incubated for 48 h with CCL39 cells led to significant cell proliferation, which was dependent on the concentration of thrombin used to prepare the clots. Thus, clots prepared with 91 nmol l−1 thrombin produced a similar proliferation (231±21%) to that obtained with 50 nmol l−1 thrombin in solution (213±29%). Rabbit plasma clots led to a 499±41% increase in cell number under identical conditions. Fibrin clot‐induced cell proliferation was inhibited by all three thrombin inhibitors with no difference in IC50 values compared to those obtained against thrombin in solution, suggesting that cell proliferation be due to thrombin leaching from the clots. We found a time‐dependent increase in thrombin release from the clots attaining a plateau at 24 h (∼61% of the total thrombin used in clot formation). Clots separated from the cells using porous cell culture chamber inserts led to similar proliferation to that of clots in contact with the cells. Thus fibrin‐clot induced CCL39 proliferation is due to thrombin released from the clots. British Journal of Pharmacology (2000) 129, 1021–1027; doi:

SummaryThe activation of rabbit platelets by rabbit plasma clots, and the inhibition of clot-associated thrombin by heparin:antithrombin III, recombinant hirudin (rHV2Lys47) and argatroban, a low molecular weight thrombin inhibitor, was studied.Plasma clots caused the aggregation of platelets suspended in a plasma-free medium as assessed by single platelet counting, and by scanning electron microscopy (platelet aggregates present on the clot surface). Platelet aggregation, induced by clot-associated thrombin, was inhibited by argatroban with an IC50 of 14 ± 3 nM compared to an IC50 of 12 ± 2 nM when human thrombin in solution titrated to give the same decrease in the platelet count as plasma clots was used. rHV2Lys47 also inhibited aggregation induced by clot-associated thrombin with an IC50 of 1.6 ± 0.4 nM compared to 1.6 ± 0.5 nM with thrombin in solution. Heparin was less active against clot-associated thrombin (IC50 = 69 ± 9 mU/ml) than against thrombin in solution (IC50 = 15 ± 5 mU/ml).This study shows that plasma clot-bound thrombin activates platelets and that direct-acting thrombin inhibitors such as argatroban and rHV2Lys47 are more effective than heparin:antithrombin III in inhibiting this phenomenon.