Method for measuring thrombin activity

FIELD: medicine.

SUBSTANCE: method is based on a method of observing turbidimetric fibrin clot formation with optical transmission of an incubation medium recorded by ultraviolet radiation band 230 to 320 nm by means of UV-spectrophotometre as a fibrin-polymer detector.

EFFECT: invention enables higher accuracy and sensitivity of the method.

4 ex, 4 dwg

 

The invention relates to the field of medical research, the pharmaceutical industry and biotechnology, and specifically for determining the activity of thrombin using optical methods.

Thrombin is a protein belonging to the class of serine proteases. According to the Second international standard for thrombin, adopted by the world Health Organization, the activity of the protein is measured in IU (international units). Direct determination of the activity of thrombin is not practiced in laboratories, more reliable is the measurement relative to a commercially available standard drug [1].

Biochemical methods for determining the activity of thrombin can be divided into two groups. First, apply a low molecular weight analogues of the substrate, which are active center of the enzyme with the formation of a colored product. Examples of such substrates are H-D-Phe-Pro-Arg-p-nitroanilide (FPR) [2], H-D-Phe-Pro-Phe-p-nitroanilide (FPF) [3]. The findings characterize the activity of only the catalytic center of thrombin, so it is better to use the methods of the second group.

The second group includes methods that take into account the effective operation of the catalytic triad, and the binding of the natural substrate recognition site of the protease. The activity of thrombin can be calculated based on the curves is aquaplane products of cleavage of PAR1, PAR4, protein C and fibrinogen [3-6]. Otsepleniya peptides are determined by chromatographic methods. However, specialized equipment HPLC are usually not available for everyday use in the laboratory, being forced to turn to the definition of modified thrombin proteins, and not derived peptides. In this area of the developed technique for the determination of fibrin, the product of the proteolysis of fibrinogen by thrombin. Fibrin spontaneous and reversible aggregates with the formation of fibrin threads. Despite the fact that for the clinical application of the developed methods of measuring the amount of fibrin-monomer (based on ELISA) [7], for laboratory studies, the degree of conversion of fibrinogen easier to evaluate opticalmechanical and spectrophotometric method for the formation of fibrin threads.

The essence of the opto-mechanical method consists in mixing the reaction mixture with a magnetic stirrer, which leads to wrap around her fibrin strands, which inhibit the rotation of the stirrer until complete stop. Time stopping the mixer is fixed as the time of the collapse. Despite some of the arbitrariness of the obtained values and the lack of correlation with the kinetic parameters, standardization of reagents and reaction conditions makes the time of collapse is well reproducible parameter. Opto-mechanical IU the od mainly finds application in clinical laboratories, because of the small range defined activity of thrombin (0,3-0,6 IU 300 ál samples), significant error results (50%) and the need for highly specialized equipment - the so-called coagulometer [8, 9]. The results of the opto-mechanical method unreliable reflect the degree of aggregation of fibrin in the case of some mutations, so it is preferable to use spectrophotometric methods aggregated fibrin [9].

In spectrophotometric methods for determining the activity of thrombin is not used, the stirring of the solution during the reaction. In the initial stages of proteolysis solution egregiousness fibrin behaves as a colloidal solution, which is over the reaction proceeds in the gel formed by three-dimensional skeleton of the aggregated fibrin and solution of low molecular weight compounds, held them. The reaction is accompanied by turbidity and viscosity increase, respectively, for monitoring, you can use nephelometry, turbidimetry and viscosimetry.

From RU 2007137972 A, 10.06.2009 known method of measuring the activity of thrombin in whole blood (in the sample), and the measurement of thrombin generation includes the following stages:

- cast layer of the specified sample in contact with fluorophenyl with what stratum thrombin, the thickness of the specified layer is from 0.05 to 5 mm, and the surface area is from 10 to 500 mm2;

- ensuring conditions for the generation of thrombin in the specified form;

- measurement of the fluorescence emitted from the surface of the specified layer fluorescent group, which releases fluorogenic substrate in the enzymatic effects of generated thrombin to the specified fluorogenic substrate.

The concentration of thrombin generated during the analysis, determine as a function of the measured fluorescence released from a fluorescent group. The specified sample of whole blood is diluted up to 10 maximum time. The thickness of the layer of the specified sample is approximately 2 mm or less. Analysis indicated a blood sample is placed in the hole of the tablet, which contains the grid hole size from 50 to 500 microns.

