An Enzyme-Linked Immunosorbent Assay (ELISA) Used to Study the Cellular Secretion of Endothelial Plasminogen Activator Inhibitor (PAI-1)

I R MacGregor; N A Booth

doi:10.1055/s-0038-1646771

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 1988; 59(01): 068-072
DOI: 10.1055/s-0038-1646771

Review Article

Schattauer GmbH Stuttgart

An Enzyme-Linked Immunosorbent Assay (ELISA) Used to Study the Cellular Secretion of Endothelial Plasminogen Activator Inhibitor (PAI-1)

I R MacGregor

¹The Scottish National Blood Transfusion Service, University of Aberdeen, Foresterhill, Aberdeen, UK

,

N A Booth

²Headquarters Unit Laboratory, Edinburgh and the Department of Medicine and Therapeutics, University of Aberdeen, Foresterhill, Aberdeen, UK

› Institutsangaben

Abstract

als PDF herunterladen

Summary

A two-site sandwich ELISA was developed to measure PAI-1 antigen and utilised a polyclonal antiserum produced against PAI-1 purified from human endothelial cell secretory products. The assay was calibrated against a preparation of pure PAI-1 whose protein concentration had been determined by amino acid analysis and the detection limit was 30 pg PAI-1 ml“³ sample. PAI-1 was detected in primate sera but not in a wide range of nonprimate sera and no cross-reactivity with α₂-antiplasmin or antithrombin III was observed.

The ELISA was used to study cellular secretion of PAI-1 which was confirmed as a major secretory protein in human umbilical vein endothelial cells (HUVEC). PAI-1 antigen accumulated in the medium in a linear fashion with time and accounted for ≃10% of total secreted protein. Specific activity of intracellular PAI1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t^½ for inactivation of secreted PAI-1 of 0.53 h was calculated. Purified endotoxin stimulated the secretion of PAI-1 antigen and raised the intracellular levels in HUVEC cultures showing that the anti-fibrinolytic actions of endotoxin are effected by increasing the rate of synthesis and secretion of PAI-1.

Key words

ELISA - Plasminogen activator inhibitor - Endothelial - Cell cultures

PDF (636 kb)

