Isolation of Multiple Types of Plasminogen Activator Inhibitors from Vascular Smooth Muscle Cells

Summary

Large arteries have a natural resistance to tumor cell invasion thought to be due to the production of protease inhibitors. Vascular smooth muscle cells (VSMC) representing the major cellular part of arteries were isolated from human aortas and grown in tissue culture. These cells were found to produce large amounts of inhibitors of plasminogen activators (PA). Fractionation of VSMC-conditioned medium by heparin-affigel chromatography separated three immunologically and functionally distinct PA inhibitors (PAI), namely PAI-1, PAI-2 and protease-nexin I. The three inhibitors were characterized by functional assays and immunoblotting. PA inhibitor 2 (PAI-2) had little affinity for heparin, whereas PA inhibitor 1 (PAI-l) bound to heparin and was eluted from the column at NaCl concentrations of 0. 1 to 0.35 M. Protease-nexin I, eluted at NaCl concentrations of 0.5 M and higher. Most of the PAI-1 was present in the latent, inactive form. PAI-1 was further purified by ion exchange chromatography on a Mono-Q column. Partial sequencing of the purified PAI-1 confirmed its nature by matching completely with the sequence deduced from the cDNA nucleotide sequence of endothelial cell PAI-1. Thus, human VSMC produce all three presently known PAI and these can be separated in a single heparin affinity purification step.

Keywords

Vascular smooth muscle cells - Protease inhibitors - Separation and purification

• References

• 1 Dano K, Andreasen PA, Grondahl-Hansen J, Kristensen P, Nielsen LS, Skriver L. Plasminogen activators, tissue degradation and cancer. Adv Cancer Res 1985; 44: 139-266
• 2 Rifkin DB, Crowe RM. Isolation of a protease inhibitor from tissues resistent to tumor invasion. Hoppe Seylers Z Physiol Chem 1977; 358: 1525-1531
• 3 Laug WE. Vascular smooth muscle cells inhibit the plasminogen activators secreted by endothelial cells. Thromb Haemostas 1985; 53: 165-169
• 4 Laug WE. Inhibition of tumor cell-associated fibrinolysis by bovine vascular smooth muscle cells. J Natl Cancer Inst 1985; 75: 345-352
• 5 Werb Z, Mainardi CL, Vater CA, Harris ED. Endogenous activation of latent collagenases by rheumatoid synovial cells. Evidence for a role of plasminogen activator N Engl J Med 1977; 296: 1017-1023
• 6 Laug WE, Weinblatt M, Jones PA. Endothelial cells degrade extracellular matrix proteins produced in vitro. Thromb Haemostas 1985; 54: 498-502
• 7 Mignetti P, Robbins E, Rifkin DB. Tumor invasion through the human amniotic membrane: Requirement for a proteinase cascade. Cell 1986; 47: 487-498
• 8 Bergman BL, Scott RW, Bajpai A, Watts S, Baker JB. Inhibition of tumor cell mediated extracellular matrix destruction by a fibroblast proteinase inhibitor, protease-nexin I. Proc Natl Acad Sci USA 1986; 83: 996-1000
• 9 Loskutoff DJ, Van Mourik JA, Erickson LA, Lawrence D. Detection of an unusually stable fibrinolytic inhibitor produced by bovine endothelial cells. Proc. Natl Acad Sci 1983; 80: 2956-2960
• 10 Kruithof EK O, Vassalli JD, Schleuning WD, Mattalino RJ, Bachmann F. Purification and characterization of a plasminogen activator inhibitor from the histiocytic lymphoma cell line U937. J Biol Chem 1986; 261: 11207-11213
• 11 Scott RW, Bergman BL, Bajpai A, Hersh RT, Rodriguez H, Jones BN, Barreda C, Watts S, Baker JB. Protease-nexin. Properties and a modified purification procedure J Biol Chem 1985; 260: 7029-7034
• 12 Kruithof EK O. Inhibitors of plasminogen activators. In: Tissue Type Plasminogen Activator: Physiological and Clinical Aspects. Kluft C. ed CRC Press; Boca Raton, FL: 1988. pp 189-210
• 13 Pannekoek H, Veerman H, Lambers H, Diergaarde P, Verweij CL, van Zonneveld AJ, van Mourik JA. Endothelial plasminogen activator inhibitor (PAI): a new member of the serpin gene family. EMBO J 1986; 5: 2539-2544
• 14 Laug WE. Glucocorticoids inhibit plasminogen activator production by endothelial cells. Thromb Haemostas 1983; 50: 888-892
• 15 Scott RW, Eaton DL, Duran N, Baker JB. Regulation of extracellular plasminogen activator by human fibroblasts. The role of protease-nexin J Biol Chem 1983; 258: 4397-5002
• 16 Fraker PJ, Speck JC. Protein and cell membrane iodinations with a sparingly soluble chloramide 1, 3, 4, 6-Terrachloro-3a, 6a-diphenyl- glycoluril. Biochem Biophys Res Comm 1978; 80: 849-857
• 17 Hewick RM, Hunkapiller MW, Hood LE, Dreyer WJ. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem 1981; 256: 7990-7997
• 18 Aebersold R, Leavitt J, Saavedra RA, Hood LE, Kent SB H. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci USA 1987; 84: 6970-6974
• 19 Hunkapiller MW, Hood LE. Analysis of phenylthiohydantoins by ultrasensitive gradient high-performance liquid chromatography. Methods Enzymol 1983; 91: 486-93
• 20 Hekman CM, Loskutoff DJ. Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants. J Biol Chem 1985; 260: 11581-11587
• 21 Ny T, Sawdey M, Lawrence D, Millan JL, Loskutoff DJ. Cloning and sequence of a cDNA coding for the human p-migrating endothelialcell-type plasminogen activator inhibitor. Proc Natl Acad Sci USA 1986; 83: 6776-6780
• 22 Levine EG, Santell L. Association of a plasminogen activator inhibitor (PAI-1) with the growth substratum and membrane of human endothelial cells. J Cell Biol 1987; 105: 2543-2549
• 23 Ross R. The pathogenesis of atherosclerosis, an update. N Engl J Med 1986; 314: 488-500