Kit for measurement of thrombin formation in sampled patient's blood or plasma

FIELD: medicine.

SUBSTANCE: specified kit contains a dried complex including tissue factor/phospholipid and a dried mixture containing thrombin substratum and CaCl2. There is disclosed method for preparing the dried complex including tissue factor (TF/phospholipid (PL). There is offered method for preparing said dried mixture containing thrombin substratum and CaCl2. Besides, there is described method for measuring thrombin formation in a sample by measuring the concentration of thrombin.

EFFECT: invention allows detecting kinetic changes in thrombin formations after introduction of agents action of which is not related to inhibitor.

22 cl, 5 dwg, 2 tbl, 6 ex

 

The SCOPE of the INVENTION

The present invention relates to a kit for measuring the formation of thrombin in blood serum or plasma of a patient or in a sample containing a blood coagulation factors. This invention also relates to methods for producing reagents for the specified set.

BACKGROUND of INVENTION

Haemophilia A is a hereditary disorder of coagulation (clotting) of blood. It occurs as a result of insufficient activity of the plasma protein factor VIII, which is involved in the blood clotting process. Usually patients with hemophilia And treated by infusion concentrates of factor VIII to replace the defective clotting factor. However, patients who develop inhibitors of factor VIII in the process of substitution therapy, unable to respond to the above therapy and treated with preparations containing activated coagulation factors (so-called parallel tools)to achieve hemostasis regardless of factor VIII, through parallel mechanisms. For the treatment of patients who develop inhibitors of factor VIII, it is preferable to use concentrates, activated prothrombin complex (APCC), such as FEIBA (Factor Eight Inhibitor Bypassing Activity-plasma-derived APCC), which triggers a natural or normal way, and activated factor VIIa (rFVIIa), such NovoSeven (recombinant FVIIa), which presumably acts through the alternate path.

In both these modes ensure immediate detection of drugs is impossible due to the fact that activated components parallel funds directly interact with proteins of the hemostatic system, causing activation of the coagulation cascade. All existing analyses focused on the detection of surrogate markers, which are of limited value for evaluating the effectiveness of parallel means, as the specificity and sensitivity depend on the specific conditions of the analysis and because these analyses do not give any information on the overall status of the activity of the hemostatic system.

The ultimate goal of the coagulation cascade is the conversion of prothrombin to thrombin, which then induces the formation of thrombus through the activation of fibrinogen. Thus, the formation of thrombin is the main function of plasma hemostasis. Currently there is no standard analysis to quantify the ability of a sample of plasma to the formation of thrombin. The clotting time of blood, such as prothrombin time (PTT), activated partial thromboplastin time (aPTT) and thrombin time (TCT), does not reflect the General education thrombin, since most of thrombin formed after SVER is ivania and therefore, these indicators are insensitive to the hypercoagulable state and possibly also to the state gipokoagulyatsii. Through analysis of PTT measure the clotting time, which is implemented in the back and the usual way; analysis measure aPTT clotting time, implemented through a natural and normal way, while using analysis TCT measure the clotting time sold only through the usual way. Hemker et al. (1986. Thromb. Haemost. 56:9-17) was first proposed to measure the formation of thrombin in the plasma of the patient by assessing the endogenous thrombin potential. The formation of thrombin is a dynamic process. The actual concentration of thrombin depends on the speed of the reactions of activation and inactivation and, therefore, reflects the effectiveness of the hemostatic system in relation to suppress bleeding. Hemker et al. (1995. Thromb. Haemost. 74:134-138) determine thrombin potential as the overall ability of the plasma to form thrombin after induction of coagulation and propose to use this option as a sensitive indicator of each form anticoagulation treatment.

Sultan and Loyer (1993. J. Lab. Clin. Med. 121:444-452) use this analysis of the formation of thrombin to assess not associated with the factor VIII activity concentrates, activated prothrombin complex concentrates, prothrombin complex is a and factor VIII in plasma of patients producing inhibitors of factor VIII. Published also other descriptions such analyses used to evaluate the effectiveness of the funds, which are not associated with factor VIII, however, since conducting these analyses due to technical difficulties, they are not suitable for routine use.

Analyses of the formation of thrombin is also described Turecek at al. (2003. Pathophysiol. Haemost. Thromb. 33:16-22), but without the use of lyophilised components. Moreover, there is a widespread belief that the lyophilization reduces the activity of tissue factor.

All known previous analyses of the prior art have the disadvantage that components can only partially be dosed before applying the analysis. Therefore, to apply these analyses need to spend a lot of stages, that makes the data analyses uncomfortable and susceptible to errors arising in the process of working with them. In addition, these analyses require a large investment of time.

Therefore, there is a need in the analytical system, which enables to detect and evaluate changes in the kinetics of formation of thrombin, such as changes associated with treatment, blood sample or plasma of the patient and which allows to overcome the above disadvantages.

BRIEF DESCRIPTION OF THE INVENTION

The aim of the present invention is the provision of a kit for measuring the formation of thrombin in blood serum or plasma of a patient or in a sample containing a blood coagulation factors. This set includes freeze-dried complex of tissue factor (TF)/phospholipid (PL) and lyophilized mixture containing the substrate of thrombin and CaCl2. In addition, the kit of the present invention may also contain any auxiliary tools, such as buffers, salts such as CaCl2standards of thrombin, standards FEIBA and other, frozen or dried form. The kit of the present invention may be present in any form, for example it can be immobilized on the carrier.

