Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma (original) (raw)
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Evidence for a Discrete Binding Protein of Plasminogen Activator Inhibitor in Plasma
Thrombosis and Haemostasis, 1988
SummaryGel-filtration experiments of mixtures of functionally active and inactive forms of plasminogen activator inhibitor (PAI) with human plasma or bovine serum albumin have provided evidence for the existence of a discrete binding protein of PAI in plasma. Most likely it is a glycoprotein with a molecular weight of approximately 150,000. The data suggest that it forms a very stable complex with functionally active forms of PAI, but not with the inactive or “latent” PAI. However, the PAI activity seems not to be significantly altered by the interaction with the binding protein. Assuming that a stoichiometric complex is formed, titration experiments suggest that a pool of normal human plasma contains about 40–50 mg of PAI-binding protein liter.
FEBS Letters, 1991
Plasmin is shown to specifically cleave vitronectin at the Arga6*-Se? bond, 18 amino acid residues upstream from the site of the endogenous cleavage which gives rise to the two-chain form of vitronectin in plasma. The cleavage site is established using the exclusive phosphorylation of Se? with protein kinase A. As a result of the plasmin cleavage, the affinity between vitronectin and the type-l inhibitor of plasminogen ac:ivatoe (PAI-1) is significantly reduced. This cleavage is stimulated by glycosaminoglycans, which are known to anchor vitronectin to the extracellular matrix. A mechanism is proposed through which plasmin can arrest its own production by feedback signalling, unleashing PAL1 from the immobilized vitronectin found in the vascular subendothelium, which becomes exposed at the locus of a hemostatic event.
Fibrinolysis, 1992
Functionally active recombinant plasminogen activator inhibitor-l (rPAI-1) has been purified from bacterial cells in the absence of any discrete binding protein. In the present study, vitronectin, which is known to stabilise the natural form of PAI-1, was evaluated for its effects on the activity and structure of rPAI-1. As assessed by PAI-activity assays, purified human vitronectin was shown to stabilise rPAI-1, by doubling its half-life at 25" and 37"C, and to enhance the activity of rPAI-1 in concentration-dependent fashion. Vitronectin also restored partial activity to a latent form of rPAI-1 prepared by a 72h incubation at 37°C. Fluorescence spectroscopy studies revealed that rPAI-1 in association with vitronectin displayed a higher tryptophan emission signal than the sum of the emissions of the individual components. These results suggest that vitronectin effects on rPAI-1 activity may result from specific conformational effects induced upon binding of the two proteins. Plasminogen activator inhibitor-l (PAI-l), a member of the superfamily of serine protease inhibitors (serpins), is the principal physiological inhibitor of both tissue-type plasminogen activator (t-PA) and urokinase (u-PA). '-' A number of observations suggest that elevated plasma levels of PAI-decrease net fibrinolytic activity and contribute to the development of thrombotic disorders including venous thrombosis,4 coronary artery disease," and myocardial infarction.6 The modulation of PAI-activity may provide a major regulatory mechanism for the fibrinolytic system. One modulator of PAI-activity appears to be the serum spreading factor vitronectin which has been shown to bind PAI-and enhance its activity and stability. 7-" The physiological relevance of the association between PAI-and vitronectin remains to be established. Recently, we described the purification and characterization of a recombinant form of PAI-(rPAI-1) from E. co/i which, unlike most reported preparations, retains a high degree of activity without the need for activation with denaturants. '* In the prese t s u y n t d , we have utilised activity assays and fluorescence spectroscopy to study the
Thrombosis Research, 1991
Molecular forms of plasminogen activator inhibitor-l (PAI-1) and tissue-type plasminogen activator (t-PA), identified by gel filtration and specific immunoassays, were studied in plasma from subjects with normal and elevated PAI-levels before and after in vitro or in vivo addition of t-PA. In normal plasma, PAI-occurs in three molecular forms, a Mr>700 KDa inactive form of heterogeneous composition, an active 450 KDa form containing PAI-l/vitronectin complex and an inactive peak at Mr 50 KDa containing free PAI-1. Stimulation of platelets results in a significant increase of the 50 KDa form and a slight increase of the 450 KDa form. Patients with increased PA1 activity levels have an increase of both the 450 KDa and the 50 KDa forms, whereas patients with thrombotic thrombocytopenic purpura have an increased 50 KDa form. In normal plasma, collected in the presence or absence of D-Phe-Pro-Arg-CH Cl, t-PA occurs primarily as a Mr > 700 KDa form containing t-PA/?AI-1 complex. Addition of high concentrations of t-PA (70 ng/ml) to plasma in vitro or t-PA injection in vivo, results in t-PA inhibitor complexes, including t-PA/cxZ antiplasmin. It is concluded that in subjects with increased PAI-levels in plasma, PAI-may occur as high molecular weight complexes with vitronectin of which 450 KDa was the most ;mportant part and as a 50 KDa inactive forms ; t-PA circulates primaril; in complex with inhibitors. Thus, some of the molecular interactions between PAI-1, t-PA and vitronectin, previously demonstrated in purified systems in vitro, also occur in plasma.
