RU2252421C1 - Method for determining tissular plasminogen adjuvant - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method involves incubating sample with plasminogen, plasminogen-to-plasmin transformation stimulator under the tissular plasminogen adjuvant action and chromogenic synthetic plasmin substrate and determining tissular plasminogen adjuvant activity from substrate hydrolysis product concentration colored with the plasmin. Lys-plasminogen pretreated with pancreatic trypsin inhibitor or Glu-plasminogen is used as the plasminogen. Specific HCO-Ala-Phe-Lys-p-ntroanilide is applied as the plasmin substrate. Soluble fibrin-monomer produced by means of thrombine-like enzyme ancistrone from snake poison of is applied as the plasminogen-to-plasmin transformation stimulator under tissular plasminogen adjuvant action. Incubation is carried out at pH=7.0-8.5. The most optimum concentration for Lys- or Glu-plasminogen is equal to 0.3 mcM, and it is equal to 0.6 mM for the specific HCO-Ala-Phe-Lys-p-ntroanilide substrate, and 0.025 mg/ml for the fibrin-monomer.

EFFECT: enhanced sensitivity and accuracy of the method.

2 cl, 9 dwg

Description

The present invention relates to medicine, in particular to a method for determining tissue plasminogen activator (TAP) in biological samples for diagnostic purposes.

TAP and urokinase are endogenous plasminogen activators. They convert plasminogen to plasmin, which dissolves a fibrin clot. TAP, with an affinity for fibrin, plays a key role in in vivo fibrinolysis. Normal plasma contains 4-6 ng / ml TAP. The level of TAP in human blood is lowered with thrombotic and increased with cancer and bleeding. In addition, recombinant TAP is administered intravenously to patients with myocardial infarction and stroke to dissolve an occluded blood clot fibrin. Thus, an accurate determination of the level of TAP in the blood is necessary for the diagnosis of thrombophilia and hemophilia, as well as for monitoring the activity of TAP during thrombolytic therapy.

TAP is secreted by endothelial cells into the circulation and is rapidly removed by the liver (τ _1/2 = 8 min). The blood contains a specific inhibitor of plasminogen activators PAI-1, α ₂ antiplasmin and C ₁ inhibitor, which form inactive TAP / inhibitor complexes with TAP. A typical basal level of active TAP in the blood is 1 ng / ml (0.5 IU / ml) and an inactive TAP / inhibitor complex is 5 ng / ml [Wiman B. et all, J. Lab. Clin. Med. 105, 265-270, 1985]. Usually, TAP is not detected in whole blood; most of the activity of TAP is found in blood plasma. To prevent the interaction of TAP with plasma inhibitors, a blood sample is acidified to pH 4.0 and plasma is secreted [Chmielewska et all, Thromb. Res., 31, 427-436, 1983]. The euglobulin fraction stabilized by citrate plasma contains the activity of TAP and does not contain inhibitors.

There are three different ways to determine TAP: enzyme-linked immunosorbent assay, direct and indirect functional methods. Enzyme immunoassay [Bergsdorf N., Nilsson T., Wallen P. Thromb. Haemost. 50, 740-744 (1993)] allows the determination of up to 1-2 ng / ml of the TAP antigen, but it does not provide information on the activity of the enzyme. The disadvantages of this method include the low availability and high cost of enzyme immunoassay. In a direct functional method, a synthetic chromogenic or fluorogenic specific TAP substrate is incubated with a sample and the rate of formation of the substrate hydrolysis product under the action of TAP is measured [USA Patent No. 4,278,762, (1981)]. However, this method is not sensitive enough. In an indirect functional method, plasminogen is activated by TAP in the presence of a plasmin substrate and the activity of TAP is determined by hydrolysis of the substrate by the resulting plasmin. Indirect functional methods that use a natural substrate of plasmin fibrin include methods based on measuring the area of the lysis zone of a fibrin plate [Astrup T., Mullerts S. Arch. Biochem. Biophys. 40, 346-351 (1952)] or the time for complete lysis of a fibrin clot [Christensen L.R. Proc. Soc. Biol. Med. 46, 674-679 (1941)]. The disadvantages of these methods are the unclear kinetics of the reactions involved in the dissolution of the solid-phase substrate, and the difficulty in interpreting the results.

