KINETIC-ANALYSIS OF INTEGRIN-DEPENDENT CELL-ADHESION ON VITRONECTIN -THE INHIBITORY POTENTIAL OF PLASMINOGEN-ACTIVATOR INHIBITOR-1 AND RGDPEPTIDES

Distinct binding interactions between cell-surface receptors and extra cellular matrix components are characteristic of multifunctional adhes ion proteins such as vitronectin. The close proximity of binding sites for alpha(v)-integrins and plasminogen activator inhibitor-1 (PAI-1) on vitronectin may have consequences for cell adhesion and migration, or for the localized inhibition of plasminogen activators. In this stu dy, the kinetics and reversibility of vitronectin-dependent cell adhes ion via alpha(v)-integrins was investigated using RGD peptides and PAI -1 as competitors. Active, but not latent or cleaved PAI-1, and RGD pe ptides were effective in preventing cell adhesion to vitronectin provi ded the inhibitor was present at the time of cell seeding. In a concen tration-dependent manner urokinase or thrombin abrogated the inhibitor y effect of PAI-1. Following cell seeding onto a vitronectin substratu m, delayed addition of RGD peptides or active PAI-1 (10-20 min post-se eding) resulted in the loss of their inhibitory potential. These data were supported by experiments in a purified system where delayed addit ion of active PAI-1 could no longer prevent vitronectin binding to imm obilized alpha(v) beta(3), while a cyclic RGD peptide gave some modera te inhibition. The apparent stabilization of vitronectin-integrin cont acts was observed with immobilized native or multimeric vitronectin bu t not with the more rigid form of denatured, aggregated multimers. The se results demonstrate that the cell adhesive properties of vitronecti n depend on its conformational flexibility and can be tightly regulate d in a spatio-temporal manner through direct competition of cellular i ntegrins by soluble or matrix-bound factors such as PAI-1.