Mats Rånby

Evidence for a rapid inhibitor to tissue plasminogen activator in plasma

An improved assay method for tissue plasminogen activator (t-PA) in plasma samples is described, which allows the plasma t-PA activity to be determined within 3 hours. The method minimizes the action of possible inhibitors by immediate acidification of blood samples prior to separation of the blood cells. In almost all plasma samples variable amounts of a rapid t-PA inhibitor could be demonstrated. The inhibitor was present in plasma and seemed not to be associated with the blood cells. A quantitative method for determination of the inhibitor content in plasma was developed. In this investigation 1 unit of inhibitor is defined as the amount that neutralized 1 unit (0.06 pmol) of t-PA in 10 minutes. The content of t-PA inhibitor in plasma samples from healthy individuals was determined as 0.6 U/ml +/- 0.5 U/ml (range 0-3.2, median 0.4). In a group of patients (n = 75) with suspected disturbance of the haemostatic system, elevated levels were found (3.8 +/- 2.7, U/ml median 3.6 U/ml, range 0 - 15 U/ml). Kinetic analysis with plasma from three patients with elevated inhibitor content showed very similar rate of t-PA inhibition and assuming the formation of a 1:1 t-PA-inhibitor complex a second order rate constant of 10(7) M(-1) s(-1) was calculated. A regulatory function may be anticipated for this inhibitor, but its exact patophysiological role has still to be established.

Studies on the kinetics of plasminogen activation by tissue plasminogen activator

The steady-state rate of plasminogen activation by tissue plasminogen activator has been determined at various plasminogen concentrations. A plasmin substrate method similar to that presented by Christensen and Müllertz (Biochim. Biophys. Acta 480 (1977) 257-281) was used. The reaction was studied using one-chain type and two-chain type tissue plasminogen activator, N-terminal glutamic acid and N-terminal lysine plasminogen in the presence and in the absence of fibrin (eight studies). The kinetic data were fitted to a general Wong-Hanes equation and the simplest equation with significant parameters was found. In the absence of fibrin N-terminal glutamic acid plasminogen activation obeyed the Michaelis-Menten rate equation (Km 4.9 and 7.6 micro M and kcat 0.0013 and 0.0078 s-1 for one-chain type and two-chain type tissue plasminogen activator, respectively. In the absence of fibrin the activation of N-terminal lysine plasminogen activation failed to obey the Michaelis-Menten rate equation. Fibrin was found to stimulate greatly (up to 1000-fold) the steady-state activation rate. A theory for the fibrin stimulating mechanism is presented.

Plasminogen activator release during venous stasis and exercise as determined by a new specific assay

A method for the specific determination of tissue plasminogen activator in plasma samples has been developed. The method is based on a recently described parabolic rate assay for tissue plasminogen activator (Rånby and Wallén, 1980) measuring plasmin activity by a chromogenic tripeptide-paranitroanilide substrate. The potential interference by plasmin inhibitors in plasma is overcome by acidification of the plasma samples to pH 4 for 15 min prior to the analysis. The baseline activity (after 10 min of rest) in 22 healthy individuals was determined to be 0.05 +/- 0.03 (SD) IU/ml (0.2 +/- 0.1 ng/ml). After venous occlusion for 10 min at 100 mm Hg the activator concentrations had increased to 1.2 +/- 1.2 (SD) IU/ml (5.2 +/- 5.2 ng/ml). Furthermore, prior to venous occlusion almost no plasmin-alpha 2-antiplasmin complex was found in the samples, whereas a pronounced increase in this complex was noted in 90% of the subjects after venous occlusion. During exercise, a gradual work-load dependent increase of tissue plasminogen activator concentration was observed. At maximal work-load the concentration was 2.3 +/- 1.9 (SD) IU/ml (9.9 +/- 8.1 ng/ml active enzyme), which after 10 min rest decreased to 26 +/- 12 (SD) % of the values at maximal work-load. In these samples only small amounts of plasmin-alpha 2-antiplasmin complex were detected, although the activator content was of the same order of magnitude as after venous occlusion.

Enzymatic properties of the one-and two-chain form of tissue plasminogen activator

Abstract The properties of the one-chain and two-chain forms of the porcine tissue plasminogen activator have been compared with respect to their amidolytic activities against a low molecular weight synthetic substrate, and their inhibition by diisopropylphosphofluoridate, α2-antiplasmin and C1-esterase inhibitor. The two-chain form is the more efficient catalyst, and is more rapidly inhibited by diisopropylphosphofluoridate and α2-antiplasmin. Physiological implications are discussed.

