The effects of the modulation of the fibronectin-binding capacity of fibrin by thrombin on osteoblast differentiation.

Fibrin is a natural provisional matrix involved in wound healing and is widely utilized for tissue regeneration. The biological performance of fibrin is largely dependent on its composition and related structures. In this study, we examined the effect of thrombin, which is engaged with fibrin, on osteoblast differentiation and its mode of action. Fibrin matrices were prepared with different concentrations of thrombin, and MC3T3-E1 pre-osteoblasts were cultured on the fibrin matrices. Thrombin-promoted fibrin-enhanced osteoblast differentiation in a dose-dependent manner, as confirmed by the extent of calcium deposition, alkaline phosphatase activity, and the level of Runx2. The synthetic activating peptide of protease-activated receptor 1 (PAR1), a prototype receptor of thrombin in osteoblast, did not alter the level of Runx2. Instead, the thrombin that was engaged with fibrin in a dose-dependent manner increased the phosphorylation of integrins β1 and β3. The integrin-blocking peptide RGDS reduced the thrombin-enhanced Runx2 in the cells grown on fibrin, whereas the non-functional peptide RGES did not change the level of Runx2. Furthermore, thrombin dose-dependently increased the fibronectin-binding of fibrin. The thrombin-induced integrin phosphorylation and Runx2 expression were also attenuated through the use of a blocking peptide to inhibit the binding of fibronectin to fibrin. The results in this study provide evidence that thrombin engaged with fibrin accelerates osteoblast differentiation via integrins but not PAR1 by modulating the fibronectin-binding capacity of fibrin.