Biochemical Journal, 1990
The structural events taking place during the reaction between PAI-1 (plasminogen-activator inhibitor 1) and the plasminogen activators sc-tPA (single-chain tissue plasminogen activator) and tc-tPA (two-chain tissue plasminogen activator) were studied. Complexes were formed by mixing sc-tPA or tc-tPA with PAI-1 in slight excess (on an activity basis). The complexes were purified from excess PAI-1 by affinity chromatography on fibrin-Sepharose. Examination of the purified complexes by SDS/polyacrylamide-gel electrophoresis (SDS/PAGE) and N-terminal amino acid sequence analysis demonstrated that a stoichiometric 1:1 complex is formed between PAI-1 and both forms of tPA. Data obtained from both complexes revealed the amino acid sequences of the parent molecules and, in addition, a new sequence: Met-Ala-Pro-Glu-Glu-. This sequence is found in the C-terminal portion of the intact PAI-1 molecule and thus locates the reactive centre of PAI-1 to Arg346-Met347. The proteolytic activity of sc...
Identification of a reversible inhibitor of plasminogen activators in blood plasma
FEBS Letters, 1985
Inhibition of tissue-type plasminogen activator (t-PA) by pooled plasma could be ascribed for only 60% to the endothelial cell type PA inhibitor. The residual inhibition is ascribed to a so-far undescribed plasma component present at 0.2 nmol/l. This component shows reversible binding to t-PA with an apparent K, of 10 pmol/l (does not hinder t-PA binding to fibrin); also reacts with urokinase, but not with DIP-t-PA; is stable at 37°C and does not occur in media of endothelial cells, hepatocytes and fibroblasts. This PA binding component in plasma adds to the regulation of plasminogen activator activities.
Biological Chemistry, 2000
Immobilized vitronectin was found to bind both purified plasminogen activator inhibitor type 1 (PAI-1) and the PAI-1 in conditioned culture medium of human sarcoma cells. Similarly, immobilized PAI-1 bound both purified vitronectin and vitronectin from normal human serum. These interactions were demonstrated using both enzyme immunoassay and radioiodinated proteins. Solid-phase vitronectin bound PAI-1 with K d 1.9 X lo" M, and the reverse interaction gave a K d 5.5 X lo-' M. Evidence was also found for a second type of binding with a K d below 10"' M. The molar ratios of the two proteins in the complex at the saturation levels were approximately one molecule of soluble PAI-1 bound per three molecules of immobilized vitronectin and approximately one molecule of soluble vitronectin being bound per one molecule of immobilized PAI-1. Binding of PAI-1 to vitronectin did not lead to an irreversible loss of the ability of PAI-1 to inhibit urokinase (u-PA) and tissue-type plasminogen activator (t-PA). Active u-PA released vitronectin-bound "'Ilabeled PAI-1 radioactivity, suggesting that u-PA interacts with the complex. The M, 50,000 urokinase cleavage product of PAI-1 also bound to vitronectin, but this bound fragment did not inhibit u-PA. Binding of PAI-1 to vitronectin did not interfere with the ability of vitronectin to promote the adhesion and spreading of cells. These results suggest that the interaction between vitronectin and PAI-1 may serve to confine pericellular u-PA activity to focal contact sites where cells use proteolysis in regional detachment.
The plasminogen activator/plasmin system
Journal of Clinical Investigation, 1991
active conformation (13). In addition, vitronectin and heparin affect the specificity of PAI-1, by enhancing its reactivity toward thrombin (14).
Detection of specific forms of plasminogen activator inhibitor type 1-by monoclonal antibodies
Fibrinolysis, 1991
Monoclonal antibodies to plasminogen activator inhibitor type-l (PAI-1) were generated and characterised for their ability to inhibit PAI-interaction with tissue plasminogen activator (t-PA) and urokinase (u-PA) and detection of the various forms of PAI-(native, complexed, and degraded) by immunoblotting. Mabl7 inhibited both complex formation between PAI-and t-PA/u-PA and PAL-1 activity in a dose dependent manner by 90%. Mab 25 was much less effective, blocking complex formation less than 30% and PAI-activity less than 20%. The Kds of Mab17 and Mab25 were 2.8~ 10-l' M and 2.6~ lo-" M, respectively. Following SDS-PAGE and immunoblotting, Mab17 detected native PAI-only; PAI-in complex and the t-PA/u-PA degraded form of PAI-(M,=42000) did not react with this antibody. In contrast, Mab25 detected all three forms of PAI-although the affinity for the native form appeared to be greater than Mabl7 or the PAI-polyclonal employed. Despite these differences, both monoclonal antibodies immunoprecipitated native and degraded PAIequally as well. These results suggest that the epitope of Mab17 is associated with the reactive site of PAI-and that this region is either missing or not accessible in the cleaved form or in complex.