Highly-selective infestin 4-based inhibitor of contact activation

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

 

Area of technology

The present invention relates to medicine, in particular to Hematology, and can be used in particular for diagnostic and research purposes in determining the coagulation characteristics of blood and its components, as well as in biotechnology and basic biological research.

Prior art

Plasma clotting

Blood clotting is a protective process of the body; one of the main components of this process - the formation of a fibrin clot. The fibrin clot is formed by activation of the enzymatic cascade of coagulation factors; many of the coagulation factors are serine proteases (VIIa, XIIa, XIa, IXa, Xa, thrombin IIa). Also in the enzymatic coagulation cascade is involved activated protein C (APC) is a protease, which is activated by thrombin and inactivates the coagulation cofactors VIIIa and Va. Activation of the enzymatic cascade is on "external" ways, which is responsible for factor VIIa (VIIa) and tissue factor (TF, or thromboplastin), or "internal" path which starts with the contact activation of factor XII (FH, or hageman factor) (Mann et al. 1990, Blood 76:1-16; Hoffman and Monroe. 2001, Thromb. Haemost. 85:958-965). Contact activation is a FHP self-activation upon contact of the plasma with the negative saragani�and surfaces. CNV is present in plasma in an inactive state, in complex with high molecular weight kininogen (HMWK) and plasma pre-kallikrein he turns into the active protease factor XIIa (FHA). Surfaces, leading to activation FH can be any foreign surfaces, including the walls of plastic or glass test tubes, the surface of the stents, catheters, etc. FHA proteoliticeski activates prekallikrein in its active form, kallikrein, the enzyme, including those responsible for the activation of fibrinolysis, and factor internal coagulation pathway XI to its active form, factor XIa (FHA) (Ichinose et al. 1986, J. Biol. Chem. 261:3486-3489; Schmaier. 2008, J. Clin. Invest. 118:3006-3009).

Inhibition of the contact activation

An important issue is the suppression of clotting via the contact path, which occurs upon contact of the blood sample or plasma (whole blood or blood plasma, including rich, poor or released from platelets, plasma) with a foreign surface and prevents the use of sample storage, transfusion and research (Sperling et al. 2009, Biomaterials 30:4447-4456; Streller et al. 2003, J. Biomed. Mater. Res. In Appl. Biomater. 66:379-390). Activation of PHPA occurs when extracting blood from the vasculature of the body, for example: 1) when the blood in the tube from the surfaces of the needles, tubes and walls of the tube; 2) storage of blood plasma in the frozen form and subsequent� plasma thawing; or 3) when using the artificial (in vitro) blood circulation, in particular when the transfusion of blood or plasma. Contact activation during blood sampling or thawing frozen plasma may initiate clotting in a test tube, which prevents the obtaining of the sample and the coagulation study (Smith et al. 2010, Blood Coagul. Fibrinolysis. 21:692-702; Suontaka et al. 2005, Vox. Sang. 88:172-180). When autotransfusion or re-infusion of blood contact activation path leads to an increased risk of thrombosis. To block blood coagulation when using the heart-lung machine tubes and other surfaces of the apparatus are covered with inhibitors FHA and thrombin, such as heparin, benzamycin etc (Hsu. 2001, Perfusion 16:417-428; Gouzy et al. 2006, Biointerphases 1:146-155). The disadvantage of this method of blocking coagulation is that the addition of inhibitors FHA and does not prevent thrombin time FHA that can initiate coagulation after infusion of blood or plasma into blood vessels of the body.

The most common way of suppressing coagulation during blood sampling is preliminary added to the test tube, which is taken blood, a chelator of calcium ions (such as sodium citrate, EDTA, etc.). The chelation of calcium ions inhibits membrane-dependent reactions of activation of pF and thrombin (Fscher. 2007, Hemodial. Int. 11:178-189). The study of coagulation in the test sample containing the chelator is added, the coagulation activator (in particular, TF or activator of the contact path) and plasma recalcified (added an excess of calcium ions), further explores the parameters of formation of a fibrin clot. Way to suppress coagulation by adding chelator has some drawbacks. In the work of Mann et al. 2007, J. Thromb. Haemost. 5:2055-2061 it has been shown that chelation of calcium ions in the blood and subsequent recalcification sample change the dynamics of the formation of a fibrin clot, the achievements of thrombin and platelet aggregation compared with samples without the addition of the chelator. The addition of the chelator does not inhibit activation reactions pH and pH that are initiated FHA generated during the conduct of blood drawing and storage of the sample (Nossel et al. 1968, J. Clin. Invest. 47:1172-1180; Oiler et al. 1976, J. Surg. Res. 20:333-340). Therefore, in the study of coagulation, in particular TF-initiated coagulation by the "external" ways (which is considered physiological and responsible for clotting in the normal state of the body), because of the resulting PHA coagulation also occurs along the contact path, which introduces unwanted distortion in the study results. Folding the contact path in such a study I�is an artifact which may prevent an adequate assessment of the effects of physiological coagulation, can lead to increased errors and inconsistency of research results, and a loss of sensitivity in relation to pathological States of coagulation. To inhibit contact activation, to avoid artifacts and to improve the quality of research coagulation, it is necessary to use an inhibitor PHA when blood or plasma preparation (Luddington and Baglin. 2004, J. Thromb. Haemost. 2:1954-1959). Used inhibitor of the contact activation must be highly selective: when the concentration added to the sample of the inhibitor sufficient to effectively suppress the contact coagulation pathway, the inhibitor has no effect on TF-initiated by the formation of a fibrin clot, i.e., did not inhibit PHA, FHA, FHA and others.

Known inhibitors of the contact activation

The number of known inhibitors FHA, in particular plasma coagulation inhibitors, such as 1-inhibitor, ALPHA2-macroglobulin. (Davis et al. 2008, Mol. Immunol. 45:4057-4063). Protein also known trypsin inhibitors that inhibit FHA and were isolated from different organisms, e.g. bacteria E. coli (Ulmer et al. 1995, FEBS Lett. 365:159-163), from the seeds of plants (Wynn and Laskowski. 1990, Biochem. Biophys. Res. Commun. 166:1406-1410). Among the inhibitors from the seeds of plants include corn trypsin inhibitor and�and LPS (Hojima et al. 1980, Thromb. Res. 20:149-162), as well as inhibitors of the family of Cucurbitaceae: Cucurbita maxima trypsin inhibitor, CMTI-III (Krishnamoorthi et al. 1990, FEBS Lett. 273:163-167), Luffa cylindrica trypsin inhibitor, LCTI-III (Ling et al. 1993, J. Biol. Chem. 268:810-814), and others. Also known nonprotein inhibitors FHA (Woodruff et al. 2013, J. Thromb. Haemost. 11:1364-1373). However, most of these inhibitors are non-selective, except FHA, they also inhibit other coagulation factors, making them impossible to use in the study of the formation of a fibrin clot.

