The adhesive glycoprotein vitronectin (VN) forms a function-stabilizing complex with plasminogen activator inhibitor-1 (PAI-1), the major fibrinolysis inhibitor in both plasma and vessel wall connective tissue. VN also interacts with two-chain high molecular weight kininogen (HKa), particularly its His-Gly-Lys-rich domain 5, and both HKa and PAI-1 are antiadhesive factors that have been shown to compete for binding to VN. In this study the influence of HKa and domain 5 on the antifibrinolytic function of PAI-1 was investigated. In a purified system, HKa and particularly domain 5 inhibited the binding of PAI-1 to VN and promoted PAI-1 displacement from both isolated VN as well as subendothelial extracellular matrix-associated VN. The sequence Gly(486)-Lys(502) of HKa domain 5 was identified as responsible for this inhibition. Although having no direct effect on PAI-1 activity itself, HKa domain 5 or the peptide Gly(486)-Lys(502) markedly destabilized the VN.PAI-1 complex interaction, resulting in a significant reduction of PAI-1 inhibitory function on plasminogen activators, resembling the effect of VN antibodies that prevent stabilization of PAI-1. Furthermore, high affinity fibrin binding of PAI-1 in the presence of VN as well as the VN-dependent fibrin clot stabilization by the inhibitor were abrogated in the presence of the kininogen forms mentioned. Taken together, our data indicate that the peptide Gly(486)-Lys(502) derived from domain 5 of HKa serves to interfere with PAI-1 function. Based on these observations potential low molecular weight PAI-1 inhibitors could be designed for the use in therapeutic interventions against thromboembolic complications.