Role of clot-associated (-derived) thrombin in cell proliferation induced by fibrin clots in vitro

Abstract

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 IC(50) 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 (approximately 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.

Figure 1

Effects of fibrin clot-associated thrombin on the proliferation of CCL39 cells. Fibrin clots were prepared using different thrombin concentrations, and were incubated with cells for 48 h. Cells incubated with 50 nM thrombin (lla) in solution and clots prepared from rabbit plasma or reptilase are shown for comparison. The results show the mean±s.e.mean per cent proliferation compared to cells incubated alone from three (reptilase clots) five (fibrin clots) or nine (plasma clots) separate experiments.

Figure 2

Effects of thrombin inhibitors on the proliferation of CCL39 cells induced by fibrin clots prepared using 91 nM thrombin and induced by 50 nM thrombin in solution. The results are expressed as the mean±s.e.mean per cent inhibition by rHV2 Lys 47 (A, n=8–10), PPACK (B, n=4) and heparin in the presence of 20 nM antithrombin lll (C, n=5).

Figure 3

CCL39 cell proliferation induced by fibrin clots or thrombin in solution incubated with the cells for different times throughout the 48 h proliferation period, after which times the clots were removed and the medium changed (thrombin in solution) and cell proliferation was estimated at 48 h. The results are expressed as the mean±s.e.mean per cent proliferation from three (0.5 h) or five separate experiments.

Figure 4

Time course of the amidolytic activity of thrombin on the clot surface and released into the supernatant. Fibrin clots were incubated for different periods and the amidolytic activity of the incubation media and the clots were determined using a synthetic chromogenic substrate (S-2238). The results are expressed as the mean±s.e.mean pmoles of thrombin determined using a thrombin standard curve in each experiment (n=3 (5 and 15 min) or five determinations). The figures in parentheses denote the per cent thrombin release into the medium based on the amount of thrombin used to prepare the clots (22.85 picomoles).