Purification and identification of two structural variants of porcine tissue plasminogen activator by affinity adsorption on fibrin

Porcine tissue plasminogen activator has been purified from delipidized heart tissue by affinity adsorption to fibrin. A crude fraction is prepared from an acid tissue extract by precipitation with ammonium sulphate. The tissue activator of this fraction is isolated by adsorption on fibrin and elution with KSCN. The procedure also includes chromatography on arginine-Sepharose and two gel-filtration steps. The final product has a specific activity of 250 000 IU/mg (+/- 16 000) as compared to an international urokinase reference preparation. The yield calculated from the active ammonium sulphate precipitate is about 28%. An approx. 7 000-fold increase of specific activity is obtained, most of which is achieved in the fibrin step. The native tissue plasminogen activator consists of a single chain molecule with a molecular weight of 64 000 as measured by SDS-polyacrylamide gel electrophoresis. In a previous report, it was claimed that the activator is composed of two disulphide-connected polypeptide chains. These results were due to a preparation artefact, caused by proteolytic activity present in the tissue extracts. The introduction of the protease inhibitor aprotinin and 6-amino-hexanoic acid in the purification procedure has abolished the effect of the protease contaminant, leading to the production of a one-chain activator. Treatment with plasmin transforms the native, one-chain tissue activator into a variant composed of two chains of about equal size (Mr 32 000) connected by disulphide bonding. This modified activator is indistinguishable from the one obtained at insufficient protection against proteolytic enzymes. The cleavage by plasmin causes about an 8-fold increase of amidolytic activity as measured on H-D-Val-Gly-Arg-p-nitroanilide. The fibrinolytic activity as measured by clot lysis in only slightly increased. The physiological significance of the cleavage is discussed.

Fibrinolytic variables are related to age, sex, blood pressure, and body build measurements: a cross-sectional study in Norsjö, Sweden.

Levels of the fibrinolytic variables, tissue plasminogen activator (tPA) antigen concentration and plasminogen activator inhibitor (PAI-1) activity, were measured in a cross sectional sample of 260 subjects aged 30, 40, 50, or 60 years. There was a significant increase of tPA with age in both sexes, but PAI-1 increased only in women. Linear regression analysis was used to assess relations between tPA or PAI-1 and the anthropometric data. In men, tPA levels were related to body mass index and waist-to-hip ratio, whereas in women, it was also related to systolic and diastolic blood pressures and with abdominal or triceps skinfold thicknesses. PAI-1 levels were related to body mass index and waist-to-hip ratio in men, and in women it was in addition related to systolic and diastolic blood pressures and to abdominal and triceps skinfold thicknesses. These data offer new insight into pathophysiological mechanisms whereby age, sex, blood pressure, and body composition variables such as body mass index or waist-to-hip ratio, might act as cardiovascular risk factors.

Isolation of two variants of native one-chain tissue plasminogen activator

The native one‐chain tissue plasminogen activator from the human melanoma cell line (Bowes) occurs in two variants as demonstrated by sodium dodecylsulphate—polyacrylamide gel electrophoresis of reduced and carboxymethylated samples. The two variants were isolated by affinity chromatography on arginine—Sepharose using a guanidinium—HCl gradient. in the order of elution, the variants were designated tissue plasminogen activator I and II. Variant I was found to be 3000 M r larger than variant II and the difference was found to reside in the N‐terminal half of the molecule. No substantial difference in carbohydrate content nor in enzymatic activity was demonstrated.

Tissue plasminogen activator, plasminogen activator inhibitor-1 and von willebrand factor in rheumatoid arthritis

SummaryTissue plasminogen activator (tPA), plasminogen activator inhibitor-1 (PAI-1) and von Willebrand factor (vWF), all of endothelial origin and active in the haemostasis, were analysed in 74 patients with rheumatoid arthritis. The concentrations were related to extra-articular disease and to the incidence of thromboembolic events (TE) registered in a 2-year follow-up period. Patients with extra-articular disease had a significant increase in PAI-1 activity and reduced tPA release in the venous occlusion test. von Willebrand factor, PAI-1 and also haptoglobin and triglycerides were significantly increased in the group of patients who later suffered from TE. In a multiple regression model, in which cholesterol, triglycerides and lipoprotein (a) showed significant association with TE, vWF had the strongest additive explanatory value. No distinct acute phase pattern of PAI-1 was found in any patient subgroup.

THE TISSUE ACTIVATOR OF PLASMINOGEN

Publisher Summary This chapter provides an overview of the tissue activators of plasminogen. The central reaction in the fibrinolytic enzyme system, the conversion of plasminogen to plasmin, is a proteolytic process. The single chain proenzyme is converted to the active enzyme consisting of two chains, the B (light) chain containing the active site and the A (heavy) chain. In the NH 2 -terminal part of the original plasminogen (glu-plasminogen), there is an internal non-covalent interaction, the dissociation of which seems to induce conformational changes of importance for activation kinetics. The highly restricted proteolysis, which leads to the activation of plasminogen, is accomplished by several more or less well defined activators. The best known activators from mammalian sources are urokinase and the tissue activator originating from kidneys and vessel walls, respectively. The activation of plasminogen may also be related to pathological processes.

Diet-induced changes in glucose and triglycerides are associated with changes in plasminogen activator inhibitor levels

Twenty-four healthy female subjects volunteered to participate in an experiment in which they maintained a high-fat/low-carbohydrate (CHO) diet followed by a low-fat/high-CHO diet or vice versa in a cross-over study design. In bivariate correlation analysis, only glucose (r = 0.52, p less than 0.05) and triglycerides (r = 0.53, p less than 0.05) correlated with changes in plasminogen activator inhibitor type 1 (PAI-1) activity. A second-degree polynomial response surface model suggested that transition from a high-fat/low-CHO diet to a low-fat/high-CHO diet is associated with reduced levels of PAI-1 provided that glucose and triglyceride levels are not elevated by more than 1.2 and 0.5 mmol/l, respectively.