Unexpectedly, it was found that freeze-dried complex TF/PL and lyophilized mixture containing the substrate of thrombin and CaCl2is a simple, effective and does not require time-consuming analytical system for measuring the formation of thrombin in the sample, which allows to obtain reproducible results. The kit of the present invention includes, at least, freeze-dried complex TF/PL and lyophilized mixture containing the substrate of thrombin and CaCl2in easy to handle dried form, so that the analysis requires only the addition of clicks is CA. Lyophilized complex TF/PL and specified lyophilized mixture containing the substrate of thrombin and CaCl2set of the present invention can also be mobilitat on the media, such as the inner surface of the bottle or the wells of a microplate, in the analysis of the formation of thrombin is translated into an analytical format, such as in the traditional analysis of ELISA, which makes it very convenient type of analysis. Analysis using the kit of the present invention has the same sensitivity when determining the formation of thrombin, which analyses the previous level of technology conducted using frozen components. Thus, the analysis of the formation of thrombin, conducted using the kit of the present invention, allows you to quickly diagnose the overall activity status of the hemostatic system of the patient. In addition, you can detect connected with the treatment of changes in the kinetics of formation of thrombin, for example after the introduction of patient medicines parallel, which allows to optimize the periods of treatment and dose of medicines and thus avoid thrombotic complications from overdose.

BRIEF DESCRIPTION of FIGURES

Figure 1 shows the changes of temperature during the cycle liofilizatsii complex TF/PL. Long cycle lyophilization with slightly the m gradient in temperature during primary drying period and a period of slow heating up to a low room temperature 20°C allow to preserve the biological activity of the complex TF/PL.

Figure 2 shows the curves of the formation of thrombin-initiated frozen or liofilizirovannami complexes TF/PL in different plasma samples. It is shown that there is no difference in the formation of thrombin (which is illustrated by the curve of the concentration of thrombin from the time when using frozen and lyophilized complexes TF/PL. There is also no difference in the formation of thrombin using complexes TF/PL, lyophilized in the absence or presence of sucrose (used as a stabilizer). (A) Normal human plasma and frozen complex TF/PL. (B) Normal human plasma and freeze-dried complex TF/PL. (C) Plasma containing FVIII inhibitor, and frozen complex TF/PL. (D) Plasma containing FVIII inhibitor, and lyophilized complex TF/PL. (E) Plasma containing FVIII inhibitor and restored to 0.5 units/ml FEIBA, and frozen complex TF/PL. (F) Plasma containing FVIII inhibitor and restored to 0.5 units/ml FEIBA, and lyophilized complex TF/PL. (G) Plasma containing FVIII inhibitor and restored to 0.5 units/ml FEIBA, and frozen complex TF/PL. (H) Plasma containing FVIII inhibitor and restored to 0.5 units/ml FEIBA, and lyophilized complex TF/PL. The symbols have the following meanings: -■- in the absence of sucrose; -∆- in the presence of 0.5% sucrose; -*- in the presence of 5% sucrose.

figure 3 shows the comparison of peak thrombin, which represents the highest concentration of thrombin observed during the period of the formation and inactivation of thrombin, measured in normal human plasma and plasma containing FVIII inhibitor, in the absence of FEIBA and restored to 0.5 units/ml FEIBA, after initiation frozen or liofilizirovannami complexes TF/PL with different compositions. (A) Normal human plasma and complexes TF/PL containing low amounts of TF. (B) Normal human plasma and complexes TF/PL containing high amounts of TF. (C) Plasma containing FVIII inhibitor, and complexes TF/PL containing low amounts of TF. (D) Plasma containing FVIII inhibitor, and complexes TF/PL containing high amounts of TF. (E) Plasma containing FVIII inhibitor, restored to 0.5 units/ml FEIBA, and complexes TF/PL containing low amounts of TF. (F) Plasma containing FVIII inhibitor, restored to 0.5 units/ml FEIBA, and complexes TF/PL containing high amounts of TF. The symbols have the following meanings: -1 - the frozen reagents; -2 - lyophilized reagents.

Figure 4 shows that if the complex TF/PL and the mixture containing the substrate of thrombin and CaCl2, lyophilized in well microtiter tablet separately or together, the difference in the obtained curves of the formation of thrombin and maximum concentrations of thrombin defined using data curves, the absence of the light. (A) Comparison of the curves of the formation of thrombin in a plasma containing FVIII inhibitor, restored to 0.5 units/ml FEIBA, initiated liofilizirovannami complexes TF/PL and measured using lyophilized mixtures containing substrate of thrombin and CaCl2(-•-), initiated by complexes TF/PL, liofilizirovannami in well microtiter tablet, and measured using lyophilized mixtures containing substrate of thrombin and CaCl2(-*-), initiated liofilizirovannami complexes TF/PL and measured using mixtures containing substrate of thrombin and CaCl2, lyophilised in well microtiter tablet (-□-), and initiated by complexes TF/PL and measured using mixtures containing substrate of thrombin and CaCl2and both complexes and mixtures lyophilizer in well microtiter tablet (-▲-). (B) Comparison of maximum concentrations of thrombin defined using the curves presented in section a, as measured in normal human plasma in a plasma containing FVIII inhibitor not containing or containing 0.5 units/ml and 1 unit/ml FEIBA, where the formation is initiated by thrombin reagents, liofilizirovannami separately or together in well microtiter plate. The symbols have the following meanings: 1 - the initiation liofilizirovannami complexes TF/P and measuring using lyophilized mixtures, containing substrate of thrombin and CaCl2; 2 - initiation complexes TF/PL, liofilizirovannami in well microtiter tablet, and measurement using lyophilized mixtures containing substrate of thrombin and CaCl2; 3 - initiation liofilizirovannami complexes TF/PL and measurement using mixtures containing substrate of thrombin and CaCl2, lyophilised in well microtiter tablet; 4 - initiation complexes TF/PL and measurement using mixtures containing substrate of thrombin and CaCl2and both complexes and mixtures lyophilizer in well microtiter plate.