Significant disadvantages of the three methods described above are corrected by the method closest in technical solution (prototype) described by Verheijen J.P. et all [USA Patent No. 4563420 (1986)]. In this method, a sample in which TAP activity is measured is incubated with Lys-plasminogen and peptide plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251) in the presence of soluble BrCN fragments of fibrinogen as a stimulator of activation of TAP plasminogen. Plasmin (Pl), formed upon activation of plasminogen (PG) TAP, catalyzes the hydrolysis of the substrate (C), and a colored product p-nitroaniline (HA) is formed, which is measured spectrophotometrically at 405 im. The reaction scheme can be represented by equations (1 and 2):

The advantages of this method are: 1) the possibility of a quantitative description of the conjugate process in solution (kinetic parameters K _m , k _CAT , K ' _M and k' _CAT can be determined from the kinetics of individual reactions) and 2) high sensitivity of the method, since fragments of fibrinogen increase catalytic efficiency (k _KAT / K _M ) of TAP plasminogen activation by 2–3 orders of magnitude due to a sharp decrease in Km and some increase in k _KAT .

The disadvantages of the method are the use of: 1) highly toxic cyanide bromine, necessary to obtain fragments of fibrinogen from fibrinogen; 2) a commercial expensive HD-Val-Leu-Lys-pNA plasmin substrate (S-2251), the hydrolysis of which by plasmin is characterized by a high Michaelis constant (K ' _M = 0.6 mM) and insufficient catalytic efficiency (k _CAT / K _M = 42 mM ^-1 s ^-1 ) [Kiss I., Biochem. Biophys. Res. Comm. 131, 928-934 (1985)]) and the TAP activity is measured not at [C] ₀ >> K ' _M (when the speed is maximum), but at [C] ₀ ≈ K' _M , which reduces the sensitivity of the method and 3) Lys-plasminogen, usually containing an admixture of plasmin, which leads to significant background hydrolysis of the substrate and increases the error in determining low concentrations of TAP.

The objective of the proposed method for determining TAP is to increase the sensitivity and accuracy of detection of TAP through the use of a catalytically more effective plasmin substrate and to reduce plasmin impurities in the plasminogen preparation, as well as to simplify the method by using a stimulator of plasminogen activation of TAP obtained without the use of cyanide bromine.

To solve this problem, a method for determining TAP is proposed, which involves incubating a TAP sample with a plasminogen solution in the presence of a chromogenic specific plasmin substrate and a TAP plasminogen activation stimulator, followed by measuring the rate of accumulation of the hydrolysis product of the substrate by the resulting plasmin. According to the invention, Glu-plasminogen, practically free of plasmin impurities, is used as a TAP substrate, or Lys-plasminogen, preferably after inhibiting plasmin impurities in it, as a plasmin substrate, a new domestic specific substrate HCO-Ala-Phe-Lys-p-mtroanilide ( APL-pNA) [Patent N 98100177/14 (1998), Russia] and a soluble Des-AA fibrin monomer obtained with the domestic thrombin-like enzyme ancistron from Agkistrodon halys snake venom is used as a stimulator of TAP plasminogen activation.

Glu plasminogen is isolated from freshly frozen human blood plasma by affinity chromatography on Lys-Sepharose 4B in the presence of a pancreatic trypsin inhibitor (to exclude plasmin formation) according to the method described in [Castellino FJ et al., Methods Enzymol., 80, 365-378 (1981)]. The drug is electrophoretically homogeneous and practically does not contain plasmin impurities.

Lys plasminogen is isolated from human plasma by affinity chromatography on Lys-sepharose 4B in the absence of a pancreatic trypsin inhibitor according to the method described in [Deutsch D.G. et al., Science, 170, 1095-1096 (1970)]. The resulting preparation of Lys-plasminogen, which usually contains 0.5-5% impurities of active plasmin, is treated with an excess of pancreatic inhibitor and purified by ultrafiltration.

To isolate the euglobulin fraction, a plasma sample (100 μl) containing TAP was diluted in 0.9 ml of distilled water, then the solution was acidified by adding 100 μl of 0.25% acetic acid and incubated for 1 h at 4 ° C. After centrifugation for 5 minutes at 2000 rpm supernatant decanted. The resulting pellet was dissolved in 100 μl of test buffer.

