Thrombin promotes aortic endothelial cell spreading and microfilament formation in nonconfluent monolayer cultures

Thrombin is present at sites of vascular injury. The objective of this study was to determine if thrombin may regulate endothelial repair. To address this our study was designed to determine the effect of thrombin on cell shape and microfilament distribution of porcine aortic endothelial cells in low-density cultures. Since these cells grow as islands of cells, low-density cultures serve as a model for conditions in which reendothelialization occurs from several foci following prominent patchy endothelial denudation. Thrombin incubation for 1, 2, and 4 hr at 0.5, 4, and 8 U/ml caused a significant increase in both the surface area of endothelial cells and the amount of microfilament bundles occupying the cell surface area. The increase in microfilament bundles was more than would be expected from the increased surface area. However, the pattern of distribution of microfilaments and of vinculin adhesion plaques within the cells was not altered by thrombin. Attempts at understanding the molecular mechanisms involved were undertaken. The role of protein kinase C was studied. Phorbol 12-myristate 13-acetate (PMA) caused cell spreading as well but no increase in microfilaments within the first 2 hr of incubation. Some cells showed mild disruption of microfilaments. Following 24 hr incubation with PMA, which reduces protein kinase C, the cells showed retraction and arborization along with thinning and disruption of microfilaments. Removal of PMA and addition of thrombin reversed these changes to normal within 2 hr while washout of PMA on its own did not alter cell shape or microfilaments. Thrombin also partially reversed the effects of H-7, a protein kinase C inhibitor, which on its own also caused cell retraction and disruption of microfilaments. Neomycin sulfate did not alter the thrombin effect suggesting that the participation of the phosphotidylinositol system is not directly required. In conclusion, thrombin promotes endothelial activities which are associated with long-term endothelial repair; however, these effects do not appear to be directly due to the involvement of conventional isoforms of protein kinase C or the phosphotidylinositol system.