Vitronectin governs the interaction between plasminogen activator inhibitor 1 and tissue-type plasminogen activator.
The "serpin" plasminogen activator inhibitor 1 (PAI-1) is the fast acting inhibitor of plasminogen activators (tissue-type (t-PA) and urokinase type-PA) and is an essential regulatory protein of the fibrinolytic system. Its P1-P1' reactive center (R346 M347) acts as a "bait" for tight binding to t-PA/urokinase-type PA. In vivo, PAI-1 is encountered in complex with vitronectin, an interaction known to stabilize its activity but not to affect the second-order association rate constant (k1) between PAI-1 and t-PA. Nevertheless, by using PAI-1 reactive site variants (R346M, M347S, and R346M M347S), we show that the binding of vitronectin to the PAI-1 mutant proteins improves plasminogen activator inhibition. In the absence of vitronectin the PAI-1 R346M mutants are virtually inactive toward t-PA (k1 less than 1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin the rate of association increases about 1,000-fold (k1 of 6-8 x 10(5) M-1 s-1). This inhibition coincides with the formation of serpin-typical, sodium dodecyl sulfide-stable t-PA.PAI-1 R346M (R346M M347S) complexes. As evidenced by amino acid sequence analysis, the newly created M346-M/S347 peptide bond is susceptible to attack by t-PA, similar to the wild-type R346-M347 peptide bond, indicating that in the presence of vitronectin M346 functions as an efficient P1 residue. In addition, we show that the inhibition of t-PA and urokinase-type PA by PAI-1 mutant proteins is accelerated by the presence of the nonprotease A chains of the plasminogen activators.
Keijer
J
J
Department of Molecular Biology, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Ehrlich
H J
HJ
Linders
M
M
Preissner
K T
KT
Pannekoek
H
H
D001483:Base Sequence; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D005337:Fibrin / Q000378:metabolism; D005786:Gene Expression Regulation; D006023:Glycoproteins / Q000502:physiology*; D006493:Heparin / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015849:Plasminogen Inactivators / Q000378:metabolism*; D010959:Tissue Plasminogen Activator / Q000378:metabolism*; D019096:Vitronectin
D006023:Glycoproteins; D015849:Plasminogen Inactivators; D019096:Vitronectin; D005337:Fibrin; D006493:Heparin; D010959:Tissue Plasminogen Activator