A steady-state competition model describes the modulating effects of thrombomodulin on thrombin inhibition by plasminogen activator inhibitor-1 in the absence and presence of vitronectin.
Thrombomodulin (TM) slows down the interaction rate between thrombin and plasminogen activator inhibitor 1 (PAI-1). We now show that the 12-fold reduced inhibition rate in the presence of TM does not result from an altered distribution between PAI-1 cleavage and irreversible complex formation. Surface plasmon resonance (SPR) revealed an over 200-fold reduced affinity of TM for thrombin-VR1tPA as compared to thrombin, demonstrating the importance of the VR1 loop in the interaction of thrombin with both TM and PAI-1. Furthermore, in contrast to ATIII, PAI-1 was not able to bind the thrombin/TM complex demonstrating complete competitive binding between PAI-1 and TM. Kinetic modeling on the inhibitory effect of TM confirms a mechanism that involves complete steric blocking of the thrombin/PAI-1 interaction. Also, it accurately decribes the biphasic inhibition profile resulting from the substantial reduction of the extremely fast rate of reversible Michaelis complex formation, which is essential for efficient inhibition of thrombin by PAI-1. Vitronectin (VN) is shown to partially relieve TM inhibitory action only by vastly increasing the initial rate of interaction between free thrombin and PAI-1. In addition, SPR established that solution-phase PAI-1/VN complexes and non-native VN (extracellular matrix form) bind TM directly via the chondroitin sulphate moiety of TM. Collectively, these results show that VR1 is a subsite of exosite 1 on thrombin's surface, which regulates exclusive binding of either PAI-1 or TM. This competition will be physiologically significant in controlling the mitogenic activity of thrombin during vascular disease.
Dekker
Rob J
RJ
Department of Biochemistry, Academic Medical Center, University of Amsterdam, the Netherlands.
Pannekoek
Hans
H
Horrevoets
Anton J G
AJ
D000818:Animals; D001665:Binding Sites; D003198:Computer Simulation; D006801:Humans; D008954:Models, Biological; D017395:Plasminogen Activator Inhibitor 1 / Q000378:metabolism*; D011485:Protein Binding; D020349:Surface Plasmon Resonance; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D018180:Thrombomodulin / Q000378:metabolism*; D019096:Vitronectin / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D018180:Thrombomodulin; D019096:Vitronectin; D013917:Thrombin