Plasminogen Activator Inhibitor Biochemical and Clinical Aspects

Abstract

Plasminogen activator (PAs) are enzymes that convert the zymogen plasminogen into the trypsin-like protease plasmin, which degrades extracellular matrix proteins and fibrin in the course of fibrinolysis, embryogenesis, tissue remodeling and in tumor metastasis. Plasminogen activator inhibitors (PAIs) are important modulators of PA activity. Several proteins have been identified which inhibit at fast rates urokinase (u-PA) and tissue-type PA (t-PA). In the order of inhibition rate constants these are: a) PAI-1, present in human plasma and platelet extracts and purified from human endothelial cell, fibrosarcoma cell and melanoma cell conditioned media; b) PAI-2, first identified in extracts of human placenta and later also in extracts and conditioned media of human granulocytes and monocytes; and c) protease nexin, a broad specificity protease inhibitor that was first identified and purified from human fibroblasts. We have chosen to use phorbol myristate acetate (30 ng/ml) stimulated histiocytic lymphoma cells (U-937) for the purification of PAI-2. The concentration of PAI-2 in the conditioned media after three days culture in the absence of fetal calf serum is 5 mg/1 and PAI-2 represents 3% of total protein. PAI-2 was purified by a two step procedure consisting of isoelectric focusing and affinity chromatography on Cibacron-Blue agarose. Two forms of PAI-2 were identified: a 47 kDa, nonglycosylated, pi 5.0 form and a 60 kDa glycosylated, pi 4.4 form. Immunctolot analysis and in vivo protein labeling studies under culture conditions that assure 100% viability of the cells showed that the glycosylated Torn is secreted, whereas the 47 kDa, nonglycosylated form remains intracellular. The glycosylation does not affect the activity of the inhibitors since both forms of PAI-2 react with the same rate with u-PA. PAI-2 is a fast inhibitor of u-PA (kl=9×l05M−1s−1) and two-chain t-PA (kl=2×l05) and a rather slow inhibitor of one chain t-PA (kl=l×l02) and of plasmin (kl×l02), but does not inhibit glandular and plasma kallikrein or thrombin. The inhibition spectrum and the kinetics of inhibition clearly distinguish PAI-2 from PAI-1 (kl of reaction with u-PA and two and one chain t-PA above 107) and from protease nexin, that is an efficient inhibitor also of thrombin and plasmin.We have cloned a 1880 Ip fragment of PAI-2 cDNA and determined its nucleotide sequence. The derived acid sequence reveals that PAI-2 is like PAI-1 and protease nexin a member of the serpin family of proteins and contains arginine at its putative active site. In an attenpt to identify parts of the inhibitor proteins that are responsible for conferring PA specificity to PAI-1 and PAI-2 we have compared the primary structures of PAI-1 and PAI-2 with each other and with antithrombin III (AT III). Surprisingly, PAI-2 exhibits no homology with PAI-1 in the region close to the active site except for the active site arginine, whereas, in that region, AT III showed three and seven conserved aminoacids when compared to PAI-1 and PAI-2, respectively. This finding suggests that other regions than those close to the active site contribute to the specificity of PAIs.Plasma concentrations of PAI-2 were measured by a specific radioimmunoassay in over 50 healthy individuals, PAI-2 levels were below detection limit (15 ng/ml) in half of the saitples. Maximal concentrations encountered were in the 30 ng/ml range. PAI-2 measurements in over 300 hospitalized patients demonstrated significantly elevated PAI-2 concentrations only in pregnant women. Measurements in various stages of pregnancy showed a steady increase of PAI-2 from below detection limit in nonpregnant women to values of 250 ng/ml at term and of PAI-1 frcm 25 ng/ml to 150 ng/ml. Unlike to PAI-1 concentrations that normalize rapidly after delivery, PAI-2 concentrations remain significantly elevated for several days.

Effect of Single Oral Dose of Aspirin on Human Platelet Functions and Plasma Plasminogen Activator Inhibitor-1

“…In this study, aspirin was shown to inhibit AA-induced platelet PAI-1 secretion and was also shown to lower the plasma levels of PAI-1 at 2 h after aspirin administration, which per sisted in part for up to 24 h. These data might suggest at least in part some explanation for impressive clinical benefits of aspirin, since high plasma levels of PAI-1 have been consid ered as a risk factor in various thromboem bolic disorders [12],…”

Fibrinolytic and inflammatory processes in pleural effusions

“…Numerous studies have correlated decreased plasma fibrinolytic activity with elevated PAI-1 in patients with septicaemia and during postoperative recovery [10]. Similarly, a positive correlation has been reported between plasma PAI-1 levels and inflammatory protein [5].…”

Fibrinolysis and fibrinogenolysis in liver disease

“…The discordance between the two may have resulted from different assay methods used for the determination of PAI-1 . PAI-1 is present in plasma not only as free PAI-1 but also as PAI-I complexed to t-PA and only a free, active form possesses the inhibitory capacity [34]. When plotted by disease categories, the magnitude of elevations of FbDP and TDP was the most prominent in patients with acute hepatitis (Fig.…”