Kinetics of inhibition of tissue-type and urokinase-type plasminogen activator by plasminogen-activator inhibitor type 1 and type 2

Highly purified plasminogen-activator inhibitors of type 1 (PAI-1) and type 2 (PAI-2), low-Mr form, were compared with respect to their kinetics of inhibition of tissue-type (t-PA) and urokinase-type plasminogen activator (u-PA). The time course of inhibition of plasminogen activator was studied under second-order or pseudo-first-order conditions. Residual enzyme activity was measured by the initial rate of hydrolysis of a chromogenic t-PA or u-PA substrate or by an immunosorbent assay for t-PA activity. PAI-1 rapidly reacted with single-chain t-PA as well as with two-chain forms of t-PA and u-PA. The second-order rate constant k for inhibition of single-chain t-PA (5.5 x 10(6) M-1 s-1) was about three times lower than k for inhibition of the two-chain activators. PAI-2 reacted slowly with single-chain t-PA, k = 4.6 x 10(3) M-1 s-1. The association rate was 26 times higher with two-chain t-PA and 435 times higher with two-chain u-PA. The k values for inhibition of single-chain t-PA, two-chain t-PA and two-chain u-PA were respectively, 1200, 150 and 8.5 times higher with PAI-1 than with PAI-2. The removal of the epidermal growth factor domain and the kringle domain from two-chain u-PA did not affect the kinetics of inhibition of the enzyme, suggesting that the C-terminal proteinase part of u-PA (B chain) is responsible for both the primary and the secondary interactions with PAI-1 and PAI-2. The k values for inhibition of single-chain t-PA and endogenous t-PA in plasma by PAI-1 or PAI-2 were identical indicating that t-PA in blood consists mainly in its single-chain form.     

Reversible interactions between plasminogen activators and plasminogen activator inhibitor-1.

We have shown that the urokinase (UK) kringle domain contains a high-affinity plasminogen activator inhibitor-1 (PAI-1) binding site, responsible for the 10-fold faster complex formation between UK and PAI-1 than between PAI-1 and low-molecular-weight urokinase (LMWUK). Complex formation between UK and PAI-1, but not between LMWUK and PAI-1, was suppressed 10-fold in the presence of peptide U-107 derived from the UK kringle domain. Peptide U-373 derived from the UK catalytic domain slowed complex formation between UK and PAI-1 and also LMWUK and PAI-1. Inactivation of tissue-type plasminogen activator (tPA) by PAI-1 was slowed 10-fold in the presence of peptides derived from the tPA finger and kringle-2 domains. DFP-inactivated (DIP) UK and both forms of DIP-tPA inhibited PAI-1 binding to U-107 and to U-373 whereas single-chain urokinase-type PA (scuPA) was unable to compete with either peptide for PAI-1 binding. These data suggest that the reversible PAI-1 binding site in the UK A-chain plays a role in the rapid association with PAI-1 as important as those that reside in the tPA A-chain and that reversible PAI-1 binding sites are expressed on the surface of UK upon conversion from scuPA, in contrast to tPA.     

The mechanism of the reaction between human plasminogen-activator inhibitor 1 and tissue plasminogen activator.

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-tPA is demonstrated by its capacity to cleave the 'bait' peptide bond in PAI-1. The complexes were inactive and dissociated slowly at physiological pH and ionic strength, but rapidly in aq. NH3 (0.1 mol/l). Amidolytic tPA activity was generated on dissociation of the complexes, corresponding to 0.4 mol of tPA/mol of complex. SDS/PAGE of the dissociated complexes indicated a small decrease in the molecular mass of PAI-1, in agreement with proteolytic cleavage of the 'bait' peptide bond during complex-formation.     

Mechanisms of plasminogen activation by mammalian plasminogen activators

Plasminogen activators convert the proenzyme plasminogen to the active serine protease plasmin by hydrolysis of the Arg560-Val561 peptide bond. Physiological plasminogen activation is however regulated by several additional molecular interactions resulting in fibrin-specific clot lysis. Tissue-type plasminogen activator (t-PA) binds to fibrin and thereby acquires a high affinity for plasminogen, resulting in efficient plasmin generation at the fibrin surface. Single-chain urokinase-type plasminogen activator (scu-PA) activates plasminogen directly but with a catalytic efficiency which is about 20 times lower than that of urokinase. In plasma, however, it is inactive in the absence of fibrin. Chimeric plasminogen activators consisting of the NH2-terminal region of t-PA (containing the fibrin-binding domains) and the COOH-terminal region of scu-PA (containing the active site), combine the mechanisms of fibrin specificity of both plasminogen activators. Combination of t-PA and scu-PA infusion in animal models of thrombosis and in patients with coronary artery thrombosis results in a synergic effect on thrombolysis, allowing a reduction of the therapeutic dose and elimination of side effects on the hemostatic system.     