Most existing selective inhibitors of LPS is considered. Known to use LPS to inhibit contact activation in the study of TF-initiated coagulation in a sample of blood or blood component (US 6,403,381, CL G01N 33/86, publ. 11.06.2002), including global tests of hemostasis, such as in a test of thrombin generation. However, there is evidence of a lack of selectivity LPS, since it is known its effect on the blood clotting system and other systems that interact with the coagulation. Thus, LPS inhibits fibrinolysis, presumably by inhibiting tissue plasminogen activator (Nielsen. 2009, Blood Coagul. Fibrinolysis 20:191-196).

Also known inhibitor of FHA infestin 4 (domain protein 4 infectin from the stomach-sucking insect Triatoma infestans), it belongs to the family of inhibitors of type of Casal (Campos et al. 2004, FEBS Lett. 577:512-516). The amino acid�tial sequence intestine 4 (Inf4) and spatial structure are listed in a public database Protein Data Bank (http://www.rcsb.org), the record number 2ERW.

Known applications infestin 4 and drawbacks of existing options infestin 4

In the work of Hagedorn, Schmidbauer et al. 2010, Circulation 121:1510-1517, it was shown that infestin 4 is a selective inhibitor FHA, blocks the activity FHA in a model of thrombosis in vivo and prevents embolism, while it does not change the parameters of hemostasis. The use infestin 4 in the form of a fused protein with albumin for the treatment and prevention of diseases associated with thrombosis, is disclosed in patent US 8,283,319, CL A61K 38/16, publ. 09.10.2012. However, a disadvantage intestine 4 is its inhibiting activity against FHA (the inhibition constant Ki 53 nm).

Known mutant intestine 4 Inf4Mutl5 (Mutl5), which has a high selectivity to PHA, because no inhibits the (Campos, Souza et al. 2012, Acta Cryst. D68:695-702). Mentioned mutant was selected as the closest analogue of the claimed solution. The prior art is unknown selectivity of inhibition indicated mutant FHA, i.e. it is unknown whether it has inhibitory activity to FHA, FHA, VIIa, thrombin and APC. The use Mutl5, including in the kit for the diagnosis of hemorrhagic and thrombolytic diseases by determining the concentration of PHPA in the blood, known from the patent BRPI 0602496-3, CL A61K 38/36, publ. 26.02.2008, however, the specified application is not associated with the diagnosis of diseases by measurement�Oia coagulation parameters. Thus, the prior art not known highly selective inhibitor PHA to block contact activation in the study of TF-initiated blood coagulation.

The object of the present invention is to provide a highly selective inhibitor PHA to block contact activation in research clotting blood or plasma, in particular initiated by the TF, as well as in the collection and storage of blood or plasma.

Summary of the invention

One aspect of the present invention is to provide a polypeptide comprising a mutant intestine 4 MutB, which is a highly selective inhibitor PHA and has activity and selectivity higher than that of the native infestin 4, inhibition of the contact activation in the tested sample.

Under the definition of "polypeptide comprising a mutant intestine 4 MutB" means a polypeptide comprising the amino acid sequence of SEQ ID NO:1. However, this definition also encompasses polypeptides with a slightly modified amino acid sequence if these polypeptides substantially retain the activity of "unmodified" polypeptide. For example, such modifications can be a change of the N - or C-Termini of the sequence, deletion or insertion of one or several amino acid residues, and she�same conservative amino acid substitutions, namely substitutions performed within groups of amino acids with similar characteristics, such as (1) small, (2) acidic, (3) polar, (4) major, (5) hydrophobic and (6) aromatic amino acids.

Polypeptide comprising a mutant intestine 4 MutB, can be obtained by chemical synthesis or recombinant protein, including in the form of a fused protein with a protein partner. Its receipt in the form of a recombinant protein can be accomplished by creating a gene expression plasmid DNA, transforming it into a host cell, expression of a polypeptide comprising a mutant intestine 4 MutB, which is encoded by the specified DNA extraction and purification of the specified polypeptide. A region of DNA that encodes essentially the same protein, can be obtained, in particular, by modifying the nucleotide sequence of the above plasmid DNA, for example by the method of site-directed mutagenesis.

Under "activity inhibitor" should be understood as the functional activity of inhibiting the blood coagulation is initiated by contact way. For example, the activity of the inhibitor can be revealed in coagulopathies test "activated partial thromboplastin time (APTT)".

Under "selective inhibition" should be understood as a property of the inhibitor FHA, consisting in inhibition tion�the stripes of PHPA with high efficiency (the inhibition constant of the order of 1 nm) and no significant inhibition (the inhibition constant is much greater than 1 nm, preferably greater than 1 μm) of other coagulation proteases such as factors FHA, FHA, FHA, VIIa, thrombin and APC. It is known that native infestin 4 inhibits the with a constant 53 nm (Campos et al. 2004, FEBS Lett. 577:512-516). Thus the inventive polypeptide comprising a mutant intestine 4 MutB, as effectively inhibits FHA as the native inhibitor, but did not inhibit PHA. Thus, the inventive polypeptide has a high selectivity to PHA compared with native intestinal 4.

"The test sample is a whole blood or blood products, e.g. platelet rich plasma, platelet-poor plasma released from the platelet plasma. Also this product can be fractionated plasma from which you deleted one or more proteins, including the method of affinity chromatography. The sample can be obtained from healthy subjects or from subjects who may be suffering or are suffering from blood clotting problems, in particular subjects with hemorrhages or thromboses who have impairments or deficits of the hemostatic system. Also the sample can be obtained from patients during surgical procedures or in the exercise of anticoagulant, drug test etc, antiplatelet, fibrinolytic, or antifibrinolytic therapy, etc. the Sample can be their or may be in a frozen state. Also, the sample may be in liofilizirovanny form. The sample may contain natural or recombinant proteins, as well as other drugs and reagents, including those that have hemostatic or fibrinolytic activity.