To measure a large number of samples in routine experiments, the reaction is carried out in 96-well strip plates using a multichannel spectrophotometer.

The reaction is carried out at 25 ° C or 37 ° C, but the reaction rate at 25 ° C is 3 times lower. Incubation of the reaction solutions at 37 ° C is carried out in closed cells to avoid evaporation.

The reaction is carried out at a pH of 7.0-8.3 in the presence of 0.1% Triton X-100 to eliminate TAP sorption to the vessel walls.

Under saturating conditions (when [Pg] ₀ >> K _M and [C] ₀ >> K ' _M ) and the absence of spontaneous hydrolysis of the substrate, the formation of the p-nitroaniline product during reactions (1 and 2), measured spectrophotometrically at 405 nm, is described the equation

where ε ₄₀₅ is the extinction coefficient of p-nitroaniline.

If plasminogen does not contain plasmin impurities ([Pl] ₀ = 0), then equation (3) is converted to equation

and the reaction rate is proportional to the concentration of TAP.

Example 1

TAPs (0.075 IU / ml) are incubated at 37 ° C in 0.2 ml Tris acetate buffer (0.1 M, pH 7.0) containing 0.4 μM Glu plasminogen, 0.05 mg / ml fibrin monomer, 0.1% Triton X-100 and 1 mM chromogenous plasmin substrate - APL-pNA, in a well of a 96-well strip plate. A control cell that does not contain TAP is used to measure background hydrolysis. During an incubation for 3 hours, the optical density of the sample (A ₄₀₅ ) and control (A ₄₀₅ °) are measured on an Antos 2020 photometer. The reaction rate is directly proportional to the concentration of TAP in the sample, which is determined either from Δ A ₄₀₅ (A _405- A) ₄₀₅ °) after 2 hours of reaction, or from the value of the slope of the dependence of Δ A ₄₀₅ on t ² . As can be seen, in the presence of TAP, the dependence of Δ A ₄₀₅ on t has a parabolic shape (Figure 1, A), and on t ² - a linear view (Figure 1, B).

Example 2

TAP (0.4 IU / ml) is incubated at 25 ° C in 0.2 ml Tris-acetate buffer (0.1 M, pH 8.5) containing 0.4 μM Lys-plasminogen, 0.05 mg / ml fibrin monomer, 0.1% Triton X-100 and 1 mM APL-pNA. The change in optical density of the sample and control (without TAP) is recorded within 9 hours. The activity of TAP is determined by the value Δ A ₄₀₅ observed after 7 hours (Figure 2).

Example 3

TAPs (0.25 IU / ml) are incubated at 37 ° C in 0.2 ml of 0.1 M Tris-acetate buffer (0.1% Triton X-100) with different pH values. The concentrations of Glu plasminogen, fibrin monomer and APL-pNA are the same as in Example 1. At each pH value, background hydrolysis of the substrate in the absence of TAP is measured (A ₄₀₅ °). Figure 3 shows the dependence of Δ A ₄₀₅ / t ² on pH. The optimal pH of the conjugated reaction is 8.3.

Example 4

TAL (0.25 IU / ml) is incubated at 37 ° C in 0.2 ml of 0.1 M Tris-acetate buffer pH 8.3 containing 0.4 μM Glu-plasminogen, 0.1% Triton X-100, 1 mM APL-pNA and different concentrations of fibrin monomer. For each stimulator concentration, background hydrolysis of the substrate in the absence of TAP is measured. From the dependence of Δ A ₄₀₅ / t ² on the concentration of fibrin monomer, it can be seen that a plateau is reached at a stimulant concentration of 0.025 mg / ml (Figure 4).

Example 5

The experiments of example 4 are carried out in the presence of 0.025 mg / ml fibrin monomer and various concentrations of glu-plasminogen. For each concentration of Glu plasminogen, background hydrolysis of the substrate in the absence of TAP is measured. The reaction rate (Δ A ₄₀₅ / t ² ) reaches a maximum at a concentration of Glu plasminogen of 0.3 μM (Figure 5).

