Evidence for a Discrete Binding Protein of Plasminogen Activator Inhibitor in Plasma (original) (raw)
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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.
Plasminogen activator inhibitor 1 (PAI) is bound to vitronectin in plasma
FEBS Letters, 1988
Functionally active plasminogen activator inhibitor 1 (PAl) is bound to a discrete binding protein in plasma [(1988) Thromb. Haemost. 59, 392-395]. The binding protein has now been partially purified using conventional chromatographic techniques. After addition of active PAl its complex with the binding protein was purified by chromatography on insolubilized monoclonal antibodies towards PAl. Dodecylsulphate (polyacrylamide gel electrophoresis revealed two main compounds with molecular masses of 50 and 75 kDa respectively. Nl-I2-terminal amino acid sequence analysis and immunoblotting analysis suggested that the two compounds were PAl (50 kDa) and vitronectin (75 kDa). We conclude that the PAl-binding protein is identical to vitronectin.
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...
A simplified estimation of tissue plasminogen activator (t-PA) inhibition in human plasma
Fibrinolysis
SC'MMAR Y. A simplified estimation of the inhibition in plasma of the tissue-plasminogen activator (t-PA) is described. The assay is based on the addition to a plasma dilution of a fixed amount of t-PA, determination after incubation for a fixed period of time of the plasmin generated from added plasminogen by an amidolytic assay, estimation of residual t-PA activity and calculation of t-PA inhibition in plasma. Experiments performed to secure optimal conditions of the assay are reported. The recommended procedure gave no indication in normal plasma of an interaction between t-PA and secondary t-P.4 inhibitors (e.g. a*-antiplasmin, a*-macroglobulin). Precision studies yielded a day-today coefficient of variation of 7",,. KE SWORDS. Fibrinolysis. Amidolytic assay. Tissue plasminogen activator activity. Tissue plasminogen activator inhibition.
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.
Biochemical Journal, 1988
The kinetics of inhibition of tissue-type plasminogen activator (t-PA) by the fast-acting plasminogen activator inhibitor-I (PAI-1) was investigated in homogeneous (plasma) and heterogeneous (solid-phase fibrin) systems by using radioisotopic and spectrophotometric analysis. It is demonstrated that fibrinbound t-PA is protected from inhibition by PAI-1, whereas t-PA in soluble phase is rapidly inhibited (K1 = I07 M-1 s-1) even in the presence of 2,uM-plasminogen. The inhibitor interferes with the binding of t-PA to fibrin in a competitive manner. As a consequence the Kd of t-PA for fibrin (1.2 + 0.4 nM) increases and the maximal velocity of plasminogen activation by fibrin-bound t-PA is not modified. From the plot of the apparent Kd versus the concentration of PAI-I a K1 value of 1.3 + 0.3 nm was calculated. The quasi-similar values for the dissociation constants between fibrin and t-PA (Kd) and between PAI-I and t-PA (K1), as well as the competitive type of inhibition observed, indicate that the fibrinolytic activity of human plasma may be the result of an equilibrium distribution of t-PA between both the amount of fibrin generated and the concentration of circulating inhibitor.
Fibrinolysis, 1995
Human recombinant PAI-1, expressed in Escherichia coil, was purified and separated into its active and latent components by chromatography on heparin-and phenyl-substituted agarose under conditions which favour the stability of the active inhibitor. Two columns, with a combined volume of less than 40 ml, were used to purify and separate up to 40mg of PAI-1 in one day. Purified fractions of PAI-1 were analysed by SDS-PAGE, fluorescence spectroscopy and thermostability measurements. A method for concentrating the inhibitor and conditions for storage of concentrated PAl-1 were established. Since PAl-1 spontaneously refolds its reactive-centre loop in a way similar to what is believed to occur in the proteinase-serpin complexes, studies with this inhibitor may play an important role in elucidating the mechanism of serpin action. The method we are presenting yields highly purified fractions of active and latent PAI-1 with relative ease and facilitates detailed investigations of its reaction mechanism.
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.
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).
EJIFCC, 2000
Background Proficiency testing of enzymatic methods for plasminogen activator inhibitor 1 (PAI-1) showed a systematic variability indicating the aspecificity of some methods. Methods To define and detect specificity of enzymatic methods for PAI-1, experts of the ISTH/SSC subcommittee defined criteria and test samples to check the criteria. 16 samples were prepared to test (a) specificity in depleted plasma, (b) interference by added PAI-2 or PAI-3, (c) interference by added tissue-type plasminogen activator (t-PA), (d) performance in dose response. To exercise the test procedure participants were recruited via the subcommittee, literature and company data. Coded samples were distributed to participants. The NIBSC standard for PAI-1 activity was included for the normalisation of results. Adherence to the predetermined criteria was judged blind in the ISTH/SSC subcommittee meeting. Results In total 17 laboratories with 15 different assay methods participated in the study. Methods were...