Alteration of serpin specificity by a protein cofactor. Vitronectin endows plasminogen activator inhibitor 1 with thrombin inhibitory properties.
Serine protease inhibitors ("serpins") are highly homologous proteins which inhibit selected "target" serine proteases by acting as a pseudo-substrate. Their specificity is primarily determined by the amino acid sequence around the carboxyl-terminally located reactive center (P1-P1'). In addition, the association rate constant between a serpin and a serine protease can be dramatically increased by non-protein cofactors, such as heparin in the case of thrombin inhibition by antithrombin III. In an attempt to alter the specificity of PAI-1 from an inhibitor of the fibrinolytic system to an inhibitor of coagulation, we replaced P1-P1' or P3 through P3' of the reactive center of PAI-1 by the corresponding residues of antithrombin III and assessed whether the mutant proteins, purified from lysates of transformed Escherichia coli cells, had acquired thrombin inhibitory properties. The experiments were performed in the presence and absence of vitronectin, a multifunctional protein which has been shown to bind PAI-1 in plasma and in the matrix of endothelial cells. The second-order rate constants for t-PA inhibition of "wild-type" PAI-1 and PAI P1-P1'ATIII, irrespective of the presence of vitronectin, were similar, whereas replacing P3-P3' resulted in a 40-fold decrease of the second-order rate constant towards t-PA, again independent of vitronectin. In the absence of vitronectin, reactivity of PAI-1 and its "antithrombin III-like" variants towards thrombin was slow; however, PAI-1 P3-P3' ATIII had a 10-fold higher k1 than wild-type PAI-1 (1.3 x 10(4) M-1 s-1 versus 1.1 x 10(3) M-1 s-1). In contrast, in the presence of vitronectin, PAI-1 and even more rapidly PAI-1 P3-P3'ATIII were found to be effective thrombin inhibitors, with k1 values of 2.2 x 10(5) M-1s-1 and 1.8 x 10(6) M-1 s-1, respectively. Thus, in the presence of vitronectin, PAI-1 P3-P3'ATIII displays a 3-fold higher k1 with thrombin than with t-PA. It is shown that vitronectin enhances, in a dose-dependent manner, the formation of sodium dodecyl sulfate-resistant complexes between PAI-1 or mutants thereof and thrombin. Therefore, vitronectin is the first protein described to function as a cofactor for serpin specificity. PAI-1 is proposed to be a versatile inhibitor which, in the presence of vitronectin, can modulate both coagulation and fibrinolysis.
Ehrlich
H J
HJ
Department of Molecular Biology, Central Laboratory of The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Gebbink
R K
RK
Keijer
J
J
Linders
M
M
Preissner
K T
KT
Pannekoek
H
H
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006023:Glycoproteins / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D015849:Plasminogen Inactivators / Q000494:pharmacology*; D011994:Recombinant Proteins / Q000494:pharmacology; D015843:Serpins / Q000235:genetics / Q000494:pharmacology*; D013917:Thrombin / Q000037:antagonists & inhibitors*; D019096:Vitronectin
D006023:Glycoproteins; D015345:Oligonucleotide Probes; D015849:Plasminogen Inactivators; D011994:Recombinant Proteins; D015843:Serpins; D019096:Vitronectin; D000990:Antithrombin III; D013917:Thrombin