Example 6

The experiments of example 4 are carried out in the presence of 0.3 μM Glu-plasminogen, 0.025 mg / ml fibrin monomer and various concentrations of the plasmin chromogenic substrate APL-pNA. Background hydrolysis is measured for each substrate concentration in the absence of TAP. The dependence of Δ A ₄₀₅ / t ² on the concentration of the substrate shows that the plateau is reached at 0.6 mm APL-pNA (Fig.6).

Example 7

0.25 IU / ml TAP is incubated in the presence of 0.3 μM Glu-plasminogen, 0.025 mg / ml fibrin monomer and 1 mM APL-pNA or S-2251 (Sigma, USA) at pH 8.3 and 37 ° C. For each substrate, measure background hydrolysis in the absence of TAP. Fig.7 shows that the rate of the conjugate reaction under the influence of TAL is significantly higher in the presence of APL-pNA used in the proposed method than in the presence of S-2251 used in the prototype. This is due to the higher catalytic efficiency of APL-pNA plasma hydrolysis (k _KAT / K _m = 82 mM ^-1 s ^-1 ) in comparison with S-2251 hydrolysis (k _CAT / K _m = 42 mM ^-1 s ^-1 [Kiss I., Biochem. Biophys. Res. Comm. 131, 928-934 (1985)]). In addition, at the used substrate concentration, APL-pNA plasma hydrolysis (K ' _M = 0.13 mM) occurs under conditions when [С] ₀ >>K' _M and the hydrolysis rate is maximum, while S-2251 (K ' _M = 0.6 mM, [Kiss I., Biochem. Biophys. Res. Comm. 131, 928-034 (1985)]) is hydrolyzed by plasmin under conditions of [C] ₀ ≈ K ' _M.

Example 8

The experiments of example 4 are carried out in the presence of 0.3 μM Glu-plasminogen, 0.025 mg / ml fibrin monomer and various concentrations of standard recombinant TAL (3rd International Standard N 98/714, National Institute of Biological Standards and Control, NIBSC, UK). There is no TAP in the control cell. Each test is repeated three times. From the kinetic curves of the change in optical density versus time, a calibration graph is built: either the dependence Δ A ₄₀₅ / 2h (absorption change over 2 hours of the reaction) on the concentration of TAP (Fig. 8, A), or the dependence Δ A ₄₀₅ / t ² on the concentration of TAP ( Fig.8, B). These line graphs are used to determine TAP in test samples.

Example 9

Different concentrations of standard TAP are added to standard plasma to a final concentration of 1-40 ng / ml. From plasma samples (100 μl) with different TAP contents, precipitates of the euglobulin plasma fraction are isolated, as described above, and dissolved in 100 μl of the test buffer. In the experiments of example 8, as a test sample, solutions of plasma euglobulin fraction with different content of TAL are pre-diluted 20 times. The activity of TAP in the samples is determined by the value Δ A ₄₀₅ / t ² using a calibration curve (Fig. 8, B). Fig.9 illustrates a linear dependence of the activity of TAP, measured by the present method, on the concentration of TAP in human plasma.

Claims (2)

1. A method for determining tissue plasminogen activator, which consists in the fact that the sample is incubated with plasminogen in the presence of a plasmin substrate and a plasminogen to plasmin stimulator under the action of tissue plasminogen activator, and the stained plasmin hydrolysis product of the substrate is measured, characterized in that Lys- is used as plasminogen plasminogen pretreated with a pancreatic trypsin inhibitor, or Glu plasminogen, a specific HCO substrate is used as a plasmin substrate -Ala-Phe-Lys-p-nitroanilide and as a stimulator of the conversion of plasminogen to plasmin by the action of a tissue plasminogen activator, a soluble fibrin monomer obtained using the thrombin-like enzyme ancistron from the venom of the snake Agkistrodon halys is used, incubation is carried out at pH 7.4-8 ,5. 2. The method according to claim 1, characterized in that Lys or Glu plasminogen is used at a concentration of 0.025-0.3 μM, a specific substrate of HCO-Ala-Phe-Lys-p-nitroanilide is used at a concentration of 0.6 mm and fibrin the monomer is used in a concentration of mg / ml.

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