Another aspect of the invention is the use of a specified polypeptide to study the coagulation of the test sample that includes at least one of the following steps, preferably, all the steps: preparation of the sample, bringing it into contact with the specified polypeptide and incubation of the resulting mixture, the bringing into contact of the activator of the coagulation of the blood sample containing the specified polypeptide, and measurement of parameters of coagulation of the sample.

Preparation of sample can be performed by drawing blood into the tube, whether or not containing anticoagulant, including sodium citrate, EDTA, an inhibitor of thrombin, inhibitor of FHA and others, and the subsequent receipt of the sample taken from the blood. The process of obtaining a sample of blood can contain stage of cleaning the blood from unwanted cells and cell components, such as erythrocytes, leukocytes, platelets, microvesicles, etc. the cleaning Stage may consist of a filtration or centrifugation. The resulting sample may be frozen for storage and then thawed prior investigated�I coagulation. Also, the study sample may pass through the stage of rekaltsifikatsii.

Polypeptide comprising a mutant intestine 4 MutB, can be driven into contact with the test sample in a test tube, in particular in a test tube for blood sampling, or analytical cell. The specified polypeptide can be in dissolved form, in dried or liofilizirovanny form also specified polypeptide can be adsorbed on the surfaces with which the blood or plasma of a subject in contact with the blood, in the preparation or conduct of the study coagulation of the test sample. In addition, the polypeptide can be pre-dissolved in a solution that contains a salt, a buffer substance, in particular, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES), excipient or stabilizer, in particular polyvinylpyrrolidone (PVP). For the effective dissolution of the specified polypeptide in the sample mixture of the sample and the polypeptide can inkubirovanija at a temperature of from +20°C to +40°C, preferably at +37°C, for a time, more than 3 minutes, preferably more than 10 minutes. For the effective inhibition of the contact activation of the coagulation sufficient amount of a specific polypeptide can be determined by measuring activity of the polypeptide in the sample obtained from a healthy donor, for example�EP, using the APTT test. So, sufficient for effective inhibition of the contact activation will be such amount of the polypeptide, wherein the APTT was increased more than 2 times, preferably more than 3 times, compared to the APTT of the sample polypeptide. Also, a sufficient amount of a specific polypeptide can be determined by means of laboratory diagnostic systems "Registrar thrombodynamics T-2 [123166 EN IN from 16.08.2012 class G01N 33/86; USPTO Pat. app. No.20100261211], in particular, by the absence of clots away from the coagulation activator in a sample obtained from a healthy donor, for more than 15 minutes, preferably more than 30 minutes after activation.

The bringing into contact of the activator of the coagulation sample containing a specified polypeptide may be carried out by adding to the specified sample solution containing the activator of coagulation, or by layering the specified solution to the sample without mixing. Also the sample can be brought into contact with the surface on which is immobilized a coagulation activator, wherein the sample is mixed with an activator (Fadeeva et 2010, Biochemistry 75:827-838). The coagulation activator may be a tissue factor, in particular re-libidinously tissue factor. Tissue factor can be obtained in the form of recombinant protein or by ejecta� of organs or tissues, such as the brain, placenta, lung, some mammals, such as man, ox, rabbit. Also, the activator may be one of the coagulation factors, such as VIIa, Xa, IIa, IXa, XIa, XIIa.

Measurement of parameters of coagulation of the test sample can be carried out, in particular, through the test of thrombin generation, test thromboelastography, known in the art, or laboratory diagnostic systems "Registrar thrombodynamics T-2. As these coagulation parameters can be defined: the delay time of the start of coagulation, clotting time, and the time delay operating time of thrombin, the time to reach maximum thrombin, the maximum concentration of thrombin, the angle of thromboelastogram, speed of clot growth, the size of the clot in a fixed moment of time, the clots away from the coagulation activator, the rate of clot lysis, and others.

Another aspect of the invention is the use of a polypeptide comprising a mutant intestine 4 MutB, as an anticoagulant that blocks the contact activation, by bringing into contact with the specified polypeptide sample for the intake and increase the retention time of the sample.

Polypeptide comprising a mutant intestine 4 MutB, it can be put in contact with the sample in a test tube, in particular, in a test tube for blood collection. Specified polypep�ID can be in dissolved form, in the dried or liofilizirovanny form also specified polypeptide can be adsorbed on the surfaces with which the blood or plasma of a subject in contact with the blood or in the preparation of the sample. In addition, the polypeptide can be pre-dissolved in a solution that contains a salt, a buffer substance, in particular, 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid (HEPES), excipient or stabilizer, in particular, polyvinylpyrrolidone (PVP).

For the effective dissolution of the specified polypeptide in the sample, the mixture of the sample and the polypeptide can inkubirovanija at a temperature of from +20°C to +40°C, preferably at +37°C, for a time, more than 3 minutes, preferably more than 10 minutes. For the effective inhibition of the contact activation of the coagulation sufficient amount of a specific polypeptide can be determined by measuring activity of the polypeptide in the sample obtained from a healthy donor, for example, using the APTT test. So, sufficient for effective inhibition of the contact activation will be such amount of the polypeptide, wherein the APTT was increased more than 2 times, preferably more than 3 times, compared to the APTT of the sample polypeptide. Also, a sufficient amount of a specific polypeptide can be determined by means of laboratory diagnostics�statistical system "Registrar thrombodynamics T-2, in particular, the absence of clots away from the coagulation activator in a sample obtained from a healthy donor, for more than 15 minutes, preferably more than 30 minutes after activation.

Blood sampling can be performed in a test tube, additionally containing or not containing an anticoagulant, including sodium citrate, EDTA, an inhibitor of thrombin, inhibitor of FHA and others. Get sample of blood may include the stage of cleaning the blood from unwanted cells and cell components.

Storage of the sample may be frozen at negative temperatures (below 0°C, preferably below -50°C, or in liquid form at a positive temperature near 0°C or at a temperature of from +20°C to +40°C. the storage process may include one or more stages of freezing and thawing of the sample. The storage time of the sample can be from 10 minutes or more, in particular, from one day or more, mostly more than one week.

