The composition of complexes between plasminogen activator inhibitor 1, vitronectin and either thrombin or tissue-type plasminogen activator.
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.
van Meijer
M
M
Department of Biochemistry, Academic Medical Center, University of Amsterdam, The Netherlands.
Stoop
A
A
Smilde
A
A
Preissner
K T
KT
van Zonneveld
A J
AJ
Pannekoek
H
H
D000911:Antibodies, Monoclonal; D006801:Humans; D007700:Kinetics; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011233:Precipitin Tests; D013917:Thrombin / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D019096:Vitronectin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D017395:Plasminogen Activator Inhibitor 1; D019096:Vitronectin; D013917:Thrombin; D010959:Tissue Plasminogen Activator