Immunoelectron microscopic localization of type 1 plasminogen activator inhibitor on the surface of activated endothelial cells.

Abstract

Abstract. Immunogold EM was employed to compare the distribution of type 1 plasminogen activator inhibitor (PAId) on the surface of agonist-activated human umbilical vein endothelial cells (HUVECs) with that of control, unactivated cells. As previously observed, (Schleef, R. R., T. J. Podor, E. Durme, J. Mimuro, and D. J. , analysis of cross-sections of nonpermeabilized control HUVEC monolayers stained first with affinity-purified rabbit antibodies to PAId and then with goldconjugated goat anti-rabbit IgG, revealed the presence of relatively few gold particles (<1-2 % of the total) on the apical cell surface. The majority of gold particles were detected primarily in the extracellular matrix between the culture substratum and the cell membrane. In contrast, treatment of HUVECs with tumor necrosis factor ot (TNFot; 200 U/ml, 24 h) or with lipopolysaccharide (LPS; 10 #g/ml, 24 h) resulted in an increased staining of PAId not only in the extracellular matrix, but also on the apical cell surface (10-fold increase). Immunoabsorption of the rabbit anti-PAid with purified PAId, or treatment of HUVECs with tissue-type plasminogen activator (2.5/zg/ml, 2 h, 4°C) reduced the amount of staining both on the apical surface and in the extracellular matrix of agonistactivated HUVECs by 80-95 %. The topographical location of PAId on the cell surface was examined further by coupling immunogold staining with high resolution surface replication. Transmission EM of surface replicas from TNFo~-or LPS-activated HUVECs revealed a general increase in PAId staining both on planar regions and within indentations of the apical cell surface. Nonactivated HUVECs revealed PAid-specific immunogold particles only in areas of exposed extracellular matrix between the cells and occasionally at regions of cell-cell contacts. Analysis of activated bovine aortic endothelial cells by immunoelectron microscopy, immunologic assays, and flow cytometry revealed similar increases in surface PAId. These increases in surface PAId could be detected by 3 h and continued over a 24-h period. The expression of PAId on the luminal surface of endothelial cells during immune or inflammatory reactions could reduce endothelial fibrinolytic activity, thus, promoting the localized, pathologic formation of intravascular thrombi.

Plasminogen Activator Inhibitor-1 Supports IL-8-Mediated Neutrophil Transendothelial Migration by Inhibition of the Constitutive Shedding of Endothelial IL-8/Heparan Sulfate/Syndecan-1 Complexes

“…However, the ability of exogenously added mutant PAI-1, which only slowly reverts to latent, to prevent solubilization of IL-8 would support the former explanation. Thus, it appears that the PAI-1-neutralizing Ab used in our experiments inhibits endothelial PAI-1, which is localized on the endothelial cell surface in active form (32,33).…”

Elevated Expression of Urokinase-like Plasminogen Activator and Plasminogen Activator Inhibitor Type 1 During the Vascular Remodeling Associated With Pulmonary Thromboembolism

“…19 Rabbit antisera against human vWF, mouse monoclonal anti-human smooth muscle ␣-actin (clone 1A4), and anti-human monocyte/macrophage CD68 (clone KP1) were purchased from DAKO. Mouse anti-human PCNA was purchased from Oncogene (Uniondale, NY).…”

Human glioma U‐251 cells contain type 1 plasminogen activator inhibitor in a rapidly releasable form

“…Immunodepletion experiments were performed using a modification of previously described procedures [30,31]. Briefly, nonimmune rabbit serum or antiserum to either urokinase [32], t-PA [32], PAI-1 [33] (20 ~tl, respectively) were added to protein A-coated Sepharose CL-4B beads (80 lag; Pharmacia, Uppsala, Sweden) that had been rehydrated and washed according to the manufacturer's instructions. U-251 conditioned media or cell lysates (100 ~tl corresponding to 105 cells) were incubated (1 h, 23°C) with the beads.…”