Brief description of figures

Fig.1 shows a graph of APTT (sec) against the concentration of inhibitor (μm), MutB, Mutl5 or Inf4 in the blood plasma. Average values and standard deviations. For MutB and Inf4 the number of iterations n=4; for Mutl5 n=2.

Fig.2 illustrates fibrin clots formed from the activator and from the walls of the cell, obtained 30 minutes after activation nor�sentatives frozen plasma without addition of an inhibitor of the contact activation.

Fig.3 illustrates fibrin clots obtained 30 minutes after activation frozen samples of normal plasma with the addition of MutB (20 μm), LPS (15 μm) and without inhibitor of the contact activation (4 repeat for each sample).

Fig.4 shows a graph of a function of time (min) area away from the activator occupied by fibrin clots in normal frozen plasma with the addition of MutB (20 μm), LPS (15 μm) and without inhibitor of the contact activation (% of the area of the cuvette) (4 repeat for each sample).

Fig.5 shows a graph of a function of time (min) area away from the activator occupied by fibrin clots in the plasma hypercoagulability, with the addition of MutB (20 μm), LPS (15 μm) and without inhibitor of the contact activation (% of the area of the cuvette) (4 repeat for each sample).

Fig.6 A-D illustrate the effect of inhibitors of the contact activation MutB (20 μm) and LPS (15 μm) on the growth of a fibrin clot activator from normal frozen and hypocoagulation plasma.

Fig.7 presents the clotting time (sec) of whole blood was taken from 3 healthy donor to a test tube containing MutB (10 μm), LPS (5 μm) or not containing the inhibitor of the contact activation (2-4 repeat for each donor).

Detailed description of the invention

Description new in�comselection inhibitor of the contact activation

The claimed polypeptide comprising a mutant intestine 4 MutB, is a highly selective inhibitor of the contact activation. Amino acid sequence of MutB presented in the sequence listing under the number SEQ ID NO:1. The mechanism of inhibition of PHPA it is assumed competitive, it is that MutB interacts with the active center of PHPA through inhibitory loop, which includes the site of the inhibitor Thr9 - Ala14, the amino acid sequence of which is shown in Table 1. Also in table 1 the corresponding sequence of the native intestine 4 (Inf4) and known mutant intestine 4 (Mutl5). Within the specified site of the inhibitory loop is reactive website Arg10-Asn11, which is cleaved in the active site of PHPA. Table 1 amino acid residues are included in this plot are numbered according to their position relative to the reactive site. MutB has been created by introducing the following amino acid substitutions at the site of inhibitory loops native infestin 4: Thr9Phe, Phel2Tyr and AlaHPro.

Table 1
ProteinThe sequence of the inhibitory loop (R2R1R1'R2'R3'R4')
MutBTRNFVA
Inf4FRNYVP
Mutl5TRRFVA

The selectivity of a new inhibitor against FHA

To assess the selectivity of inhibition FHA a polypeptide comprising a mutant intestine 4 MutB, was defined by its inhibiting activity in buffer against purified coagulation factors. For comparison with the polypeptide were taken known inhibitors FHA: LPS, infestin 4 mutant-based infestin 4 Mutl5, LCTI-III. Table 2 shows the average values of inhibition constants (Ki) indicated some proteins purified proteases of the coagulation (FHA, FHA, FHA and APC). The standard deviation was about 50% from the average value; for each value held 2-3 repeat.

Table 2
ProteinFHAFHAFHAARS
MutB1 nmN. I.N. I.N. I.
LPS1 nm 15 µmN. I.20 µm
Inf41NmN. I.2 µmN. I.
Mutl51 nmN. I.N. I.25 µm
LCTI-III15 nm30 µmN. I.N. I.
where "N. I." (from "not inhibit") - inhibits the activity of an enzyme.

From Table 2 it follows that LPS, native infestin 4 and its mutants equally effectively inhibit the activity of protease FHA in a buffer solution. It was found that in high concentrations, of the order of tens micromol per liter, LPS has inhibitory activity to FHA and APC, i.e. not a highly selective inhibitor PHA. Native infestin 4 and its mutants and MutB Mutl5 not inhibit PHA. Also, mutants intestine 4 MutB and Mutl5 not inhibit PHA, unlike native infestin 4. However Mutl5, like LPS, possesses inhibitory activity against ARS, i.e., too, is not highly selective. From the above Table 2 shows that of all the decree�nnyh inhibitors FHA, MutB has the greatest selectivity of inhibition FHA, because they do not have inhibitory activity to these other factors.

Evaluation of inhibiting activity of a new inhibitor for the inhibition of the contact activation in plasma

Evaluation of the activity of the polypeptide comprising a mutant intestine 4 MutB, inhibition of the contact activation of coagulation, as described later with reference to Fig.1. In three separate samples are free from platelet plasma (PFP) of healthy donor were added, respectively, native infestin 4, mutants and MutB Mutl5, and measured the clotting time upon activation of the contact path through APTT test. According to the results of the tests were plotted APTT (sec) against the concentration of added inhibitor (μm), from which it follows that APTT increased to 120 seconds when added to a sample of 10 microns MutB, 10 ám native infestin 4 or 20 μm Mutl5, with no added inhibitor of the contact activation APTT was 45 sec. Thus, it was found that MutB has about 2 times greater activity in the blood plasma than Mutl5.

The application of a new inhibitor for the inhibition of the contact activation in the research on TF-initiated coagulation