Currently in medical practice is widely used optical turbidimetric method (G.V.Born, V.J.Cross. The aggregation of platelet. // J Physiol, 1963, V.16, P.178-195), based on detection of light transmission suspension of platelets (platelet-rich plasma, obtained by centrifugation of blood). A measure of the aggregation process is graphically recorded an increase in the transmittance of the plasma, due to a decrease in light scattering is robotito as a result of their absorption units, formed under the influence of aggregation inducers.

Turbidimetry method of quantitative chemical analysis that is based on measuring the intensity of light transmitted through the investigated dispersion system.

To date, most of the developed technique based on turbidimetry, which is the closest analogue of the invention.

The closest analogue of the invention is conventional turbidimetric method for determining fibrin strands presented in [10], figure 1. As a source of fibrinogen used pool estramboticos citrate plasma of healthy donors. For activation of coagulation was used bovine thrombin. Registration absorption incubation medium was performed using fotoelektrokalorimetry MKMF-02M with filter 340 nm in a cell with a thickness of 1 cm, thermostatted at 37°C. Graphic registration process was carried out using the recorder CAL-4-003. The standard procedure consisted in the following. In a cuvette with optical layer thickness of 1 cm was made to 1.79 ml of 0.02 M veronlough buffer containing 0,13 NaCl and 1 mm CaCl2; 0.1 ml of plasma and 0.1 ml of the working solution of thrombin, quickly mixed and recorded the changes in absorption over time. Latent period, when no change in turbidity of the medium, estimated time Tlag. This is elicina characterized by the formation of soluble fibrin-polymer and dunacity of protofibril. During this period, the medium remains liquid. The most clear and simple patterns are identified for Tlag: figure 1 presents, as in the investigated range of the inverse of Tlag, linearly dependent on the amount of thrombin added to the sample, and the resulting line goes through the origin (the activity is proportional to the concentration, is determined relative to the standard drug thrombin).

Undoubted advantage of such techniques is the use for detection of spectrophotometers devices, compact and available for laboratory use. Scattering of light by the sample egregiousness fibrin is recorded by the spectrophotometer as the absorption of radiation. The resulting value is a complex function of the shape, size and concentration of particles aggregated fibrin, however, the resulting dependence of the absorption on reaction time allow you to select multiple options installed dependence on the concentration of enzyme, fibrinogen and temperature. Traditionally, the wavelength of the incident radiation is chosen 330-660 nm, i.e. in the area of transparency unpainted proteins. As a parameter, linearly dependent on the concentration of thrombin in the range of 0.03 to 0.3 IU/sample (accurate to about 10%), choose the so-called "lag" time Tlag, i.e. the time during which there is no absorption of the sample [10].

Those who practical objective of the claimed invention is to improve the accuracy and sensitivity of the method, i.e. the ability to define thousandths IU activity of thrombin, as well as simplifying the process.

The goal of the project is achieved by the method for determining the activity of thrombin using the turbidimetric method, including observation of the formation of the fibrin clot by hydrolysis of fibrinogen and registration of transmission incubation medium with ultraviolet band radiation by UV spectrophotometer as a detector fibrin-polymer.

The invention consists in using the UV range for turbidimetric measurements. Organic molecules rapidly absorb UV radiation, but the decreasing wavelength gives the gain in sensitivity turbidimetric method.

Preferably in the process according to the invention uses a temperature of 27°C. All solutions were heated at operating temperature for 15 min the solution Composition is preferably as follows:

0,1±0,005% human fibrinogen;

10x buffer mixture (pH of 7.4±0.1 and 200±10 mm Tris-acetate, 1,4±0.05 M NaCl, 50±5 mm KCl, 10±1 mm MgCl2, 10±1 mm CaCl2);

the human thrombin (0.2 to 50 microns).

The time of addition of a solution of thrombin fix a stopwatch and are really measuring the optical density of the sample at the operating wavelength of 200-300 nm (typically used from the doctrine 230 nm). The measurement stops when reaching the plateau curve of optical density is approximately 40-100 seconds after addition of a solution of thrombin (depending on quality protein and specific activity of thrombin). The quality of protein - specific activity for the enzyme (thrombin), the ability to perform its specific function for fibrinogen, hirudin. The quality of protein depends on technology for protein, storage conditions, shelf-life, the number of cycles of freezing/thawing, etc.