Fluorometric microassay of plasminogen activators.

A new method for determining plasminogen activator levels has been developed. Data are presented which demonstrate measurements of trypsin, plasmin, urokinase, and plasminogen activation. The assay is based on the digestion of N-terminal-blocked protamine and subsequent measurement of the exposed amino groups using the fluorogenic amine reagent, Fluram. The soluble substrate provides an assay which is linear with respect to both time and concentration and which is sensitive enough to allow measurements on a microscale. As little as 1 ng of trypsin, 0.002 CTA units (established by the Committee on Thrombolytic Agents of the NIH) of plasmin, and 0.01 CTA units of urokinase can be detected under the conditions described.Interference with the amine determination due to Fluram-positive material found in biological samples is minimized with the high dilutions attainable with the system. Plasminogen activator in the urine of the female mouse can be detected using 1 μl of urine in a 200-μl test system.     

Identification of a reversible inhibitor of plasminogen activators in blood plasma

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_i 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.

Fibrinolysis Tissue-type plasminogen activator Urokinase Blood plasma Endothelial cell type plasminogen activator inhibitor Protease inhibitor     

Production of three plasminogen activators and an inhibitor in keratinocyte cultures

Keratinocyte function in extracellular proteolysis was investigated. Keratinocytes derived from rat tongue ventral epithelium were maintained and serially propagated under conditions which support continuous expansion of epithelial colonies but are restrictive to fibroblast proliferation (30-32 degrees C and pH 6.8-7.0). These cultures, and cultures of an established, terminally differentiating keratinocyte line, also derived from the ventral epithelium of the rat tongue, released substantial plasminogen activator activity as visualized by the fibrin-agar overlay technique. In addition, keratinocytes grown directly on 3H-labeled fibrin lysed this substrate in a plasmin-assisted process. The presence of serum modulated the kinetics of the reaction in a manner which suggests that a constant inhibitor tonus serves to contain the proteolytic reaction in the tissues and to prevent a chain reaction. Electrophoretic resolution of keratinocyte secretory proteins and of cell lysates revealed three distinct activators migrating at molecular weights of 48 000, 66 000 and 95 000. The keratinocytes also manufactured inhibitor(s) of the fibrinolytic reaction mainly directed against the activation step. The inhibitory activity was present in serum-free culture harvest media in quantities sufficient to completely annihilate the endogenous activators.     

Characterization of a macrophage-derived plasminogen-activator inhibitor. Similarities with placental urokinase inhibitor.

Human and mouse macrophages release a fibrinolytic inhibitor after stimulation by endotoxin in vitro. The released mouse inhibitor was indistinguishable in size by molecular-sieve chromatography from an intracellular form (approx. 50 kDa), and both inhibitors blocked urokinase directly as judged by a 125I-plasminogen conversion assay. The intracellular inhibitor was found mostly to dissociate from 125I-urokinase during sodium dodecyl sulphate/polyacrylamide-gel electrophoresis under reduced conditions, but a dodecyl sulphate-stable complex at 65-67 kDa was observed. Because of similarities in the reported size, stability and urokinase-binding properties of a placental urokinase inhibitor, the kinetic properties of the two inhibitors were compared. Under the reaction conditions employed (37 degrees C at pH7.4 in the presence of 0.2% Triton X-100), the association rate constants and equilibrium dissociation constants of the two inhibitors were indistinguishable, 3 X 10(5) M-1 X s-1 and 4 X 10(-10) M respectively. These data show that peritoneal macrophages contain a plasminogen-activator very similar to a previously recognized placental inhibitor. Although the inhibitor appears to be a trace protein in macrophages, placental macrophages may account for the accumulation of the inhibitor in placental tissue.     