The possibility of use of said polypeptide as an inhibitor of the contact path was pokazane test coagulation of blood plasma through laboratory diagnostic system "Registrar thrombodynamics T-2. The test involved the following steps: thawing the frozen sample PFP, the addition of an inhibitor of the contact activation and incubation of the resulting mixture, recalcification sample and placing it in the measuring channel of the cell, bringing it into contact with TF immobilized on the butt paste special-activator, and registration of the emergence and growth of fibrin clots in the sequential photography. Frozen sample PFP was previously prepared by drawing blood into tubes with sodium citrate at several (at least five) of healthy donors, the next two stages of centrifugation and freezing of the obtained plasma at -80°C. the test was the growth of a fibrin clot from the insert-activator, as well as possibly causing additional clots in the area of the cuvette away from the activator, as shown in Fig.2. Clots in the area of the cuvette away from the activator can be a result of hypercoagulation state of the sample either artifacts of the contact activation of the sample with the walls of the cuvette (artifact clots) that must be suppressed, so that the specified test has allowed to distinguish between normal and hypercoagulability state of coagulation system. To do this, before activating the sample by bringing it into contact with TF, the sample was added an effective amount inhib�torus contact activation in our case it is a polypeptide comprising a mutant intestine 4 MutB-To compare the effectiveness of the inventive polypeptide comprising a mutant intestine 4 MutB, are known in the art as an inhibitor of the contact activation of LPS, in two separate sample of normal frozen PFP was added the indicated inhibitors. Used such concentrations of these inhibitors in which APTT is increased about 4 times: 20 µm in the case of MutB and 15 μm in the case of LPS. As a negative control, the third plasma sample not added any inhibitor of the contact activation. For each sample was performed 4 re-survey. 30 minutes after the start of the study were received image of a fibrin clot in the measuring cuvettes, 4 cuvettes for each sample, shown in Fig.3. Fig.3 shows that the addition or polypeptide comprising a mutant intestine 4 MutB, or LPS, leads to almost complete suppression of the contact activation and prevents the occurrence of artifact clots.

4 repeated experiments for each inhibitor were determined area of the cell occupied by artifact clots (in % of the total area of the cell), for three samples of normal frozen plasma in a separate time points were plotted according to the specified area from time to time (Fig.4, used�military values in each time point). Without inhibitor of the contact activation artifact clots in the area of the cuvette away from the activator appear about 15 minutes after activation and fully occupy the entire area of the cell in 30 minutes. However, if you add LPS or MutB these clots take no more than 15% of the area of the cuvette 30 minutes after activation.

In order to evaluate the effect of inhibitors of the contact activation on the appearance of clots away from the activator-induced hypercoagulation, and not by the contact activation of the sample with the walls of the cuvette, in two separate sample hypercoagulability PFP was added MutB (20 μm) and LPS (15 μm). Hypercoagulability PFP was prepared from normal frozen PFP by the addition of 100 PM FHA. As a negative control, the third plasma sample not added any inhibitor of the contact activation. Fig.5 shows the average of 4 repeated experiments, the time dependence of the square of the cell occupied by the clumps away from the activator (in % of the total area of the cell), for the three plasma samples. Fig.5 shows that in the samples of plasma hypercoagulability without inhibitor of the contact activation and addition of MutB clots away from the activator arise after about 5-8 minutes after activation, while in the sample with LPS they occur in 10-15 minutes. Thus, the authors set�eno, the addition of MutB or LPS leads to almost complete suppression of the contact activation and prevents the occurrence of artifact clots in normal plasma. However, in the plasma hypercoagulability add MutB does not affect the appearance of clots away from the activator, which arise not due to contact activation FHA, while the addition of LPS delays the appearance of the clots away from the activator, presumably due to inhibition of PHA, and can reduce the sensitivity of the diagnostic system to a hypercoagulable state.

Therefore, the polypeptide comprising a mutant intestine 4 MutB may be applied to efficiently block the contact activation and coagulation by contact way.

The effect of inhibitors of the contact activation on the dynamics of fibrin clot formation initiated by TF

The effect of inhibitors of the contact activation on the dynamics of coagulation can be shown by the example of the growth of the fibrin clot from the surface with immobilized TF. The growth dynamics of fibrin clot from the activator may be described by the following parameters: time delay clot formation after contact of the plasma with tissue factor-lag-time (Tlag, min); average speed of clot growth in the range of 2-6 minutes after the start of the drawdown is the initial velocity (Vin, µm/min); and average� the rate of clot growth in the range of 15-25 minutes after the start of the coagulation - fixed speed (Vst, µm/min) (Balandina et al. 2011, Biophys. J. 101:1816-1824; Dashkevich et al. 2012, Biophys. J. 103:2233-2240). According to these parameters can determine the status of blood coagulation in subjects who had taken the test model ("Practical coagulation research" Panteleev M. A., Vasiliev S. A., Sinauridse E. I. et al. - Ed. M. Ed. "Practical medicine", 2011, 192 p., ISBN: 978-5-98811-165-8). So when hypocoagulation States of different nature (deficiency of factors VII, V, X, thrombin, hemophilia A, b and C, treatment with anticoagulants, such as unfractionated and low molecular weight heparin, vitamin K antagonists, antithrombin III) the lag-time increased, and the initial and/or stationary velocity of clot growth is decreased relative to normal values for healthy donors (Parunov et al. 2011, J. Thromb. Haemost. 9:1825-1834). When hypercoagulability States of different nature arise clots in the area of the cuvette away from the activator, and increased primary and/or fixed speed relative to normal values. When using the inhibitor of the contact activation is preferred such inhibitor has no effect on the occurrence of clots away from the activator in the sample plasma hypercoagulability and does not affect the growth dynamics of fibrin clot activator from in any plasma sample.

To compare the impact of Inga�of itoro contact activation MutB and LPS sensitivity to anticoagulation parameters of growth of a fibrin clot activator from, in two samples of normal frozen PFP and in two samples hypocoagulation PFP was added MutB (20 μm) and LPS (15 μm). Hypocoagulation PFP was prepared from normal frozen PFP by adding 0.1 IU/ml of unfractionated heparin. Fig.6A shows the average of 4 repeated experiments, versus time (min) the size of the fibrin clot along the direction of its growth (μm), for samples hypocoagulation and normal plasma with added inhibitors MutB and LPS. Also bar charts show the mean +/- standard deviation (S. D.) of 4 repeated experiments for the parameters of clot growth: lag-time (Fig.6B), the initial velocity (Fig.6C) and fixed speed (Fig.6C). For statistical comparisons was used the student's t test. Statistical significance of differences of the parameters of clot growth in hypocoagulation and normal plasma marked "p<0.001". Non-significant differences are marked with "R>0.5".

Fig.6A shows that in the samples of hypocoagulation and normal plasma growth dynamics of fibrin clot very different from each other, the addition of MutB or LPS does not affect this difference. Parameter lag time (Tlag) is not changed when adding heparin plasma (Fig.6B), however, the speed of clot growth is reduced by about two times (Fig.6C, D).

