Plasminogen activator inhibitor type‐1 : reactive center and amino‐terminal heterogeneity determined by protein and cDNA sequencing

Both the urokinase‐type and tissue‐type plasminogen activator can convert their 5̃4 kDa type‐1 inhibitor (PAI‐1) to an inactive form with a lower apparent molecular mass. We have determined the amino‐terminal amino acid sequences of human native and converted PAI‐1, and isolated PAI‐1 cDNA and determined the nucleotide sequence in regions corresponding to the amino‐terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg‐Met bond 33 residues from the carboxy‐terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino‐terminus, with a Ser‐Ala‐Val‐His‐His form and a two‐residue shorter form (Val‐His‐His‐) occurring in approximately equal quantities.

[1]  F. Sanger,et al.  DNA sequencing with chain-terminating inhibitors. , 1977, Proceedings of the National Academy of Sciences of the United States of America.

[2]  R. Stephens,et al.  Minactivin: a human monocyte product which specifically inactivates urokinase-type plasminogen activators. , 1983, European Journal of Biochemistry.

[3]  A. Berger,et al.  On the size of the active site in proteases. I. Papain. , 1967, Biochemical and biophysical research communications.

[4]  V. V. van Hinsbergh,et al.  Evidence for the presence of two different fibrinolytic inhibitors in human endothelial cell conditioned medium. , 1984, Biochimica et biophysica acta.

[5]  K. Robbins,et al.  The peptide chains of human plasmin. Mechanism of activation of human plasminogen to plasmin. , 1967, The Journal of biological chemistry.

[6]  D. Loskutoff,et al.  Cultured granulosa cells produce two plasminogen activators and an antiactivator, each regulated differently by gonadotropins. , 1985, Endocrinology.

[7]  Gunnar von Heijne,et al.  How signal sequences maintain cleavage specificity. , 1984 .

[8]  K. Danø,et al.  Plasminogen activators, tissue degradation, and cancer. , 1985, Advances in cancer research.

[9]  H. Jörnvall,et al.  The site in human antithrombin for functional proteolytic cleavage by human thrombin , 1981, FEBS letters.

[10]  J. Messing,et al.  Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. , 1983, Gene.

[11]  D. Loskutoff,et al.  The Dexamethasone-Induced Inhibitor of Plasminogen Activator in Hepatoma Cells Is Antigenically-Related to an Inhibitor Produced by Bovine Aortic Endothelial Cells , 1986, Thrombosis and Haemostasis.

[12]  H. Okayama,et al.  A cDNA cloning vector that permits expression of cDNA inserts in mammalian cells. , 1983, Molecular and cellular biology.

[13]  R. Lawn,et al.  Cloning and expression of the cDNA for human antithrom, bin III , 1982 .

[14]  K. Robson,et al.  Sequence homology between human alpha 1-antichymotrypsin, alpha 1-antitrypsin, and antithrombin III. , 1983, Biochemistry.

[15]  J. Millán,et al.  Cloning and sequence of a cDNA coding for the human beta-migrating endothelial-cell-type plasminogen activator inhibitor. , 1986, Proceedings of the National Academy of Sciences of the United States of America.

[16]  J. Catanese,et al.  Primary structure of the reactive site of human C1-inhibitor. , 1985, The Journal of biological chemistry.

[17]  D. Crutchley,et al.  Endotoxin induction of an inhibitor of plasminogen activator in bovine pulmonary artery endothelial cells. , 1986, The Journal of biological chemistry.

[18]  R. Carrell,et al.  Structure and variation of human α1–antitrypsin , 1982, Nature.

[19]  R. Mattaliano,et al.  Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U-937. , 1986, The Journal of biological chemistry.

[20]  G. Salvesen,et al.  Human plasma proteinase inhibitors. , 1983, Annual review of biochemistry.

[21]  A. Lundblad,et al.  Purification of a Specific Placental Plasminogen Activator Inhibitor by Monoclonal Antibody and Its Complex Formation with Plasminogen Activator , 1985, Thrombosis and Haemostasis.

[22]  V. Turk,et al.  Human Leucocyte Urokinase Inhibitor - Purification, Characterization and Comparative Studies Against Different Plasminogen Activators , 1985, Thrombosis and Haemostasis.

