An enzyme-linked immunosorbent assay (ELISA) used to study the cellular secretion of endothelial plasminogen activator inhibitor (PAI-1).
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-1 sample. PAI-1 was detected in primate sera but not in a wide range of non-primate sera and no cross-reactivity with alpha 2-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 approximately equal to 10% of total secreted protein. Specific activity of intracellular PAI-1 was typically 20-fold greater than that of PAI-1 in 24 h conditioned medium and a t1/2 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.
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D010960:Plasminogen Activators / Q000037:antagonists & inhibitors*; D015849:Plasminogen Inactivators
D006023:Glycoproteins; D015849:Plasminogen Inactivators; D010960:Plasminogen Activators