Application� new inhibitor as an anticoagulant for collection and storage whole blood

The possibility of applying the polypeptide comprising a mutant intestine 4 MutB, as an anticoagulant in the collection and storage of the sample of whole blood was shown by measuring the time of spontaneous blood clotting in a test tube containing MutB (10 μm), LPS (5 μm) or not containing the inhibitor of the contact activation. Were used such concentrations of inhibitors of the contact activation, which lead to an increase in APTT in normal plasma by 2.5 times. The fence of whole blood was performed in 3 healthy donors without the addition of anticoagulant, such as citrate, in addition to the inhibitors of the contact activation. Blood, mixed with the indicated inhibitors were mixed using a rotator at a temperature of +22°C, and measured the formation of clots in the blood.

Fig.7 the results of the measurement are shown by icons corresponding to each dimension; a line or a rectangle shows the area of provision of medians of the values obtained clotting times. Fig.7 shows that the use of an inhibitor of the contact activation or MutB LPS during blood sampling without chelator of calcium ions allows to increase the storage time of whole blood in a test tube about 2-3 times.

Thus, it has been shown the advantage of using the polypeptide comprising a mutant intestine 4 MutB, a new highly selective inhibitor of the contact list�and, to prevent coagulation by contact paths in a plasma sample and to increase the diagnostic sensitivity of the system to deviations of the hemostatic system from the normal state.

Aspects of the present invention are described in the following examples, which are presented only to illustrate the present invention and to help ordinary skilled in the art in its implementation and use. Examples are in no way intended to in other cases to limit the scope of the invention.

Examples

Hereinafter, the present invention is illustrated but not limited by the following examples. However, the materials, methods, etc. described in the present description, only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. Therefore, the materials, methods, etc., similar or equivalent described in the present description, can be used for practical implementation or testing of the present invention.

Example 1. Selectivity MutB to FHA in a buffer solution

To determine the inhibitory activity of the proteins MutB, LPS, Inf4, Mutl5 and LCTI-III in relation to various coagulation proteases in the buffer was used chromogenic test: a mixture of protease and inhibitor in the wells of innumerables� for 15 minutes at a temperature of +37°C, next, the mixture was added a solution of chromogenic substrate specified proteases, and protease activity was measured by formation of a chromogenic product at +37°C. table 3 shows the names and concentrations of chromogenic substrates and related proteases.

Table 3
ProteaseThe concentration of protease in the test, nmSubstrateThe concentration of substrate in the dough, mmKm of the substrate to the protease, mm
FHA1S-23020,20,2
FHAODS-23660,51,0
FHA0,5S-27650,50,25
ARS1Spectrozyme pCa0,20,4

Chromogenic substrates include p-nitroaniline group, cat�Paradise was useplease relevant proteases. After cleavage of the p-nitroanilide group from a substrate molecule the group is changing absorption spectrum, and the change in the concentration of the product in time, one can detect the wavelength of light of 405 nm using a spectrophotometer tablet. The generation of protease activity on the concentration of the inhibitor, in multiple samples with the same concentration of protease was added different concentrations of inhibitor. It was further determined the concentration of inhibitor that reduces the activity of the protease by 50% (IC50). The inhibition constant (Kiwas calculated according to the equation Chang-Prusoff (Ki=IC50/(1+S/KM)), where S is the initial substrate concentration, km is the constant of Michaelis-Menten substrate to the protease. The results of a calculation of Kishown in Table 2.

Native infestin 4 and its mutants and MutB Mutl5 were in the form of a fused protein with thioredoxin I of E. coli containing polyhistidine clusters. LPS was a protein isolated from corn and purified chromatographically. LCTI-III was obtained by chemical synthesis. The reaction of cleavage of the substrate was carried out in a buffer of 50 mm Tris-HCl, 130 mm NaCl, and 0.5% bovine serum albumin, pH 8.3.

Example 2. Evaluation of inhibitory activity of MutB by inhibition of the contact activation in plasma

The blood plasma of healthy donors g�touili in accordance with the standard rules of drawing blood for koagulologicheskih tests (Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline-Fifth Edition by Clinical and Laboratory Standards Institute (CLSI) H21-A5 Vol.28 No.5). For blocking ions in the blood added of 0.38% sodium citrate.

The APTT test was carried out using reagents Coagula-test (NGO "Rena", Moscow, Russia) and various concentrations of inhibitors of the contact activation: native infestin 4, MutB and Mutl5. For this, 50 µl of blood plasma of a healthy person was mixed with 5.6 μl of a tenfold solution of the inhibitor in different concentrations and incubated for 15 minutes at +37°C. Further, in accordance with the guidelines Coagulo test, 25 µl of the mixture was transferred into a cuvette of coagulometer Helena-S2 and added 25 µl of kaolin-kefallineou mixture, activating FH in plasma. After incubation at +37°C for 3 minutes was added 25 μl of a solution of calcium chloride in the initial concentration of 25 mm, and photometrically determined the formation of the fibrin clot in a ditch of coagulometer.

According to APTT concentration of native infestin 4, MutB and Mutl5 shown in Fig.1. It was found that the mutant intestine 4 MutB has a high activity in plasma, compared with Mutl5 and native intestinal 4.

Example 3. The influence of MutB on the appearance of the artifact clots due to contact activation and growth dynamics of fibrin clot initiated by TF

Effect�s polypeptide, comprising a mutant intestine 4 MutB, on the growth of fibrin clots in the samples hypocoagulation, normal, and hypercoagulability of blood plasma, was investigated using laboratory diagnostic systems "Registrar thrombodynamics T-2" and "a Diagnostic kit for research thrombodynamics in plasma" (OOO Hemacore, Moscow, Russia) in accordance with the guide to "Diagnostic kit for research thrombodynamics in plasma" (OOO Hemacore, Moscow, Russia). 15 minutes before the study measuring cuvette, insert-activator with immobilized TF and reagents were maintained at +37°C. the Standard temperature research +37°C. For 15 minutes prior to study 120 µl of plasma were mixed with the test substances, MutB, or LPS for comparison, and was placed in a microtube. Produced through the mixing of plasma with substances, and after that, the tube of plasma was placed for 15 minutes in a thermostat at +37°C. Immediately before the study was conducted recalcification plasma were selected 120 μl of the mixture of plasma with the test substances were added to the microtube with the Reagent P (freeze-dried solution of calcium acetate with additive) and was stirred until complete dissolution of the Reagent II. Next, 120 μl of a mixture of micro tubes with Reagent II instant� were transferred into a cuvette. Next, insert-activator gently lowered into the cell so that the surface of the immobilized tissue factor came into contact with the plasma. Directly after bringing into contact of the activator of the coagulation of the blood plasma started taking pictures of the process of growth of a fibrin clot in plasma, placed in a cuvette. Insert the activator was a plate on the lower end surface of which is immobilized activator of coagulation tissue factor (Fadeeva et 2010, Biochemistry 75:827-838). Calculation of results the study was performed automatically by the software (user manual "Software system diagnostic laboratory "Registrar thrombodynamics T-2", LLC "Hamakor", Moscow, Russia).

