THROMBIN PROMOTION OF ISOMETRIC CONTRACTION IN FIBROBLASTS - ITS EXTRACELLULAR MECHANISM OF ACTION

Isometric force generated by fibroblasts plays an essential role in tissue contraction during normal wound healing and pathologic contractures. Thrombin, a serine protease present in all wounds, has been shown to promote wound healing. The purpose of this study was to determine the extracellular mechanism by which thrombin promotes isometric contraction by fibroblasts in an in vitro collagen lattice model of tissue contraction. The amount of isometric force generated by human fibroblasts can be measured directly with a stabilized collagen lattice attached to a force transducer. Thrombin promoted isometric contraction by human fibroblasts in a dose-dependent manner. In addition, thrombin-promoted isometric contraction is dependent on the enzymatic and anionic binding activity of thrombin, as demonstrated by inhibition with specific enzymatic and anionic binding inhibitors. These results suggest that thrombin may promote isometric contraction by fibroblasts through the enzymatic cleavage of its cell surface receptor, resulting in a ne iu amino terminus that serves as a ''tethered ligand'' to activate the receptor directly. To test this mechanism of action, a synthetic peptide (SFLLRN) representing the ''tethered ligand'' region of the activated thrombin receptor was synthesized and examined for its ability to promote isometric contraction by fibroblasts. This peptide promoted fibroblast contraction in a dose-dependent manner. In contrast, a control isomer peptide (FSLLRN), in which the two amino-terminal amino acids were reversed, failed to promote this response. These findings demonstrate that human alpha-thrombin promotes isometric contraction by human fibroblasts and that binding to and cleavage of its cell surface receptor are integral to this response.