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

Isometric force generated by fibroblasts plays an essential role in ti ssue contraction during normal wound healing and pathologic contractur es. Thrombin, a serine protease present in all wounds, has been shown to promote wound healing. The purpose of this study was to determine t he extracellular mechanism by which thrombin promotes isometric contra ction by fibroblasts in an in vitro collagen lattice model of tissue c ontraction. The amount of isometric force generated by human fibroblas ts can be measured directly with a stabilized collagen lattice attache d to a force transducer. Thrombin promoted isometric contraction by hu man fibroblasts in a dose-dependent manner. In addition, thrombin-prom oted isometric contraction is dependent on the enzymatic and anionic b inding activity of thrombin, as demonstrated by inhibition with specif ic enzymatic and anionic binding inhibitors. These results suggest tha t thrombin may promote isometric contraction by fibroblasts through th e enzymatic cleavage of its cell surface receptor, resulting in a ne i u amino terminus that serves as a ''tethered ligand'' to activate the receptor directly. To test this mechanism of action, a synthetic pepti de (SFLLRN) representing the ''tethered ligand'' region of the activat ed thrombin receptor was synthesized and examined for its ability to p romote isometric contraction by fibroblasts. This peptide promoted fib roblast 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 demonst rate that human alpha-thrombin promotes isometric contraction by human fibroblasts and that binding to and cleavage of its cell surface rece ptor are integral to this response.