Thrombospondin-1 (TSP1) Promotes Thrombin Generation on the Surface of Fibroblasts (HS-68) and Induces Up-Regulation of Connective Tissue Growth Factor (CTGF) Gene and Protein Expression.

Abstract

TSP1 has the ability to bind to human fibroblasts, to form a complex with coagulation factor V/Va (Thrombosis Research 116:533, 2005), to promote thrombin generation on the surface of a monocytic cell line and to neutralize tissue factor pathway inhibitor (TFPI) (J Biol Chem 275:31715, 2000). Disruption of TSP1 binding to neutrophils was associated with beneficial effects in an experimental animal model of inflammation, in part, by down regulating CTGF gene and protein expression (Arthritis Rheum 54:2415, 2006). CTGF is a novel potent cysteine-rich heparin-binding growth factor and is highly expressed by fibroblasts. CTGF plays a major role in angiogenesis and fibrosis. There is also growing evidence that CTGF may be the downstream autocrine mediator responsible for some of the cellular effects of TGF-beta. Since fibroblasts express tissue factor (TF) on their surface, and purified thrombin and TF-VIIa complex have been shown to up-regulate the gene expression of CTGF (J Biol Chem 275:14632, 2000) experiments were conducted to evaluate the ability of HS-68 to support assembly of the prothrombinase complex, TF-FVIIa, thrombin generation and the effect of thrombin generation on CTGF expression. The role of TSP1 in these reactions was assessed as well. Thrombin generation was measured by the chromogenic substrate S-2238. Although the initial rates of the reactions are available we are presenting the end-point values of the reaction expressed in umol/L of pNA released per minute. All reaction mixtures were performed in the presence of 2mM Ca++. When HS-68 cells were preincubated with FVII (5 nM) prior to the addition of activated factor V (FVa, 45nM)), FX (5nM) and prothrombin (FII, 1.4 uM), thrombin was efficiently generated (282 umol/L pNA/min), indicating that FVII was activated by TF expressed by the cell and that the HS-68 cell membrane provided an ideal surface for the reaction to occur. The addition of FII, FV, FVII and FX to the reaction mixtures was an absolute requirement. When the reaction mixture was evaluated in the presence of FII, FV, FVII, FX and TFPI (8nM), there was a 70% reduction in thrombin production (86 umol/L pNA released) confirming the important role of TFPI in regulating the activity of the TF-FVIIa complex. The addition of TSP1 to the reaction mixture containing FII, FV, FVII and FX at concentrations found in plasma during the inflammatory response (20nM) enhanced the production of thrombin (327 umol/L pNA released per min) and neutralized the inhibitory effect of TFPI by 50% (171 umol/L pNA released per min). Therefore, TSP1 promotes thrombin generation by participating in the assembly of the prothrombinase complex on the surface of HS-68 cells and by neutralizing, in part, the inhibitory effect of TFPI on TF-VIIa complex. Finally, thrombin generation on the surface of HS-68 cells was associated with up-regulation of CTGF gene expression from the baseline value by 67% at 1hr and 72% by 2 hrs. In summary, we have identified on human fibroblasts a pathway previously shown to play an important role on human neutrophils and in an experimental model of inflammation. Our laboratory is currently characterizing the binding of TSP1 to this cell line and silencing the gene for TSP1 to test its potential therapeutic benefit in an experimental model of erosive arthritis and to further determine the role of TSP1 in this pathway.