Assembly of the Prothrombinase Complex on Human Fibroblast Surface Is Enhanced by TSP1 and Leads to CTGF Upregulation by a Different Mechanism from the One Used by FXa and Thrombin.

Introduction: There is evidence in the literature indicating that activated factor X (FXa), thrombin and the complex of tissue factor (TF)-activated factor VII (VIIa) can induce upregulation of gene expression of connective tissue growth factor (CTGF) on human fibroblasts [

Objectives: We hypothesized that human fibroblasts due to their ability to express constitutively TF on their cell surface may be able to assemble the prothrombinase complex leading to cell signaling associated with upregulation of CTGF through the TGFβ pathway.

Methods: Cultured human HS-68 fibroblasts, between four to ten passages, were incubated systematically with components of the prothrombinase complex, namely purified FVII (5nM), factor X (5nM), Factor V (45nM) prothrombin (1.4μM) and Ca⁺⁺ (2mM). Generation of FXa and thrombin were measured with specific chromogenic substrates for FXa and thrombin. In addition, the reactions were assessed in the presence and absence of exogenous purified TSP1 (20nM). The signaling pathway potentially triggered by serine proteases generated (FXa and thrombin) were evaluated by the use of Western-blotting Analysis with specific antibodies directed to phosphorylated transcription factors.

Results: Pre-incubation of FVII with cultured fibroblasts lead to its activation by cell-surface expressed TF, which in turn in the presence of FX, FV, FII (prothrombin) and Ca⁺⁺ lead to the generation of FXa (9.7 ± 0.77 nM) and thrombin (7.99 ± 0.04 U/mL x10⁻³) as judged by efficient hydrolysis of specific chromogenic substrates. Addition of activated FV (FVa) but not FX after pre-incubation of FVII with cultured fibroblasts (under these experimental conditions) was an absolute requirement for the reactions to take place. Addition of TSP1 significantly enhanced thrombin generation (23.3 ± 0.69 U/mL x10⁻³ p<0.05) but not FXa generation (8.47 ± 0.62 nM), an interesting phenomenon currently under investigation in our laboratory. FXa and thrombin generation lead to upregulation of CTGF an effect mediated by a different pathway used by transforming growth factor (TGFβ). TSP1 alone was capable to up-regulate CTGF, an effect mediated via the TGFβ pathway as judged by phosphorylation of the SMAD pathway and blocked by a specific inhibitor directed to TGFβ-II cell surface receptor.

In summary, TF expressed by normal human fibroblasts activates FVII and subsequently FX in the presence of FVa, prothrombin and Ca⁺⁺. Generation of FXa and thrombin is associated with a downstream effect leading to upregulation of CTGF, independently of the TGFβ pathway. TSP1 not only promotes thrombin generation on the surface of human fibroblast but alone can induce CTGF up-regulation via the TGFβ pathway. This mechanism may play an important role in human diseases associated with fibrosis and with atherosclerosis.