As a parameter obtained curve of optical density is proportional to the activity of thrombin, you can use the slope of the tangent at the inflection point of the curve. This parameter can be interpreted as the maximum rate of accumulation of fibrin-polymer Wmax. The use of preparations of thrombin with a standardized activity allows to obtain the calibration curve is linear for a relatively broad range protease activity.

The method is illustrated by the following examples.

Example 1. Determination of the optimal operating wavelength (figure 2)

The data in figure 2 are consistent with the fact that the scattering of light increases with decreasing wavelength. During the experimental determination of the wavelength of the used solution comprising fibrinogen 2,15 (μm) and thrombin 7,15 (nm). On the larger response and the smallest error points were observed at 230 nm, therefore, this wavelength was used in subsequent experiments. At the same time, proteins absorb in this spectral region, this limitation was overcome by measuring the absorption of the working solution relative to the same solution of fibrinogen, but without the addition of thrombin (fibrinogen was always present in 103-104-fold excess by weight).

Example 2. The calibration curve

As a standard the activity of the protease used thrombin human activity 110 IU/ampoule (NIBSC, UK). As a parameter of the curve is proportional to the activity of thrombin, you can choose the tangent of the slope portion of the curve. The tangent of an angle α is denoted as Wmax(MoE/s, mile optical units per second), showing compliance with this parameter the maximum rate of change of absorption of the sample in time.

Figure 3 presents the calibration curve of the enzymatic activity of thrombin. Thus, demonstrated a linear correlation between drug activity of thrombin in the sample and the parameter Wmax. The obtained calibration curve is approximated by equation (Wmax=524*A+0.5, where A is the activity of thrombin, IU.

The above equation of the calibration curve obtained in the program Excel (Microsoft Office) by linear approximation based Wmax on the number of activity units Tr is Mina, included in the reaction mixture, there is Wmax (MoE/s), x is the activity of thrombin (IU), R2- the value of the accuracy of the approximation.

Example 3. The determination of the activity of an unknown drug thrombin

The determination of the activity of an unknown drug thrombin

In microdelivery UV-permeable cell for spectrophotometer put preheated for 15 minutes at 27°C solutions:

0,1±0,005% human fibrinogen (50 µl);

10x buffer mixture (pH of 7.4±0.1 and 200±10 mm Tris-acetate, 1,4±0.05 M NaCl, 50±5 mm KCl, 10±1 mm MgCl2, 10±1 mm CaCl2) (7 μl);

Water (bidistillate) (to a total volume of the reaction mixture to 70 μl);

the human thrombin (0.01 to 50 μm) (from 1 to 13 μl).

The time of addition of thrombin fix a stopwatch and are really measuring the optical density of the sample at the working wavelength (230 nm).

The measurement stops when reaching the plateau curve of optical density of approximately 40-100 seconds.

Next, add the quantity of the preparation of thrombin with unknown activity so that the resulting value of Wmaxwas within the calibration curve, constructed in example 2. For the reliability of recurrent experimentally selecting the quantity of the preparation of thrombin so that Wmaxwas within the range of values of the calibration curve (see figure 3). The activity made thrombin in what Islaam based on the equation of the calibration curve, in this case, A=(Wmax-0.5)/524, IU. The result can be expressed as a volumetric activity of aThr=A/V (IU/ál), where V is the volume of listed drug (Microlitre).

For example, in the cuvette of a spectrophotometer made 2 μl of the preparation of thrombin with unknown activity, Wmaxwas 8 MoE/s whereas the activity of the deposited amount of thrombin was:

A=(8-0 .5)/524=0,014 IU, and volume activity of αThr=0,014/2-0,007 IU/µl.

Example 4. Determination of the activity of thrombin inhibitors on the example of hirudin.

Hirudin, a specific thrombin inhibitor is a polypeptide (65 amino acids). In microdelivery UV-permeable cell for spectrophotometer put preheated for 15 minutes at 27°C solutions:

0,1±0,005% human fibrinogen (50 µl);

10x buffer mixture (pH of 7.4±0.1 and 200±10 mm Tris-acetate, 1,4±0.05 M NaCl, 50±5 mm KCl, 10±1 mm MgCl2, 10±1 mm CaCl2) (7 μl);

Hirudin (100±10Nm) (1-5 µl);

Water (bidistillate) (to a total volume of the reaction mixture to 70 μl);

The human thrombin (0.1 ám) (from 1 to 8 ál).

