PAI-1 synthesis in the human hepatoma cell line HepG2 is increased by cytokines--evidence that the liver contributes to acute phase behaviour of PAI-1.

The acute phase behaviour of the fast inhibitor of tissue-type plasminogen activator (PAI-1) in vivo has been attributed to increased synthesis by endothelial cells. However, most other acute phase proteins in vivo are synthesized in the liver, which process is regulated by cytokines and can be studied in the hepatoma derived cell line HepG2. In this study, we investigated whether the synthesis of PAI-1 by HepG2 cells is regulated by the cytokines recombinant IL-1, rIL-6 and rTNF. Recombinant IL-1 and rTNF each increased PAI-1 synthesis by HepG2 cells two to three fold, whereas rIL-6 hardly had an effect. Mixtures of rIL-1, rIL-6 and rTNF increased PAI-1 synthesis up to eleven fold. The effects observed were not due to non-specific effects on HepG2 cell metabolism, since synthesis of alpha-2-antiplasmin was not effected by any of those cytokines, whereas fibrinogen synthesis was increased three to four fold by rIL-6, but was unaffected by rIL-1. Thus, our results demonstrate that synthesis of PAI-1 by HepG2 cells is regulated by cytokines and implicate that the acute phase behaviour of PAI-1 in vivo at least in part may be due to an increased synthesis by the liver.

Activation of human endothelial cell-type plasminogen activator inhibitor (PAI-1) by negatively charged phospholipids.

The endothelial cell-type plasminogen activator inhibitor (PAI-1) may exist in an inactive, latent form that can be converted into an active form upon treatment of the protein with denaturants, such as sodium dodecyl sulfate, guanidine HCl, or urea. The present paper demonstrates that latent PAI-1 can be activated by lipid vesicles containing the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol. The presence of a net negative charge on the phospholipid headgroup is essential for activation, since lipid vesicles consisting exclusively of zwitterionic phospholipids, such as phosphatidylcholine and phosphatidylethanolamine, do not activate PAI-1. In the presence of PS vesicles, PAI-1 inhibited tissue-type plasminogen activator 50-fold more effectively than in the absence of phospholipids, whereas sodium dodecyl sulfate enhanced PAI-1 activity by 25-fold. In mixed phospholipid vesicles containing PS and phosphatidylcholine in various molar ratios, the extent of PAI-1 activation was directly related to the PS content of the phospholipid membrane. Ca2+ ions interfered with the inhibitory activity of PS-activated PAI-1, suggesting that Ca2+ ions may regulate PAI-1 activity in the presence of negatively charged phospholipids. An important consequence of these findings is that, as in blood coagulation, negatively charged phospholipids may play an important regulatory role in controlling the fibrinolytic system by activating an inhibitor of tissue-type plasminogen activator.

Inactivation of chlorophyllase by negatively charged plant membrane lipids.

Chlorophyllide combines spontaneously not only with phosphatidylcholine (PC) liposomes but also with various other (plant) lipids dispersed in an aqueous medium. The lipid-associated chlorophyllide is highly fluorescent and the fluorescence yield is virtually independent of the nature of the lipid. Chlorophyllase (chlorophyll chlorophyllidohydrolase, EC 3.1.1.14) activity assays that are based on the determination of this chlorophyllide fluorescence show that phosphatidylglycerol (PG), and also sulphoquinovosyldiacylglycerol (SQDG), associate with isolated chlorophyllase, thereby inactivating the enzyme in a co-operative way. The extent of this inactivation depends on the pH and ionic strength of the reaction medium and can be completely reversed by divalent cations (Mg2+). The inhibition of chlorophyllase effected by free PG liposomes can be counteracted by electrically neutral lipids at relatively high concentration (PC and also chloroplast lipids). Digalactosyldiacylglycerol (DGDG) is not effective in this respect. When PG has been incorporated in PC or DGDG liposomes, its ability to inhibit chlorophyllase activity is reduced. Whereas the remaining chlorophyllase-inactivating effect of PG, incorporated in PC, can still be reversed by Mg2+, this is not found when enzyme inactivation is caused by PG incorporated in DGDG. The results reported here are consistent with those obtained earlier concerning the stabilization of chlorophyllase by PG and PG/galactolipid mixtures (Lambers, J.W.J., Verkleij, A.J. and Terpstra, W. (1984) Biochim. Biophys. Acta 786, 1-8). They are discussed in terms of the regulation of chlorophyllase activity by lipids surrounding the enzyme and by divalent cations.