The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator (original) (raw)
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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.
Data in Brief, 2016
Thrombosis is a leading cause of death worldwide [1]. Recombinant tissue-type plasminogen activator (tPA) is the FDA-approved thrombolytic drug for ischemic strokes, myocardial infarction and pulmonary embolism. tPA is a multi-domain serine protease of the trypsin-family [2] and catalyses the critical step in fibrinolysis [3], converting the zymogen plasminogen to the active serine protease plasmin, which degrades the fibrin network of thrombi and blood clots. tPA is rapidly inactivated by endogenous plasminogen activators inhibitor-1 (PAI-1) [4] (Fig. 1). Engineering on tPA to reduce its inhibition by PAI-1 without compromising its thrombolytic effect is a continuous effort [5]. Tenecteplase (TNK-tPA) is a newer generation of tPA variant showing slower inhibition by PAI-1 [6]. Extensive studies to understand the molecular interactions between tPA and PAI-1 have been carried out [7-18], however, the precise details at atomic resolution remain unknown. We report the crystal structure of tPA Á PAI-1 complex here. The methods required to achieve these data include: (1) recombinant expression and purification of a PAI-1 variant (14-1B) containing four mutations (N150H, K154T, Q319L, and M354I), and a tPA serine protease domain (tPA-SPD) variant with three mutations (C122A, N173Q, and S195A, in the chymotrypsin numbering) [19]; (2) formation of a tPA-SPD Á PAI-1 Michaëlis Contents lists available at ScienceDirect
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.
Involvement of residues 296–299 in the enzymatic activity of tissue-type plasminogen activator
"Protein Engineering, Design and Selection", 1992
The tetra-alanine substitution variant KHRR 296-299 AAAA of tissue-type plasminogen activator (t-PA) was previously shown to have enhanced fibrin specificity and enhanced activity in the presence of fibrin compared with the wild-type form of the molecule. The structural requirements for these alterations in enzymatic activity were investigated by constructing several amino acid substitution variants at each of the positions from 2% to 299 and evaluating their activities under a variety of conditions. Effects on plasminogen activator activity were common among the point mutants at positions 296-299; nearly all had a phenotype similar to the KHRR 296-299 AAAA variant. The greatest effects on enzymatic function were found with multiple substitution variants, but some single charge reversals and proline substitutions had substantial effects. The enhanced fibrin specificity of KHRR 296-299 AAAA t-PA results in less fibrinogenolysis than seen with wild-type t-PA. Approximately four times greater concentration of KHRR 296-299 AAAA compared with wild-type t-PA was required to consume 50% of the fibrinogen in human plasma.
Heterogeneity of human tissue-type plasminogen activator
FEBS Letters, 1988
Tissue-type plasminogen activator (t-PA) from human melanoma cells (Bowes) was purified by immunosorbent chromatography on affinospecific polyclonal antibodies and gel filtration in the presence of KSCN. The immunosorbent eluate contained three major components of >200, 85 and 65 kDa, respectively. The 65 kDa t-PA component could be separated by gel filtration on Ultrogel AcA44 in the presence of KSCN to a pure preparation yielding a unique N-terminal amino acid sequence. Immunoblot analysis, using affinospecific antibodies against t-PA, was a specific and sensitive method to identify different types of t-PA (I-IV), as well as t-PA-inhibitor complexes and degradation products in unstimulated melanoma cell culture fluids. Furthermore, the t-PA preparations, produced by phorbol ester-treated melanoma cells, were free of type IV and thus differed physicochemically from the constitutively produced t-PA preparations. The composition of t-PA from mammalian cell cultures is thus more complex than hitherto described.
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, 1986
Both the urokinase-type and tissue-type plasminogen activator can convert their -54 kDa type-l inhibitor (PAI-1) to an inactive form with a lower apparent molecular mass. We have determined the amino-terminal amino acid sequences of human native and converted PAI-1, and isolated PAI-I cDNA and determined the nucleotide sequence in regions corresponding to the amino-terminus and the cleavage site. The data show that the conversion of the inhibitor consists of cleavage of an Arg-Met bond 33 residues from the carboxy-terminus, thus localizing the reactive center of the inhibitor to that position. In addition, a heterogeneity was found at the amino-terminus, with a Ser-Ala-Val-His-His form and a two-residue shorter form (Val-His-His-) occurring in approximately equal quantities.
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).
Phosphorylation of human plasminogen activators and plasminogen
FEBS Letters, 1995
Plasminogen (PC), urokinase-type plasminogen activator (u-PA) and tissue-type PA (t-PA) are the main molecules involved in fibrinolysis and in many other physiological and pathological processes. In the present study we report that human t-PA, purified from human melanoma cells, and PG, purified from human plasma, both contain P-Tyr residues, as revealed by immunoblotting analyses with monoclonal anti-P-Tyr antibodies. In addition HPLC amino acid analysis of acid-hydrolyzed t-PA, PG and u-PA, shows that: (i) P-Ser and P-Tyr residues are present in t-PA; (ii) P-Thr and P-Tyr are present in PG; (iii) P-Ser, P-Thr and P-Tyr are present in u-PA. The utilization of monoclonal anti-P-Ser and anti-P-Thr antibodies in immunoblotting experiments has confirmed these data which indicate that phosphorylation is a common feature of PAS and of PG.
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.