Figure 5 shows the sensitivity analysis of the formation of thrombin in relation to the activity of FVIII and IX. (A) change the maximum concentration of thrombin-related changes in the activity of factor VIII. In the larger section of the figure shows the values when FVIII activity below 0.1 units/ml (B) change the maximum concentration of thrombin-related changes in the activity of factor IX. In the larger section of the figure shows the values when the activity of the FIX below 0.1 units/ml

DETAILED description of the INVENTION

In one embodiment of the present invention features a kit for measuring the formation of thrombin in a sample containing freeze-dried complex of tissue factor (TF)/phospholipid (PL) and liof the lysed mixture, containing substrate of thrombin and CaCl2.

The term "sample" in this description refers to the biological fluid of a human or animal, such as blood or plasma, such as plasma, enriched blood cells, or plasma that does not contain cells. The sample can be obtained from healthy subjects or from subjects who may suffer or suffer from blood clotting disorders, particularly disorders associated with the presence of FVIII inhibitors are not exposed or subjected to treatment. The sample can be their or it may be in a frozen state, for example, in the case of samples that do not contain cells. The sample may also contain a mixture of purified natural, synthetic or recombinant proteins and/or other drugs/reagents having hemostatic activity.

The mass ratio of TF and PL in dried complex TF/PL varies depending on destination. To analyze the formation of thrombin, as a rule, it is preferable to use a low number of TF and low number of PL. In the preferred embodiment of the present invention, the concentration of TF in complex TF/PL varies approximately from 5 to 1000 PM and/or the concentration of PL in the complex TF/PL varies from approximately 1 to 100 microns.

TF in complex TF/PL is either a full-sized cloth is a new factor, or, at least, its functional part. Tissue factor can be natural or recombinant. The expression "at least its functional part" refers to any part of tissue factor, which performs the same function as the full-size tissue factor. In the preferred embodiment use a full-sized human recombinant tissue factor.

PL of the complex TF/PL can be synthetic or natural. The composition of the vesicles PL depends on their importance for coagulation, i.e. their role in the physiological process of blood clotting. In the preferred embodiment of the present invention, the phospholipids are selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and mixtures thereof. Preferably, the phospholipids are selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS) and 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE). In the preferred embodiment of the present invention, the mass ratio PC/PS is in the range from about 60/40 to 95/5 in relation to the total number of phospholipids, and the mass ratio PC/PS/PE is in the range from about 60/20/20 to 78/17/5 in relation to the total amount of phospholipids.

The complex TF/PL, and the mixture containing the substrate of thrombin and CaCl 2you can mobilitat on the media separately or together. The term "immobilized" covers both simple immobilization on the carrier by lyophilization, and immobilization by interaction with the media or by attaching to the media, such as covalent joining the carrier directly or through a linker molecule. Preferably, the lyophilized complex TF/PL and lyophilized mixture containing the substrate of thrombin and CaCl2, lyophilizer together on the media. Immobilization spend so essentially to preserve the biological activity of the components, for example, TF, PL and the substrate of thrombin. The term "media" has no particular limitation, and include, for example, to the surface of an inert material, such as polymeric material, which can be an organic polymer, such as polyamide or vinyl polymer (e.g. poly(meth)acrylate, polystyrene and polyvinyl alcohol or derivatives thereof), or a natural polymer such as cellulose, dextran, agarose, chitin and polyaminoamide, or an inorganic substance such as glass. The carrier may be of any type and shape, for example it may be an internal surface and the bottom of the bottles, Micronesian, particles, membranes, strips, paper, foil, granules or tablets, such as ICRI the title tablets, with holes.

The substrates of thrombin used in the present invention, well-known in this field and, preferably, should be highly specific with respect to thrombin, i.e. essentially, they shall not enter or can enter into a slight degree of cross-reaction with other coagulation enzymes and, preferably, they should have a low affinity for thrombin (high Km)to ensure the long-term kinetics. The substrate of thrombin contains labeled fragment, where the labeled fragment can be chipped off by the action of thrombin. The specified portion may contain a fluorescent or radioactive label. In the preferred embodiment of the present invention labeled fragment of the thrombin substrate contains a fluorophore. In addition, the labeled fragment preferably contains a peptide, such as di - or Tripeptide.

In accordance with the present invention, the kit also contains at least one standard thrombin, as the reference sample.

The present invention also relates to a method for producing a lyophilized complex TF/PL, which allows to obtain high-level TF, the activity of which is stored in the lyophilization process.

The method of producing complex TF/PL includes the following stages:

(a) obtaining phospholipid vesicles having a diameter in the range will bring the flax from 200 to 300 nm, preferably using any method known in this field, such as extrusion or sonication;

(b) lyophilization of phospholipid vesicles with obtaining powder;

(c) conversion of lyophilized powder with water for injection and mixing it with tissue factor;

(d) freezing and thawing the mixture obtained in stage (c) obtaining a complex TF/PL;

(e) stabilization of the complex TF/PL by incubation at approximately 4°C for approximately 24 to 72 hours and optional dilution of the complex TF/PL to a suitable concentration "ready to use" product;

(f) freeze-drying complex TF/PL.