Example 4. Increase the storage time of whole blood at the fence without calcium chelator using MutB

Blood sampling was performed from the median vein of the elbow of his left hand into a sterile syringe Master UN RE a volume of 10 ml with needle 21G x 1½"; 0,8×40 mm (Parmelin, USA). In the blood didn't add the calcium chelator or another anticoagulant, also inhibitors of the contact activation MutB or LPS. Taken the blood was poured into a test tube with a volume of 15 ml was stirred briefly 2 times and placed in 0.7 ml of blood in polypropylene microcentrifuge tubes MaxyClear Snaplock (Axygen, USA) in a 1.5 ml, direct� then starts counting time using the timer. In these tubes was previously added by 78 μl of a solution MutB with a concentration of 100 μm solution of LPS at a concentration of 50 μm or buffer solution of 30 mm Hepes pH 7,4. Tubes containing the mixture of whole blood and these solutions were recorded in the rotator and mixed by turning with a speed of 10 rpm.minutes Recorded the time of occurrence of clots or fibrin strands in the whole blood with stirring.

1. Polypeptide for inhibiting contact activation in the sample of blood or blood product, characterized by the sequence of the mutant intestine 4 MutB (SEQ ID NO : 1) and essentially corresponds to it, where the specified sequence can have modifications beyond inhibitory loop, substantially retains the activity of the specified polypeptide, which is a highly selective inhibitor PHA, the selectivity is higher than that of the native intestine 4 and Mut15, or activity of which is higher than that of the native intestine 4 and Mut15.

2. The polypeptide according to claim 1, where modifying the amino acid sequence beyond inhibitory loops constitute a variation of the N - or C-Termini of the sequence SEQ ID NO: 1, deletion or insertion of one or several amino acid residues, as well as conservative amino acid substitutions.

3. The use of a polypeptide according to claim 1 for the study of Saint�of stevania in the sample of blood or blood product, which includes:
(a) obtaining a sample for coagulation studies,
(b) inhibition of the contact activation of coagulation in the sample by bringing the sample into contact with said polypeptide and incubation of the resulting mixture,
c) bringing into contact of the activator of the coagulation sample, prepared at the stage (b); and
(d) measurement of coagulation parameters in the sample.

4. The use according to claim 3, wherein the obtaining of the test sample of blood or blood product comprises blood in a tube containing an anticoagulant selected from the group: sodium citrate, EDTA, an inhibitor of thrombin or inhibitor of FHA, and the subsequent blocking of the anticoagulant, in particular recalcification sample.

5. The use according to claim 4, wherein obtaining a sample of blood or blood product additionally consists of cleaning the blood from unwanted cells and cell components, in particular by filtration or centrifugation.

6. The use according to claim 3, where the sample of blood or blood product is their, is liofilizirovanny or frozen form, which is converted into liquid form before conducting the study.

7. The use according to claim 6, where the sample of blood or blood product is whole blood or blood products such as platelet rich plasma, platelet-poor plasma, released from platelets, plasma, coat�jodirovannaja plasma.

8. The use according to claim 3, where the sample of blood or blood product obtained from a healthy subject or from a subject who suffers from impaired blood clotting, in particular from the subject with hemorrhages or thromboses who have impairments or deficits of the hemostatic system.

9. The use according to claim 3, where the sample of blood or blood product obtained from a patient during surgical procedures or in the exercise therapy.

10. The use according to claim 3, where the sample of blood or blood product contains natural or recombinant proteins or other drugs, which have a haemostatic or fibrinolytic activity.

11. The use according to claim 3, where the specified polypeptide is in a dried form or liofilizirovanny form, or adsorbed on the surface which contacts the sample of blood or blood product, or pre-dissolved in a solution containing salt, a buffer substance, excipient or stabilizer.

12. The use according to claim 3, wherein the mixture of step b) is incubated at a temperature of +20°C to +40°C, preferably at +37°C, for a time at least 3 minutes.

13. The use according to claim 3, where the specified polypeptide is taken in a quantity sufficient to effectively block contact activation.

14. The use according to claim 13, where sufficient is the amount of the specified polypeptide, wherein ACTV n�malnoy plasma increased more than 2 times, mostly more than 3 times compared to the APTT of plasma without polypeptide.

15. The use according to claim 14, where sufficient is the amount of the specified polypeptide, which lacks artifact clots away from the activator of coagulation in a sample of normal frozen plasma, for more than 15 minutes, preferably more than 30 minutes after activation.

16. The use according to claim 3, wherein the bringing into contact of the activator of the coagulation specimen of blood or blood product in step C) is carried out by adding to the specified pattern and subsequent mixing of the activator of coagulation in dissolved form.

17. The use according to claim 3, wherein the coagulation activator dissolved in the form of a layer on a specimen of blood or blood product without stirring, or the sample is brought into contact with the activator, which is immobilized on the surface.

18. The use according to claim 3, wherein the activator of coagulation is tissue factor or one of the coagulation proteases: VIIa, XA, II, IXa, Pendant, Hiia.

19. The use according to claim 3, wherein the coagulation parameters are: delay time start coagulation, clotting time, the delay time of the operating time of thrombin, the time to reach maximum thrombin, the maximum concentration of thrombin, the angle of thromboelastogram, speed of clot growth, the size of the clot in a fixed moment of time, aliciashouse.com away from the activator of coagulation, the speed of clot lysis.

20. The use of a polypeptide according to claim 1 as an anticoagulant that blocks the contact activation, by bringing into contact with said polypeptide of a sample of blood or blood product for intake and increase the retention time of the sample.

