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