In the method of producing complex TF/PL of the present invention to add preservatives optional. The addition of preservatives makes getting set for the analysis more time consuming and expensive. In addition, some of these preservatives, such as albumin, are not appropriate, since it is known that albumin interacts with many proteins and thereby negatively affects the process of analysis.

At stage (d) the process of freezing and thawing preferably carried out by freezing the tissue factor and phospholipid vesicles at approximately -20°C over night and then by thawing for about 30 minutes when on the th temperature.

The present invention encompasses a method of obtaining a mixture containing the substrate of thrombin and CaCl2this method is simple and suitable for holding in aqueous solution. Methods for producing thrombin substrate, known from the previous prior art includes first dissolving in a suitable buffer, often containing DMSO, and then diluted with water. Subsequent addition of CaCl2drugs substrate of thrombin, obtained using the methods of the previous prior art, lead to the precipitation, which is poorly soluble and, therefore, complicates the application.

A method of obtaining a lyophilized mixture containing the substrate of thrombin and CaCl2includes the following stages:

(a) dissolution of the substrate of thrombin in a suitable solvent;

(b) adding CaCl2and dissolving the resulting precipitate containing the substrate of thrombin and CaCl2usually, obtaining a clear solution;

(c) freeze-drying a mixture containing the substrate of thrombin and CaCl2.

The dissolution stage (b) is preferably carried out at a temperature of approximately 37°C to obtain a clear solution.

In addition, the present invention relates to a method for measuring the formation of thrombin in a sample, for example, obtained from the patient, which includes the following stages

(a) obtaining a lyophilized complex TF/PL and lyophilized mixture containing the substrate of thrombin, as described above, and CaCl2;

(b) bringing into contact of the sample with the specified liofilizirovannam complex TF/PL and the freeze-dried mixture containing the substrate of thrombin and CaCl2;

(c) measuring the formation of thrombin in the sample.

When you apply the present invention includes a substrate of thrombin containing fragment with a fluorescent label, the development of the fluorescence intensity of the released fluorophore can be monitored continuously. The development rate of the fluorescence intensity (measured in fluorescence units (FU)) are calculated for each reading (FU/min), and it can be converted to an equivalent concentration of thrombin (nm) using a calibration curve obtained by measuring the rate of conversion of substrate by using a substrate of thrombin.

The kit and method of the present invention allow high sensitivity to analyze one or more factors of the coagulation cascade coagulation by measuring the formation of thrombin. Accordingly, the kit and method of the present invention can control the results of any treatment that affect hemostasis by increasing or decreasing the Akti the activity of any coagulation factor, for example, to monitor the results of treatment means, mechanism of action which is not associated with FVIII or antagonists of vitamin K. the Control treatment, concurrent medications also allows you to optimize the periods of treatment and dose of drugs and helps to avoid thrombotic complications from overdose.

In addition, lyophilized reagents of the present invention have a longer shelf life and allow you to get more reproducible results than frozen ingredients used in other analyses. Moreover, when applying the set of the present invention does not require stages of dilution, making this set more simple and convenient in use.

Further, the present invention is illustrated using the following examples, but is not limited to them.

EXAMPLES

Example 1: Getting frozen and lyophilized complex TF/PL

Tissue factor containing phospholipid vesicles (complex TF/PL), receive, using recombinant full-TF (American Diagnostica Inc. Greenwich, CT, USA) and synthetic PL (Avanti Polar Lipids, Alabaster, AL, USA). The method of obtaining includes the following stages:

Phospholipid vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphoserine (POPS) and 1,2-dileo the l-sn-glycero-3-phosphoethanolamine (DOPE) (Avanti Polar Lipids, Alabaster, AL), receive by way of extrusion Hope et al. (Hope MJ, Bally MB, Webb G, Cullis PR: "Production of large unilamellar vesicles by a rapid extrusion procedure. Characterization of size distribution, trapped volume and ability to maintain a membrane potential." Biochim Biophys Acta 812: 55, 1985), using the device for the extrusion of Lipex Biomembranes, Inc. (Vancouver, Canada), equipped with two stacked on top of each polycarbonate filters (pore size 1000 nm). Preparation of vesicles diluted with 20 mm Tris buffer, pH of 7.4, containing 150 mm NaCl (TBS), to the concentration of 1.27 mm and after addition of 5% (wt./about.) the sucrose solution is dried from the frozen state. After conversion dried from the frozen state of the powder with distilled water vesicles have an average diameter of 260 nm, determined by dynamic light scattering (Zetasizer 4, Malvern Instruments, Worcestershire, UK).

The formation of the complex with TF PL vesicles: the Initial mixture containing from 2 to 700 nm TF and 850 µm PL vesicles, frozen at -20°C overnight, then thawed for 30 minutes at room temperature and diluted with 6.7 times in TBS. TF/PL vesicles balance at 4°C for 48-168 hours and freeze in the aliquot or lyophilizer in the absence or in the presence of 0.5 and 5% sucrose solution. Or diluted 40 times, getting the appropriate working concentration of PL 3.2 mm and different concentrations of TF, and freeze in the aliquot, or lyophilizer in the absence or in the presence of 0.5 and 5% rest the RA sucrose.

The figure 1 shows the temperature changes in the process of lyophilization cycle.