21. The use according to claim 20, where the specified polypeptide is in a dried form or liofilizirovanny form, or adsorbed on the surface which contacts the sample, or pre-dissolved in a solution containing salt, a buffer substance, excipient or stabilizer.

22. The use according to claim 20, where blood sampling is carried out in a tube containing an anticoagulant selected from the group: sodium citrate, EDTA, an inhibitor of thrombin or inhibitor of the.

23. The use according to claim 20, wherein the storage sample of blood or blood product is frozen at negative temperatures below 0°C, preferably below -50°C, or in liquid form at a positive temperature more than 0°C, preferably from +20°C to +40°C.

24. The use according to claim 20, wherein the storage time is more than 10 minutes, preferably from one day or more, mostly more than one week.

25. The use according to claim 20, wherein the sample of blood or blood product obtained from the subject, not having a blood coagulation disorder, or a subject who suffers from impaired blood clotting in castnet� from the subject with hemorrhages or thromboses have impairments or deficits of the hemostatic system, or from the subject during surgical procedures or in the exercise therapy.



 

Same patents:

FIELD: medicine.

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and to determine initial Ti and final Tf blood coagulation parameters wherein Ui, Uf are the normalised initial and final blood coagulation thresholds.

EFFECT: invention enables reducing a procedure error by tens orders, increasing a coagulation time accuracy by 4 orders, and reducing the efficiency three times as much that eventually provides higher metrological effectiveness of computer analysers applicable for the purpose of automating the process of detecting the individuals having a risk of haemocoagulation complications.

4 dwg

FIELD: chemistry.

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2 dwg, 1 tbl, 2 ex

FIELD: medicine.

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2 ex, 1 dwg

FIELD: medicine.

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EFFECT: method provides higher information value of biochemical tests, reducing consumption of the biological material.

9 tbl, 2 ex

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.

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4 tbl, 3 ex, 2 dwg

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.

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32 cl, 1 dwg

FIELD: physics.

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4 cl, 2 dwg

FIELD: medicine.

SUBSTANCE: blood plasma is examined in 4 minutes after the beginning of spontaneous red blood cell aggregation for free red blood cell count and cell count in aggregates. A percentage of non-aggregated red blood cells (PNA RBC) by formula PNA RBC=FRBSC×100/(TRBCA+FRBSC) wherein FRBSC is the free red blood cell count, TRBCA are total red blood cells in aggregates. If the PNA RBC is 56 to 30%, I degree of severity is stated, 30% to 4% - II degree of severity, less than 4% - III degree of severity.

EFFECT: use of the invention enables objectifying and increasing precision of evaluation of red blood cell aggregation, evaluating an intensity of patient's microcirculation disorders in a relatively short time, and thereby ensuring well-timed adequate complex of therapeutic measures or corrected therapy.

3 ex

FIELD: medicine.

SUBSTANCE: quantity of fibrin-monomers, dissolved in 0.5 N sodium hydroxide, is determined spectrophotometrically with application of ethanol test. Claimed method of quantitative determination of fibrin-monomers in blood makes it possible to reveal pathological process in organism with 95% reliability.

EFFECT: increase of determination accuracy.

2 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: thrombosis monitor comprises: a thrombosis chamber, at least in a part of which there is a thrombogenic material; an inlet tube connected to the thrombosis chamber through which blood flows into the thrombosis chamber; a blood supply container connected to the inlet tube; a feed pump for the container; a pressure sensor for measuring pressure applied to the container. A method of thrombosis monitoring consists in the fact that after introduction of an anticoagulant, blood is supplied from the container to the thrombosis chamber by pressing on a fluid placed on a blood layer and having density less than that of the blood layer; it is combined with anticoagulation blood processing or blood coagulation stimulation, and measurement of pressure applied to the container; the thrombogenic material is placed at least in a part of the thrombosis chamber.

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11 cl, 15 dwg, 23 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.

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15 cl, 5 dwg, 1 tbl, 2 ex

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: 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: 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: 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: 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: 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: medicine, pharmaceutics.

SUBSTANCE: invention refers to polypeptides having hydrolysable covalent bonds with therapeutic agents for drug delivery. The invention also refers to pharmaceutical compositions which contain the compounds according to the invention, to using them in methods of treating cancer.

EFFECT: polypeptide conjugates can be used as vectors for a therapeutic agent transport through the blood-brain barrier (BBB) and for delivery into specific types of cells, such as the ovaries, liver, lungs or kidneys.

6 cl, 16 dwg, 8 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biochemistry, particularly to an isolated version of a Bowman Birk protease inhibitor (BBPI), where said BBPI version has a frame containing amino acid sequences 1-40 and 50-66 of SEQ ID NO: 187 and cysteine residues in amino acid positions 41 and 49 of SEQ ID NO: 187, between which is included a binding peptide selected from a binding VEGF peptide, a binding FGF-5 peptide, a binding TGFb peptide and a binding TNFα peptide, where said BBPI version further includes a substituted amino acid in an amino acid position selected from positions equivalent to positions 1, 4, 5, 11, 13, 18, 25, 27, 29, 31, 38, 40, 50, 52, 55 and 65 of SEQ ID NO: 187, where said substituted amino acid in position 1 is A, in position 4 is V, in position 5 is selected from P and A, in position 11 is 11G, in position 13 is selected from 13Y, 13I, 13F, 13M, 13L, 13V, 13K and 13R, in position 18 is selected from 18I and 18V, in position 25 is selected from 25K, 25N, 25W, 25I, 25A and 25R, in position 27 is selected from 27H, 27K, 27V, 27A and 27Q, in position 29 is selected from 29R, 29K and 29P, in position 31 is selected from 31Q, 31H, 31E, 31A, 31R, 31W, 31K and 31T, in position 38 is selected from 38N, 38K and 38R, in position 40 is selected from 40H, 40K, 40Q, 40R and 40Y, in position 50 is selected from 50R, 50Q, 50K, 50T, 50V, 50M and 50S, in position 52 is selected from 52K, 52T, 52R, 52Q, 52L, 52H, 52A, 52M, 52S and 52E, in position 55 is 55M, in position 65 is selected from 65E and 65D.

EFFECT: invention enables to effectively inhibit trypsin compared with a non-modified variant.

6 cl, 18 dwg, 7 tbl, 18 ex

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