[23]  K. Welinder,et al.  Amino acid sequence analysis of the glycopeptides from human complement component C3 , 1986, FEBS letters.

[24]  P. Verde,et al.  The human urokinase-plasminogen activator gene and its promoter. , 1985, Nucleic acids research.

[25]  M. Kozak.  Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. , 1984, Nucleic acids research.

[26]  E. Levin.  Latent tissue plasminogen activator produced by human endothelial cells in culture: evidence for an enzyme-inhibitor complex. , 1983, Proceedings of the National Academy of Sciences of the United States of America.

[27]  J. Zeuthen,et al.  Immunological Relationship Between the Fast-Acting Plasminogen Activator Inhibitors from Plasma, Blood Platelets and Endothelial Cells Demonstrated with a Monoclonal Antibody Against an Inhibitor from Placenta , 1986, Thrombosis and Haemostasis.

[28]  K. Danø,et al.  Monoclonal Antibodies to Human 54,000 Molecular Weight Plasminogen Activator Inhibitor from Fibrosarcoma Cells - Inhibitor Neutralization and One-Step Affinity Purification , 1986, Thrombosis and Haemostasis.

[29]  S. Thorsen,et al.  Human endothelial cells produce a plasminogen activator inhibitor and a tissue-type plasminogen activator-inhibitor complex. , 1984, Biochimica et biophysica acta.

[30]  H. Lambers,et al.  Endothelial plasminogen activator inhibitor (PAI): a new member of the Serpin gene family. , 1986, The EMBO journal.

[31]  K. Robbins,et al.  The specific mechanism of activation of human plasminogen to plasmin. , 1967, The Journal of biological chemistry.

[32]  R. Carrell,et al.  α1-Antitrypsin and the serpins: variation and countervariation , 1985, Trends in Biochemical Sciences.

[33]  P. Verde,et al.  Identification and primary sequence of an unspliced human urokinase poly(A)+ RNA. , 1984, Proceedings of the National Academy of Sciences of the United States of America.

[34]  D. Loskutoff,et al.  Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. , 1985, The Journal of biological chemistry.

[35]  T. Steitz,et al.  Sequencing a protein by x-ray crystallography. I. Interpretation of yeast hexokinase B at 2.5 A resolution by model building. , 1978, Journal of molecular biology.

[36]  K. Danø,et al.  Plasminogen activators catalyse conversion of inhibitor from fibrosarcoma cells to an inactive form with a lower apparent molecular mass , 1986, FEBS letters.

[37]  K. Danø,et al.  Plasminogen activator inhibitor from human fibrosarcoma cells binds urokinase-type plasminogen activator, but not its proenzyme. , 1986, The Journal of biological chemistry.

[38]  R. Lathe.  Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. , 1985, Journal of molecular biology.

[39]  T. Gelehrter,et al.  Characterization of the dexamethasone-induced inhibitor of plasminogen activator in HTC hepatoma cells. , 1986, The Journal of biological chemistry.

[40]  D. Loskutoff,et al.  An Inhibitor Of Plasminogen Activator In Rabbit Endothelial Cells , 1981, Thrombosis and Haemostasis.

[41]  H. Baumann,et al.  Plasma protease inhibitors in mouse and man: divergence within the reactive centre regions , 1984, Nature.

[42]  D. Loskutoff,et al.  The primary plasminogen-activator inhibitors in endothelial cells, platelets, serum, and plasma are immunologically related. , 1985, Proceedings of the National Academy of Sciences of the United States of America.

[43]  H. Chapman,et al.  Characterization of a macrophage-derived plasminogen-activator inhibitor. Similarities with placental urokinase inhibitor. , 1985, Biochemical Journal.

[44]  J. Vieira,et al.  A new pair of M13 vectors for selecting either DNA strand of double-digest restriction fragments. , 1982, Gene.

[45]  R. Huber,et al.  Human C1 inhibitor: primary structure, cDNA cloning, and chromosomal localization. , 1986, Biochemistry.

[46]  L. Hood,et al.  A gas-liquid solid phase peptide and protein sequenator. , 1981, The Journal of biological chemistry.

[47]  B. Wiman,et al.  Inactivation of tissue plasminogen activator in plasma. Demonstration of a complex with a new rapid inhibitor. , 1984, The Journal of biological chemistry.