Example 2: the Formation of thrombin-initiated frozen or liofilizirovannam complex TF/PL in different plasma samples

The formation of thrombin initiate complex TF/PL obtained by the above method and containing 18 PM TF and 3.2 ám PL, where PL contains 80% by weight of DOPC and 20% by weight of POPS. Lyophilized complex TF/PL dissolved in water for injection (to a final concentration 18 PM TF and 3.2 μm PL) and 10 μl of the resulting aqueous solution is added to 50 μl of 1 mm solution of thrombin substrate Z-Gly-Gly-Arg-AMC (Bachem AG, Bubendorf, Switzerland), previously mixed with 15 mm CaCl2. To obtain the reference sample 10 ál frozen complex TF/PL (18 PM TF and 3.2 μm PL) is mixed with 50 μl of the solution above the substrate of thrombin. The reaction is initiated by adding 40 μl of the plasma sample. Components are incubated at 37°C.

Under the action of the generated thrombin is the splitting of the substrate of thrombin and release flurforderzeuge substrate. The increase in fluorescence intensity, which is proportional to the concentration of the formed thrombin, continuously recorded at 37°C by automatically reading every minute for 120 min using a fluorescent reader for a FL600 microplate (Bio-TEK Instruments, Winoski, Vermont, USA) at a wavelength of excitation of 360 nm and the wavelength of emission of 460 nm.

The development rate of the fluorescence intensity [units of fluorescence (FU)] are calculated for each reading (FU/min) and convert to an equivalent concentration of thrombin (nm) using a calibration curve obtained by measuring the rate of conversion of substrate using purified thrombin added instead of the sample of plasma.

Trigger effects of frozen and freeze-dried complexes TF/PL (containing 18 PM TF and 3.2 μm PL) compare when using normal human plasma (FACT, George King Bio-Medical Inc. Overland Parks, KS, USA) and plasma containing FVIII inhibitor, in the absence and in the presence of 0.5 units/ml FEIBA (both products get from Baxter, Vienna, Austria).

Curves of formation of thrombin is shown in figure 2.

Example 3: Comparison of the initiating action is frozen and lyophilized complexes TF/PL with different composition on the formation of thrombin

Complexes TF/PL get by the method of example 1, but they are composed of different phospholipids in a concentration of 3.2 mm and 18 PM 89 PM TF. Complexes TF/ML freeze in the aliquot or lyophilizer in the absence of sucrose, using the lyophilization cycle described in example 1.

Table 2
The complex TF/PL
The PL composition (mass ratio)The PL concentration (µm)The concentration of TF (PM)
PC:PS80/203,218
PC:PS60/403,218
PC:PS95/53,218
PC:PS:PE78/17/53,218
PC:PS:PE60/20/203,218
PC:PS80/203,289
PC:PS60/403,289
PC:PS95/53,289
PC:PS:PE78/17/53,289/td>
PC:PS:PE60/20/203,289
PC: 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC)
PS: 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS)
PE: 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE)

Curves of education thrombin receive the above-described method. The most characteristic parameter, peak thrombin, i.e. the maximum concentration of thrombin, measured over the period of the formation and inactivation of thrombin, calculated and depicted as a function of concentration and composition of the complex TF/PL. The figure 3 shows the maximum concentration of thrombin, measured in normal human plasma and plasma containing FVIII inhibitor, in the absence and in the presence restored to 0.5 units/ml FEIBA, after initiation frozen or liofilizirovannami complexes TF/PL.

The difference in any of the plasma samples after initiating the formation of thrombin frozen or liofilizirovannami complexes TF/PL is missing.

Example 4: Lyophilization complexes TF/PL and mixtures containing substrate of thrombin and CaCl2in the hole titration microplate

Analysis of the formation of thrombin is carried out in the wells titration microplate. The complex TF/PL and/or the mixture containing abstrat thrombin and CaCl 2directly lyophilizer, separately or jointly, in the wells titration microplate. If two components lyophilizer together in well tiralongo microplate, then, ready to use, the embodiment need only add the analyzed plasma samples. The figure 4 shows that there are no differences in the obtained curves of the formation of thrombin and maximum concentrations of thrombin obtained using complex TF/PL and mixtures containing substrate of thrombin and CaCl2, lyophilized separately or together in well tiralongo microplate.

Example 5: Sensitivity analysis of the formation of thrombin

Analysis of the formation of thrombin is very sensitive towards any individual coagulation factors or their groups. Therefore, this analysis can be used to determine the effectiveness of substitution and comprehensive therapy using, for example, funds which are not associated with FVIII. As can be seen from figure 5, the analysis is most sensitive in the low interval of the activity of coagulation factors, even below 0.01 units/ml, a concentration that, as a rule, is the limit of sensitivity of conventional coagulation tests and pigmentation. Because there are differences in the pattern of bleeding in severe hemo is Ilijkov, i.e. patients in whom the activity of FVIII or FIX below 0.01 units/ml, the possibility of measuring the activity of the factor in the low range allows you to avoid the risk of spontaneous bleeding in such patients.

Example 6: Getting frozen and freeze-dried water-soluble compound containing a thrombin substrate and CaCl2

The thrombin substrate Z-Gly-Gly-Arg-AMC/HCl dissolved in 25 mm HEPES-buffer pH of 7.35, containing 175 mm NaCl and 10% DMSO, stirring on a magnetic stirrer for 5 minutes, followed by addition of CaCl2. At this point you receive the sludge, which can be dissolved by vigorous shaking for 15 minutes at 37°C, followed by stirring for one hour at room temperature. The obtained clear solution contains a substrate of thrombin to a final concentration of 5 mm CaCl2with the final concentration of 75 mm. Then the solution was diluted with HEPES-buffer pH of 7.35,Containing 175 mm NaCl (without DMSO)to a final concentration of thrombin substrate 1 mm CaCl215 mm, freeze in the aliquot or lyophilizer, getting "ready to use" solution after dissolution in water for injection. A concentrated solution (i.e. a solution containing 5 mm substrate of thrombin and 75 mm CaCl2can freeze in the aliquot or liofilizirovanny and need not be diluted to a suitable concentration before use.