Referenzen

References
1 Collen D. On the regulation and control of fibrinolysis. Thromb Haemostas 1980; 43: 77-89
2 van Mourik JA, Lawrence DA, Loskutoff DJ. Purification of an inhibitor of plasminogen activator (antiactivator) synthesised by endothelial cells. J Biol Chem 1984; 259: 14914-14921
3 Booth NA, MacGregor IR, Hunter NR, Bennett B. Plasminogen activator inhibitor from human endothelial cells: purification and partial characterization. Eur J Biochem 1987; 165: 595-600
4 Erikson LA, Hekman CM, Loskutoff DJ. The primary plasminogen activator inhibitors in endothelial cells, platelets, serum and plasma are immunologically related. Proc Natl Acad Sci USA 1985; 82: 8710-8714
5 Booth NA, Anderson JA, Bennett B. Platelet release protein which inhibits plasminogen activators. J Clin Pathol 1985; 38: 825-830
6 Philips M, Juul AG, Thorsen S, Selmer J, Zeuthen J. 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. Thromb Haemostas 1986; 55: 213-217
7 Sprengers ED, Princen HM G, Kooistra T, van Hinsbergh VW M. Inhibition of plasminogen activators by conditioned medium of human hepatocytes and hepatoma cell line Hep G2. J Lab Clin Med 1985; 105: 751-758
8 Collen D. Report of the meeting of the subcommittee on fibrinolysis, Jerusalem, Israel, June 2, 1986. Thromb Haemostas 1986; 56: 415-416
9 Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ. Cloning and sequence of a cDNA coding for the human p migrating endothelial cell type plasminogen activator inhibitor. Proc Natl Acad Sci USA 1986; 83: 6776-6880
10 Pannekoeck H, Veerman H, Lambers H, Diergaarde P, Verweij CL, Van Zonneveld AJ, van Mourik JA. Endothelial plasminogen activator inhibitor-a new member of the serpin gene family. Embo J 1986; 5: 2539-2544
11 Andreasen PA, Riccio A, Welinder KG, Douglas R, Sartorio R, Nielsen LS, Oppenheimer C, Blasi F, Dano K. Plasminogen activator inhibitor type-1: reactive center and amino-terminal heterogeneity determined by protein and cDNA sequencing. Febs Letter 1986; 209: 213-218
12 Sprengers ED, Kluft K. Plasminogen activator inhibitors. Blood 1987; 69: 381-387
13 Verheijen JH, Chang GT G, Kluft C. Evidence for the occurrence of a fast-acting inhibitor of plasminogen activator in human plasma. Thromb Haemostas 1984; 51: 392-395
14 Chmielewska J, Wiman B. Determination of tissue plasminogen activator and its “fast” inhibitor in plasma. Clin Chem 1986; 32: 482-485
15 Levin EG. Quantitation and properties of the active and latent plasminogen activator inhibitors in cultures of human endothelial cells. Blood 1986; 67: 1309-1313
16 SciileCi RR, Sinku M, Loskuluff DJ. Immunoiudiomeliie assay to measure the binding of a specific inhibitor to tissue-type plasminogen activator. J Lab Clin Med 1985; 106: 408-415
17 Urden G, Johansson U, Chmielewska J, Brandt J, Wiman B. Monoclonal antibodies towards the fast inhibitor of tissue plasminogen activator from human plasma and serum. Thromb Haemostas 1986; 55: 383-387
18 Rijken DC, Sprengers FD, van der Hoeven RJ, Jansen RG. An enzyme immunoassay for endothelial cell type plasminogen activator inhibitor (PAI). Fibrinolysis 1986. Vol. 1 (Suppl. 1) Abst No 144
19 Guesdon J-L, Ternynck T, Avrameas S. The use of avidin-biotin interaction in immunoenzymatic techniques. J Histochem Cytochem 1979; 27: 1131-1139
20 Deutsch DG, Mertz ET. Plasminogen: purification from human plasma by affinity chromatography. Science 1970; 170: 1095-1096
21 Jaffe EA, Nachman RL, Becker CG, Minick CR. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 1973; 52: 2745-2756
22 Hunter NR, Dawes J, MacGregor IR, Pepper DS. Quantification by radioimmunoassay of thrombospondin synthesised and secreted by human endothelial cells. Thromb Haemostas 1984; 52: 288-291
23 Colucci M, Paramo JA, Collen D. Generation in plasma of a fastacting inhibitor of plasminogen activator in response to endotoxin stimulation. J Clin Invest 1985; 75: 818-824
24 Emeis JJ, Kooistra T. Interleukin I and lipopolysaccharide induce an inhibitor of tissue-type plasminogen activator in vivo and in cultured endothelial cells. J Exp Med 1986; 163: 1260-1266
25 Kooistra T, Sprengers ED, van Hinsbergh VM. Rapid inactivation of the plasminogen-activator inhibitor upon secretion from cultured human endothelial cells. Biochem J 1986; 239: 497-503
26 Lawrence DA, Loskutoff DJ. Inactivation of plasminogen activator inhibitor by oxidants. Biochem 1986; 25: 6351-6355
27 Wagner OF, Binder BR. Purification of an active plasminogen activator inhibitor immunologically related to the endothelial type plasminogen activator inhibitor from the conditioned media of a human melanoma cell line. J Biol Chem 1986; 261: 14474-14481
28 McArthur MM, MacGregor IR, Prowse CV, Hunter NR, Dawes J, Pepper DS. The use of human endothelial cells cultured in flat wells and on microcarrier beads to assess tissue plasminogen activator and factor VIII related antigen release. Thromb Res 1986; 41: 581-587