van Meijer M, Stoop A, Smilde A, Preissner K T, van Zonneveld A J, Pannekoek H
Department of Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
Thromb Haemost. 1997 Mar;77(3):516-21.
Vitronectin (VN) is an obligatory cofactor for the inhibition of thrombin by plasminogen activator inhibitor 1 (PAI-1). It accelerates the rate of association between thrombin and PAI-1 more than two orders of magnitude. In contrast, VN does not accelerate the association between tissue-type plasminogen activator (t-PA) and PAI-1. Previously, we reported that the anti-PAI-1 monoclonal antibody (MoAb) CLB-2C8 binds to a short stretch of amino acids of PAI-1, located between residues 128 and 145, and prevents PAI-1 binding to VN. Furthermore, MoAb CLB-2C8 fully blocks the inhibitory activity of PAI-1 towards t-PA, emphasizing the importance of this area for the interaction with t-PA. Here, we show that this area is also required for the interaction between thrombin and PAI-1, since MoAb CLB-2C8 fully prevents inhibition of thrombin by PAI-1. In spite of similar structural requirements for the interaction between t-PA, PAI-1 and VN and between thrombin, PAI-1 and VN, the intermediate reaction products are clearly distinct. By employing surface plasmon resonance (SPR), using the BIAcore equipment, and by immunoprecipitation we demonstrate that, in the presence of VN, t-PA and PAI-1 form exclusively equimolar binary t-PA/PAI-1 complexes. Thrombin, PAI-1 and VN generate equimolar, binary thrombin/PAI-1 complexes and in addition equimolar, ternary complexes and multimers.
纤连蛋白（VN）是纤溶酶原激活物抑制剂1（PAI - 1）抑制凝血酶的必需辅因子。它将凝血酶与PAI - 1之间的结合速率提高了两个多数量级。相比之下，VN不会加速组织型纤溶酶原激活物（t - PA）与PAI - 1之间的结合。此前，我们报道抗PAI - 1单克隆抗体（MoAb）CLB - 2C8与PAI - 1位于第128至145位残基之间的一小段氨基酸结合，并阻止PAI - 1与VN结合。此外，MoAb CLB - 2C8完全阻断了PAI - 1对t - PA的抑制活性，强调了该区域对于与t - PA相互作用的重要性。在此，我们表明该区域对于凝血酶与PAI - 1之间的相互作用也是必需的，因为MoAb CLB - 2C8完全阻止了PAI - 1对凝血酶的抑制。尽管t - PA、PAI - 1与VN之间以及凝血酶、PAI - 1与VN之间的相互作用有相似的结构要求，但中间反应产物明显不同。通过使用BIAcore设备进行表面等离子体共振（SPR）以及免疫沉淀，我们证明，在VN存在的情况下，t - PA和PAI - 1仅形成等摩尔的二元t - PA/PAI - 1复合物。凝血酶、PAI - 1和VN生成等摩尔的二元凝血酶/PAI - 1复合物，此外还生成等摩尔的三元复合物和多聚体。
