Activation of Human Endothelial Type Plasminogen Activator Inhibitor (Pai-1) by Negatively Charged Phospholipids

Abstract

The endothelial cell type plasminogen activator inhibitor (PAI-1) may exist in an active, latent form that can be converted into an active form upon exposure to denaturants such as sodium dodecyl sulphate (SDS), guanidine-HCl or urea. Here we show that latent PAI-1 can be activated with lipid vesicles, consisting of the negatively charged phospholipids phosphatidylserine (PS) or phosphatidylinositol (PI). The presence of a net negative charge on the phospholipid headgroup is essential for activation. Incubation with lipid vesicles, consisting of the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanol-amine (PE), does not result in activation of the inhibitor. In the presence of PS vesicles, the capacity of PAI-1 to inhibit tissue type plasminogen activator (t-PA) is 50-fold higher than that of the untreated protein. For comparison, the activity of PAI-1 was enhanced 25-fold by treatment of the protein with SDS. PS induces activation of the inhibitor at much lower concentrations than SDS. For example, to achieve 50% inhibition of t-PA with a more than 100-fold excess of PAI-1, 0.25 nmoles of PS are required, whereas L.60 nmoles of SDS are necessary to reach half maximal inactivation of t-PA. Activation of PAI-1 by PS can be reversed by the addition of Ca2+-ions, suggesting that Ca2+-ions interfere with the interaction of PAI-1 with the negatively charged lipid surface, thus preventing its activation. The lipid-induced activation of PAI-1 points to a possibly important role of phospholipids in fibrinolysis; regulation of the fibrinolytic activity in blood plasma may ultimately be determined by the extent to which these phospholipids activate the inhibitor of t-PA.This study was supported by the Netherlands Thrombosis Foundation.

Plasminogen activator inhibitor activity in diabetic and nondiabetic survivors of myocardial infarction.

“…In addition to the complexed and free forms, PAI-1 also exists in a latent form in plasma. 25 This molecule is immunologically similar to the active form but has no antiactivator activity in its native state, 26 although it can be activated by negatively charged phospholipids 27 and chemical denaturants. 28 One possible explanation for the finding of increased PAI-1 activity but not antigen levels in diabetic compared with nondiabetic subjects is that a greater proportion of latent PAI-1 is in an active form in diabetic subjects.…”

Fibrinolytic System of Vascular Endothelial Cells

“…These dramatic differences in the ra tio of active to latent PAI-1 in various sam-pies suggest that additional factors may exist which regulate the balance between these two activity forms. The recent demonstra tion that negatively charged phospholipids convert latent PA1-1 into its active form [Lambers et al, 1987] raises the possibility that cell membranes, perhaps from activated platelets, may convert latent PAI-1 into its active form in vivo and thus modulate this balance. The recent and unexpected observa tion that PAI-1 is a component of the ECM of fibroblasts [Laiho et al, 1986;Pollanen et al, 1987], mesothelial cells [Rheinwald et al, 1987], smooth muscle cells [Knudsen et al, 1987], and ECs provides additional support for the conclu sion that a previously undescribed family of molecules exists which bind to and modulate the activity of PAI-1.…”

Inhibition of SERPINE1 Function Attenuates Wound Closure in Response to Tissue Injury: A Role for PAI-1 in Re-Epithelialization and Granulation Tissue Formation

“…PAI-1 is initially synthesized in the active conformation but spontaneously converts to a latent form with a rather short active half-life of 1-2 h that can be extended 2-to 10-fold upon binding of PAI-1 to vitronectin [2][3][4][5]. Furthermore, the latent conformation can be reversed by denaturants [6] and negatively-charged phospholipids [7]. In the active conformation, reactive center residues (Arg 346 /Met 347 ) reside in a "strained loop" region acting as "bait" for the urokinase and tissue-type serine protease activators (uPA and tPA, respectively).…”