<> 1. Set for measuring the formation of thrombin in a sample by measuring the concentration of thrombin generated in the sample containing the freeze-dried complex of tissue factor (TR)/phospholipid (PL) and lyophilized mixture of thrombin substrate containing a fluorescent label, and CaCl2.

2. The kit according to claim 1, where the concentration of TF in dried complex TF/PL varies approximately from 5 to 1000 PM.

3. The kit according to claim 1, where the concentration of PL in dried complex TF/PL varies from approximately 1 to 100 microns.

4. The kit according to claim 1, where the specified TF, or at least its functional part is a natural or recombinant.

5. The kit according to claim 1, where the specified PL is natural or synthetic.

6. The kit according to claim 1, where the specified PL selected from the group consisting of phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE) and mixtures thereof.

7. The kit according to claim 6, where the mass ratio PC/PS is in the range from about 60/40 to 95/5.

8. The kit according to claim 6, where the mass ratio PC/PS/PE is in the range from about 60/20/20 to 78/17/5, relative to the total amount of phospholipids.

9. The kit according to claim 1, additionally containing at least one standard thrombin.

10. The kit according to claim 1, where the freeze-dried complex TF/PL immobilized on the carrier.

11. The kit according to claim 1, where dried of socialstate thrombin, containing a fluorescent label, and CaCl2immobilized on the carrier.

12. The kit of claim 10 or 11, where the carrier is an inner surface of the vial, or wells, or strips ELISA.

13. A method of obtaining a lyophilized complex of tissue factor (TF)/phospholipid (PL), comprising the following stages:
(a) receiving, by extrusion of phospholipid vesicles having a diameter in the range from about 200 to 300 nm;
(b) lyophilization of phospholipid vesicles with obtaining powder;
(c) the conversion of this powder with water for injection and mixing it with tissue factor;
(d) freezing and thawing the mixture obtained in stage (C) obtaining a complex TF/PL;
(e) stabilization of the complex TF/PL by incubation at approximately 4°C for approximately 24-72 hours; and
(f) freeze-drying complex TF/PL.

14. The method according to item 13, where between stages (e) and (f) the complex TF/PL diluted to a suitable "ready to use" concentration.

15. The method according to item 13, where the complex TF/PL on the stage (f) immobilized on the media by lyophilization.

16. The method according to clause 15, where the carrier is an inner surface of the vial, or wells, or strips ELISA.

17. The method according to item 13, where the complex TF/PL on the stage (f) lyophilizer in the absence of preservatives.

18. A method of obtaining a lyophilized mixture of the substrate t is ombine, containing a fluorescent label, and CaCl2that includes the following stages:
(a) dissolution of the substrate of thrombin containing a fluorescent label, in a suitable solvent;
(b) adding CaCl2and dissolution of the precipitate formed, which includes a substrate of thrombin containing fluorescent label, and CaCl2;
(c) freeze-drying the mixture of the substrate of thrombin containing fluorescent label, and CaCl2.

19. The method according to p, where the phase (s) mixture immobilized on the media by lyophilization.

20. The method according to claim 19, where the carrier is an inner surface of the vial, or wells, or strips ELISA.

21. The method of measuring the formation of thrombin in a sample by measuring the concentration of thrombin, resulting in a sample, comprising the following stages:
(a) obtaining dried complex of tissue factor (TF)/phospholipid (PL) and lyophilized mixture containing the substrate of thrombin and CaCl2;
(b) bringing the sample into contact with a specified liofilizirovannam complex TF/PL and the freeze-dried mixture containing the substrate of thrombin and CaCl2;
(c) measuring the formation of thrombin in the specified pattern.

22. The method according to item 21, where the sample is selected from the group consisting of whole blood, plasma and mixtures containing purified natural, synthetic or R is combinatie proteins, having hemostatic activity.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to haemostasis and fibrinolysis system analysis methods. Coagulation properties of two citrated blood samples are analysed simultaneously by re-calcification with kaolin added. One blood sample is added with plasmin in amount 1.5 times extending coagulation time as compared to the reference thus calculating the coagulation prolongation percentage by formula: where twith plasm is coagulation time with plasmin, twithout plasm is coagulation time without plasmin. The coagulation prolongation percentage is compared to the reference results in hyperfibrinolysis prediction and diagnostics.

EFFECT: method provides improved quality of treatment ensured by higher diagnostic accuracy and possibility to predict thromboses and haemorrhages in surgical, therapeutic and neurologic patients.

3 ex

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine and gynaecology, and can be applied to probability prediction of severe destructive process development in pyoinflammatory conditions of uterine appendages. For this purpose, viscosity indices of blood are determined: reaction period (r), spontaneous platelets aggregation intensity (Ar), thrombin constant (k), maximal amplitude (MA), formation time of fibrin-thrombocyte clot structure (T), total retraction index, and spontaneous lysis of clot (F). Subject to values obtained, high, medium, or low probability of severe destructive process development is predicted.

EFFECT: more informative and exact probability prediction of severe destructive process development in different clinical presentations of pyoinflammatory conditions of uterine appendages.

3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention can be used for prediction of autointoxication (AI) severity associated with suppurative inflammatory diseases of uterine appendages. Method of prediction implies testing of blood serum for erythrocyte sorption property (ESP) and soluble fibrin-monomeric complexes (SFMC). Autointoxication ratio is calculated by formula RAI=ESP/SFMC. If values RAI=2.48±0.10, severe autointoxication is predicted. Values RAI=3.29±0.29 indicate medium autointoxication, while RAI=4.60±0.08 indicates slight autointoxication associated with suppurative inflammatory diseases of uterine appendages.