Endothelial cells produce a latent inhibitor of plasminogen activators that can be activated by denaturants

Conditioned medium from cultured bovine aortic endothelial cells contains an inactive plasminogen activator inhibitor (PAI). This latent PAI can be "activated" with denaturants. For example, less than 0.01 units/microliter of PAI activity was detected in untreated conditioned medium, but medium treated with sodium dodecyl sulfate (1.7 mM), guanidine HCl (4 M), urea (12 M) or KSCN (6 M) contained 0.9, 1.9, 0.8, and 0.5 units/microliter, respectively. This effect was dose-dependent with respect to the particular reagent used, and the same concentration of reagent which induced PAI activity also stimulated the ability of a component in conditioned medium to form sodium dodecyl sulfate-stable complexes with exogenously added plasminogen activators. Neither activity was stimulated by extensive dialysis or by treatment with NaCl (5 M), Na2SO4 (2.8 M), or dicetyl phosphate (0.1%). Analysis of treated and untreated conditioned medium by gel filtration revealed that the latent and active PAIs migrated with apparent Mr values of 30,000 and 50,000, respectively. Thus, "activation" is associated with an increase in the apparent Mr of the molecule. These observations suggest that activation does not result from the removal of either a small dialyzable component from the medium, or of a large Mr component that is bound to the latent PAI. Other possible mechanisms of activation are discussed. We recently isolated an active PAI from bovine endothelial cells (van Mourik, J.A., Lawrence, D.A., and Loskutoff, D.J. (1984) J. Biol. Chem. 259, 14914-14921). Monospecific antiserum to this active PAI selectivity immunoprecipitated the latent PAI from conditioned medium. These results indicate that the two PAIs are immunologically related and suggest that the latent form is converted into the active form by the sodium dodecyl sulfate present during the purification.     

Concentration of plasminogen activators and inhibitor in the human endometrium at different phases of the menstrual cycle.

The concentrations of tissue plasminogen activator (t-PA), urokinase plasminogen activator (u-PA) and plasminogen activator inhibitor (PAI-1) have been determined in endometrial curettings obtained from 46 subfertile women during proliferative, early or late secretory phases of the menstrual cycle. t-PA activity and antigen concentrations was significantly higher (P < 0.001) in late secretory endometrium than in proliferative or early secretory endometrium. Higher concentrations of PAI-1 antigen (P < 0.05) were also noted in late secretory phase than in proliferative and early secretory endometrium. However, u-PA concentration was not significantly different and no PAI activity could be demonstrated in the menstrual phases studied. Zymography studies confirmed the presence of both t-PA and u-PA in the endometrium. Ovarian hormonal patterns may therefore influence the activity of plasminogen activators especially of t-PA in the endometrium during various phases of the menstrual cycle.     

Activation of plasminogen by pro-urokinase. II. Kinetics

The kinetics of the activation of plasminogen by recombinant pro-urokinase obtained by expression of human urokinase cDNA in Escherichia coli was studied. The conversion of pro-urokinase (U) and plasminogen (P) to urokinase (u) and plasmin (p) is represented by a sequence of three reactions which each obey Michaelis-Menten kinetics, i.e. (Formula: see text). In this model, pro-urokinase formally behaves as an enzyme in Reaction I and as a substrate in reaction II. The experimentally measured overall rates of formation of urokinase and plasmin are in good agreement with those calculated from the kinetic parameters and the initial concentrations of pro-urokinase and plasminogen, confirming the validity of the model. It appears that recombinant pro-urokinase is an equally potent activator of plasminogen (k2/Km = 0.05 microM-1 s-1), as in urokinase (k"2/K"m = 0.02 microM-1 s-1). This is due to the fact that the proenzyme, which is virtually inactive toward low Mr substrates for urokinase, forms an intermediate of the Michaelis-Menten type with plasminogen, with a much higher affinity than that of the active enzyme with its substrate. This is an exceptional phenomenon among the serine proteases.     