Every time you make a certain number of units of activity of thrombin, and the amount of drug hirudin with every experience increase until reaching the minimum value of Wmaxgraduated curve of example 2.

The time of addition of thrombin fix a stopwatch and carry out the measurement of the optical density of the specimen PR is the working wavelength (230 nm).

The measurement stops when reaching the plateau curve of optical density is approximately 40-300 sec.

Figure 4 shows the dependence of the parameter Wmaxthe number of added hirudin. Extrapolating the dependence of Wmax=0, we obtain the number of hirudin with activity equal to the activity made thrombin, which he completely inhibited (i.e. 10 mIU in this case). The result can be represented in the form αHir=A/m (IU/pmol), where m is the amount of substance of hirudin (pmol). Ahir=0.01/0.2=0.05 (IU/pmol).

Optical density measurement stops when reaching the plateau curve of optical density. In the above experiments for the plateau took the change in optical density at 230 nm less than 1 MoA per second.

The composition of the buffer solution and the amount of fibrinogen can be varied in a wide range of concentrations. In the description above are the best option ratio of reagents. The change in the ratio of the reactants affects the sensitivity of the method.

Thus, the use of UV-range enabled in order to increase the sensitivity of the method, i.e. to determine thousandths IU activity of thrombin. Reproducibility, low cost of reagents and simplicity of technical equipment make the way easy for the laboratory determination of the activity of preparations of thrombi is a, the degree of inhibition of autolysis and other processes involving changes in the activity of the protease.

Literature

1. Whitton, C., Sands, D., Lee, T., Chang, A., and Longstaff, C. (2005). Thromb Haemost., 93, 261-266.

2. Papaconstantinou, M.E., Bah, A. and Di Cera, E. (2008). Cell. Mol Life Sci., 65, 1943-1947.

3. Bush, LA., Nelson, R.W. and Di Cera, E. (2006). J. Biol. Chem., 281, 7183-7188.

4. Nieman, M.T. and Schmaier, A.H. (2007). Biochemistry, 46, 8603-8610.

5. Mullin, J.L., Gorkun, O.V., Binnie, C.G. and Lord, S.T. (2000). J. Biol. Chem., 275, 25239-25246.

6. Pineda, A.O., Cantwell, A.M., Bush, L.A., Rose, T. and Di Cera, E. (2002). J. Biol. Chem., 277, 32015-32019.

7. Schutgens, R. E., Haas, F.J., Agterof, M.J., Vos, M. and Biesma, D.H. (2007). Thromb Haemost., 97, 807-813.

8. Spiridonov V.A., horn E.V., Y. Baranov, Dugin T.N., Strukova S.M., Kopylov A.M. (2003). Bioorg. Chem., 29, 1-4.

9. Lefkowitz, J.B., DeBoom, T., Weller, A., Clarke, S. and Lavrinets, D. (2000). Am J Hematol., 63, 149-155.

10. Shcherbak IG, Subbotina TF, Penkova VP, Rumin E.V. (2001). The matters. the honey. chem., 47, 80-89.

The method for determining the activity of thrombin, characterized in that use turbidimetric method, including observation of the formation of the fibrin clot and registration of transmission incubation medium with ultraviolet radiation band from 230 to 320 nm by UV-spectrophotometer as a detector fibrin-polymer.



 

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1 dwg

FIELD: medicine, clinical neurology, neurosurgery.

SUBSTANCE: one should study both activation and aggregation of thrombocytes in blood of carotid artery, at the quantity of thrombocytic active forms being above 70% and the number of aggregated thrombocytes being above 9.0% one should predict the development of cerebral ischemic lesion along with stable focal neurological symptomatology, and at the quantity of thrombocytic active forms being below 30% and the number of aggregated thrombocytes being below 8.0% it is possible to predict positive dynamics in the course of the disease mentioned without developing cerebral ischemic lesion.

EFFECT: higher accuracy of prediction.

2 ex

FIELD: medicine, clinical neurology, neurosurgery.

SUBSTANCE: one should study the level of von Willebrand's factor in patient's carotid artery blood. At its content being below 105% one should predict the development of repeated AICH. The innovation improved information value of testing due to possibility to obtain reliable prediction in latent period, as well.

EFFECT: higher accuracy of prediction.

2 ex, 1 tbl

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