EFFECT: higher information value and accuracy of prediction, reduced time of prediction.

3 ex, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to medicine area, namely, to nephrology, and can be used for predicting development of chronic renal insufficiency at patients with postcapillarotoxic glomerulonephritis. Tap clinical signs of an arterial hypertonia and a daily proteinuria more than 1 g. in the anamnesis is determined. A combination of a debut of a hemorrhagic vasculitis with the years the patient is defined, the creatinine maintenance in blood serum in the beginning of a glomerulonephritis, indicators of a plasma link of a hemostasis, and also the analysis of therapeutic treatment in a disease debut is carried out. If revealing debut of a hemorrhagic vasculitis at the age of 31-45 years, the content in blood serum of a creatinine above 130 micromole, a blood hypercoagulation in a plasma link of a hemostasis and absence of anticoagulant therapy in a disease debut, the forecast of development of chronic renal insufficiency is considered as adverse, in other cases the forecast is favourable.

EFFECT: allows providing more objective and early forecasting of development of chronic renal insufficiency at patients with postcapillarotoxic glomerulonephritis.

3 ex, 6 dwg

FIELD: medicine; cardiology.

SUBSTANCE: for this purpose vascular wall antiaggregatory activity, antithrombin III plasma level, euglobulin lysis time, nitrate plasma content are determined prior to and after venous occlusion for each characteristics. Based on measured values vascular wall antiaggregatory activity index, antithrombin III vascular synthesis index, vascular wall fibrinolytic activity index and nitrate index are calculated. Further arithmetic mean value of given indexes is resulted in calculated vascular antithrombotic index of vessels. Value of this index indicates necessity of individual low-calorie diet combined with graduated physical exercise, introduction of methphormin combined with low-calorie diet and physical exercises.

EFFECT: decreased risk of thrombotic complications.

2 tbl, 4 ex, 1 dwg

FIELD: medicine; cardiology.

SUBSTANCE: patients are examined for activity and formation time of thromboplastin during three days running provided these systems are tested within multivariate analysis combined with evaluation of general thromboplastin potential. If value XBiFTP=0.020 and less thromboplastin-formation is considered to be normal, and if value XBiFTP within 0.021 to 0.030 start of increased thromboplastin-formation is diagnosed proceeding with evident thromboplastin-formation at value XBiFTP=0.031 and higher.

EFFECT: application of invention enables to avoid complications for various patients.

3 ex

FIELD: medicine.

SUBSTANCE: invention concerns glycoforms of VII factor and compositions of VII factor, characterized by modified configurations on basis of asparaginic oligosaccharide chains. In addition invention includes detection method applied for polypeptide glycoforms of VII factor, receiving method and disease treatment method as well.

EFFECT: identification of biologically active forms of recombinant VII factor.

53 cl, 5ex

FIELD: medicine, orthopedics and traumatology.

SUBSTANCE: method can be useful for early diagnostics and prevention of thromboembolism in hip and knee replacement in traumotologic, orthopedic, surgery hospital departments, clinics and research institutes. In preoperational period, antithrombin III is evaluated, sodium thiosulfate 10 ml is injected intravenously daily, at the same time cuff test is taken by blood sampling following tonometer's cuff application and pumping pressure equal to systolic, antithrombin III is evaluated in blood samples and if its level is not changed against the baseline preoperational preparation is taken with sodium thiosulfate injection until antithrombin III is elevated during cuff test. And at that, sodium thiosulfate can be injected during 5-8 days up to the moment when antithrombin III is increased 80% from baseline and positive dynamics in cuff test is maintained.

EFFECT: method increases impartiality and precision of preoperational diagnostics of thromboses and embolism.

3 cl, 3 tbl, 1 ex

FIELD: medicine, surgery.

SUBSTANCE: one should detect hemocoagulation potential (P) by the following formula: where t - time of plasma recalcification (min), i - prothrombin index, f - the quantity of fibrinogen (mg/l), a - a patient's age (yr), moreover, additionally it is necessary to detect the volume of infusion therapy (ml) (V) and the volume of operational blood loss (ml) (W), and the time for the onset of thromboembolism of pulmonary artery (T) should be determined by the following formula:

EFFECT: higher accuracy of prediction.

1 ex

FIELD: medicine.

SUBSTANCE: method involves determining the following hemostasis parameters before beginning, in a month and one year later after 0 mg/kg during 10 min after glucocorticoid therapy when indications to applying steroid therapy are available. The parameters are procoagulant activity as activated partial thromboplastin time, s, prothrombin index; anticoagulant activity as antithrombine III, %, protein-C as well as fibrinogen, g/l and fibrinolytic blood activity, min and their changes on the background of additional respiratory resistance action 40% Pmmax during 3 min. If procoagulant activity as response to additional respiratory resistance falls before and during the treatment with prothrombin index decreasing and activated partial thromboplastin time growing, anticoagulant activity as antithrombine III, protein-C grows whereas fibrinogen and fibrinolytic blood activity remain unchanged especially towards the year end, favorable clinical course of sarcoidosis is to be predicted. If procoagulant activity as response to additional respiratory resistance grows before and in a month after the glucocorticoid therapy with prothrombin index growing and activated partial thromboplastin time decreasing, one year later their activity falling, anticoagulant activity as antithrombine III, protein-C grows as well as fibrinogen and fibrinolytic blood activity remain unchanged during the whole investigation time, disease regress retardation and high probability of fibrous changes being occurred in lungs is to be predicted.