Platelet plasminogen activator inhibitor: purification and characterization of interaction with plasminogen activators and activated protein C

Plasminogen activator inhibitor (PAI) was purified in active form from porcine platelets under nondenaturing conditions. The purified inhibitor (Mr 47,000) reacts with tissue-type plasminogen activator (t-PA), urokinase (UK), and activated protein C (APC) to yield both SDS-stable complexes and a modified PAI of slightly reduced molecular weight. The second-order rate constants for the inhibition of t-PA and UK by PAI are 3.5 X 10(7) and 3.4 X 10(7) M-1 s-1, respectively. Activated protein C reacts with PAI with a second-order rate constant of 1.1 X 10(4) M-1 s-1. This rate is not accelerated by protein S, phospholipid, and calcium, or heparin. It is concluded that (1) PAI can function as both inhibitor and substrate of its target proteases, (2) if APC promotes fibrinolysis via inactivation of PAI, then APC must be present in concentrations several orders of magnitude greater than t-PA, or the interaction of APC and PAI must be accelerated by presently unknown mechanisms, and (3) in the absence of heparin, platelet PAI is the most rapid inhibitor of APC yet described.     

人子宫内膜纤蛋白溶酶元激活因子及其抑制因子...

Two types of plasminogen activator (PAs) are present in human endometrium, and their contents vary with the different phases of menstrual cycle, i.e. high in the proliferative phase and low in the secretory phase. In the present study by immunohistochemical technique, both uPA and tPA antigens were demonstrated in the stromal and glandular cells of the endometrium. In cell culture, tPA was released only from stromal cells and uPA only from glandular cells as determined by SDS-PAGE followed by fibrin overlay technique, but PA inhibitor type-1 (PAI-1) was secreted by both stromal and glandular cells. Furthermore, secretion of PAs from endometrial cells was enhanced by adding estradiol and markedly inhibited by progesterone in a dose dependent manner, while the PAI reacted just in the opposite way. The effect of the peptide hormones, hCG, GnRH, PRL, as well as cAMP in cell culture on the secretion of PAs and PAI was similar to that of estradiol, while forskolin demonstrated definitely more stimulative effect on tPA than uPA. Taking into account of the finding of the present study, it appears that, under hormonal control, a balance between PAs and PAI in the endometrium exists. The physiological roles of the PAs and PAI in the endometrium were discussed.     

Production of human plasminogen activators by cell culture

Plasminogen activators are a group of enzymes which play a crucial role in the breakdown of blood clots. The plasminogen activators currently used in medicine to remove clots from veins and arteries suffer from several disadvantages, but new cell culture techniques or cloned genes could make available safer and cheaper enzymes.     

Evidence for a plasma inhibitor of the heparin accelerated inhibition of factor Xa by antithrombin III.

The ability of heparin fractions of different molecular weight to potentiate the action of antithrombin III against the coagulation factors thrombin and Xa has been examined in purified reaction mixtures and in plasma. Residual thrombin and Xa have been determined by their peptidase activities against the synthetic peptide substrates H-D-Phe-Pip-Arg-pNA and Bz-Ile-Gly-Arg-pNA. High molecular weight heparin fractions were found to have higher anticoagulant activities than low molecular weight heparin when studied with both thrombin and Xa incubation mixtures in purified mixtures and in plasma. The inhibition of thrombin by heparin fractions and antithrombin III was unaffected by other plasma components. However, normal human plasma contained a component that inhibited the heparin and antithrombin III inhibition of Xa particularly when the high molecular weight heparin fraction was used. Experiments using a purified preparation of platelet factor 4 suggested that the platelet-derived heparin-neutralizing protein was not responsible for the inhibition.     

Detection of cathepsin B, plasminogen activators and plasminogen activator inhibitor in human non-small lung cancer cell lines

Human non-small lung cancer cell lines HS-24 (established from a primary squamous cell carcinoma) and SB-3 (established from a metastasis of a primary adenocarcinoma of the lung into the adrenal gland) were analysed for the proteinases tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator inhibitor (PAI-1). The proteinases were characterized by activity measurements, inhibition studies, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis. Cell-associated proteinases were determined in cell lysates, secreted proteinases in cell conditioned culture media. Both cell lines were found to secrete uPA and PAI-1, whereas tPA could be detected only in HS-24 conditioned media. No cathepsin B activity could be detected in media of both cell lines. However, activation experiments and western blot analysis showed, that at least HS-24 secrete an inactive precursor. Cell lysates of HS-24 and SB-3 show PA activity, but on a low level. Cathepsin B activity was also found to be low in HS-24 lysates. However, SB-3 lysates show high cathepsin B activity. Further characterization of the proteinases by their sensitivity against several inhibitors suggests that they are similar to the corresponding proteinases of normal, nonmalignant cells.