Assembly of the prothrombinase complex on fibroblast surface, promoted by TSP1, results in cytokine release and CTGF upregulation

Abstract

Activated factor X (FXa) and thrombin can upregulate the gene expression of connective tissue growth factor (CTGF) on fibroblasts. Since TF is expressed on fibroblasts, we hypothesized that fibroblasts may assemble the prothrombinase complex leading to CTGF upregulation. In addition, the effect of thrombospondin‐1 (TSP1) was evaluated. HS‐68 fibroblasts were incubated with purified FVII, FX, FV, FII and Ca++ in the presence and absence of TSP1 and generation of FXa and thrombin assessed with chromogenic substrates. SMAD pathway phosphorylation was detected via Western‐blot analysis. Pre‐incubation of FVII with fibroblasts lead to its activation by cell‐surface expressed TF, which in turn in the presence of FX, FV, FII and Ca++ lead to the generation of FXa (9.7 ± 0.8 nM) and thrombin (7.9 ± 0.04 U/mL x10‐3). Addition of TSP1 significantly enhanced thrombin generation (23.3 ± 0.7 U/mL x10‐3 p<0.05) but not FXa generation (8.5 ± 0.6 nM). FXa and thrombin generation lead to upregulation of CTGF. Interestingly TSP1 alone upregulated CTGF, TNFα and IL‐6. The upregulation of CTGF and IL‐6 was mediated via the TGFβ pathway as judged by phosphorylation of the SMAD pathway, which was blocked by a TGFβI receptor antagonist. FXa and thrombin‐ induced upregulation of CTGF was not blocked by TGFβI receptor antagonist. This mechanism may play an important role in human diseases associated with fibrosis and with atherosclerosis.