EFFECT: high accuracy of prognosis.

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

The invention relates to immunology

The invention relates to the diagnosis and treatment of diseases such as atherosclerosis and thrombosis

FIELD: production methods.

SUBSTANCE: method is based on the capability of defibrotide to increase the fermentation activity of plasmin and foresee the stages: a) making the contact in reactional area defibrotide, plasmin and substrate specific for plasmin which, because of reaction, provides the defined product b) the definition of the amount of obtained product in temporary points.

EFFECT: invention allows to define the biological activity of defibrotide in comparison with standard etalon with height accuracy and big repeatability.

9 cl, 6 dwg, 4 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: specified kit contains a dried complex including tissue factor/phospholipid and a dried mixture containing thrombin substratum and CaCl2. There is disclosed method for preparing the dried complex including tissue factor (TF/phospholipid (PL). There is offered method for preparing said dried mixture containing thrombin substratum and CaCl2. Besides, there is described method for measuring thrombin formation in a sample by measuring the concentration of thrombin.

EFFECT: invention allows detecting kinetic changes in thrombin formations after introduction of agents action of which is not related to inhibitor.

22 cl, 5 dwg, 2 tbl, 6 ex

FIELD: medicine.

SUBSTANCE: for thrombin production measurement, a layer of said sample contacts with a fluorogenic substratum of thrombin where the thickness of said layer is 0.05 to 5 mm, while the surface area is 10 to 500 mm2. Further, the thrombin production environment in said sample is provided. It is followed by measuring the fluorescence emitted from the layer surface by a fluorescent group released by the fluorescent substratum as a result of an enzymatic action of produced thrombin on said fluorogenic substratum. Besides, the invention ensures a kit for measuring the thrombin activity in the sample.

EFFECT: higher measuring accuracy.

29 cl, 12 dwg, 5 ex

FIELD: medicine.

SUBSTANCE: method for thrombin activity test in an initially non-reacted mixture of thrombin and fibrinogen (versions) involving the stages: (a) reversible thrombin inhibition by adding an inhibitory solution having pH varying within the range of 8.5 to 11.5; (b) addition of the known amount of fibrinogen to the mixture (or the known amount of a chromogenic or fluorogenic thrombin substrate), (c) reversible thrombin activation by pH reduction to approximately 6.0 to less than 8.5, (d) enabling thrombin reacting with fibrinogen, (e) thrombin activity test initially found in the dry mixture. The method for fibrinogen functionality test in an initially non-reacted mixture of thrombin and fibrinogen (versions) involving the stages: (a) reversible thrombin inhibition by adding an inhibitory solution having pH varying within the range of 8.5 to 11.5; (b) addition of the known amount of thrombin to the mixture (or a thrombin-like enzyme), (c) reversible thrombin activation by pH reduction to approximately 6.0 to less than 8.5, (d) enabling thrombin reacting with fibrinogen, (e) fibrinogen functionality test initially found in the dry mixture.

EFFECT: group of inventions enables higher accuracy of thrombin and fibrinogen activity test.

32 cl, 1 dwg

FIELD: medicine.

SUBSTANCE: invention includes determination of content of soluble fibrin and D-dimers, formed in the process of fibrinolysis, activated in blood sample. In method, in accordance with the claimed invention, level of D-dimers, corresponding to destruction of soluble fibrin and level of D-dimers in sample with border values of the norm, are compared.

EFFECT: test in accordance with the claimed invention can be applied for determining whether resistance to blood coagulation in patient is sufficient.

4 tbl, 3 ex, 2 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to medical microbiology and a method of determining activation of plasminogen with bacteria. The method involves adding protamine sulphate to a prepared supernatant fluid, incubating the obtained mixture, depositing cells by centrifuging, incubating the supernatant fluid with the protamine sulphate, depositing protein and detecting activation of plasminogen with bacteria from the amount of split arginine, content of which is determined by Sakaguchi method from the red colour of the sample.

EFFECT: invention enables to detect activation of plasminogen with bacteria in vitro using protamine.

4 tbl, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention is a method of controlling autoactivation of factor VII or its analogue and a method of prevention of cleavage of activated factor VII or its analogue. The methods comprise measuring the initial concentration of factor VII/VIIa or its analogue. The initial part of activated factor VII or its analogue is measured. The reaction time of activation of factor VII or its analogue is calculated by correlating the values measured at the abovementioned steps, with the value of the required part of activated factor VII or its analogue. The reaction of activation of the factor VII is carried out at pH of from 6.0 to 8.0. The reaction is terminated by reducing pH to the value below about 6.0.

EFFECT: invention enables to determine the optimum reaction time to achieve the desired levels of the modified factor VII or its analogue, to obtain a purer protease product, to reduce the cost of manufacturing base.

15 cl, 5 dwg, 1 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: claimed invention relates to biotechnology, namely to obtaining infestin-4 mutants, and can be used for diagnostic purposes in the determination of characteristics of clotting of blood and its components. A polypeptide is characterised by a sequence of the infestin 4 mutant MutB SEQ ID NO: 1. The said sequence can have modifications outside inhibiting the loop part, which essentially preserve the activity of the said polypeptide. The polypeptide is used for the inhibition of contact activation in a tested sample of blood or its product in order to increase the time of the sample storage.

EFFECT: invention makes it possible to obtain the highly-selective inhibitor fXIIa, selectivity or activity of which is higher than in the native infestin-4 and Mut15.

25 cl, 7 dwg, 3 tbl, 4 ex

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