Abstract
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.
Highlights
After vascular injury, pathological thrombosis is the critical event leading to acute myocardial infarction, unstable angina pectoris, or stroke [1]
plasminogen activator inhibitor-1 (PAI-1) is a central regulator of the fibrinolytic system, and increased levels of the inhibitor result in reduced fibrinolytic activity associated with a prothrombotic state
Because VN has a major impact on stabilizing the activity of PAI-1 [13,14,15,16], any disturbance of PAI-11⁄7VN complexes may lead to a rapid decay of the inhibitor associated with a decrease of antifibrinolytic function
Summary
Pathological thrombosis is the critical event leading to acute myocardial infarction, unstable angina pectoris, or stroke [1]. An elevated PAI-1 level constitutes an important thrombotic risk factor for, e.g. myocardial infarction [7, 8] or deep venous thrombosis because of an overall increased antifibrinolytic potential [9]. PAI-1 serves as an important antiadhesive factor for VN-dependent cell adhesion reactions involving either integrins or the urokinase receptor [21, 22]. Similar to PAI-1, the six domain-containing high molecular weight kininogen (HK), and especially the kinin-free two-chain form (HKa) [23], binds to VN and serves as an additional antiadhesive protein for both integrin- and urokinase receptordependent cellular interactions [24]. Our results indicate that HKa, domain 5, and especially a particular peptide Gly486-Lys502 can inhibit the function of PAI-1 as a primary inhibitor of fibrinolysis thereby providing a novel plausible mechanism for the recently described antithrombotic properties of kininogen. Based on the sequence Gly486Lys502 within domain 5 of HKa, potential low molecular weight
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