Interaction of plasminogen activator inhibitor type-1 (PAI-1) with vitronectin.
The serpin plasminogen activator inhibitor type 1 (PAI-1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue-type (tPA) and urokinase-type (uPA) plasminogen activators, PAI-1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI-1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn-binding site of PAI-1 appear to reside between amino acids 110-147, which includes alpha helix E (hE, amino acids 109-118). Ten different PAI-1 variants (mostly harboring modifications in hE) as well as wild-type PAI-1, the previously described PAI-1 mutant Q123K, and another serpin, PAI-2, were recombinantly produced in Escherichia coli containing a His(6) tag and purified by affinity chromatography. As shown in microtiter plate-based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114-118, in which all amino-acids at positions 114-118 of PAI-1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild-type PAI-1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI-1 mutants, the inhibitory properties of A114-118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild-type sequence of the region around hE in PAI-1 is not a prerequisite for binding to Vn.
Arroyo De Prada
Nuria
N
Klinische Forschergruppe der Frauenklinik der Technischen Universität München, Klinikum rechts der Isar, German.
Schroeck
Florian
F
Sinner
Eva-Kathrin
EK
Muehlenweg
Bernd
B
Twellmeyer
Jens
J
Sperl
Stefan
S
Wilhelm
Olaf G
OG
Schmitt
Manfred
M
Magdolen
Viktor
V
D006493:Heparin / Q000378:metabolism; D017395:Plasminogen Activator Inhibitor 1 / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D017396:Plasminogen Activator Inhibitor 2 / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D013917:Thrombin / Q000494:pharmacology; D014568:Urokinase-Type Plasminogen Activator / Q000037:antagonists & inhibitors; D019096:Vitronectin / Q000378:metabolism*
D017395:Plasminogen Activator Inhibitor 1; D017396:Plasminogen Activator Inhibitor 2; D011994:Recombinant Proteins; D019096:Vitronectin; D006493:Heparin; D013917:Thrombin; D014568:Urokinase-Type Plasminogen Activator