JPH06506605A - Method for measuring fibrinolytic capacity in human whole plasma - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

[Detailed description of the invention] Method for measuring fibrinolytic capacity in human whole plasma Honsujiri mutual tray The present invention relates to a method for measuring m-solubility in whole plasma. In particular, the present invention provides fibrin clots. A table that mimics the in vivo surface of blood cells and thus allows a better determination of plasma fibrinolytic capacity. An immobilized plasminogen binding material is utilized to provide a surface.

Vehicle volume Blood flow or blood clots usually form in response to signals associated with a rupture in the blood vessel wall; to prevent excessive bleeding from the circulation. However, excessive growth of thrombi is itself It occludes systemic blood vessels, causing a lack of local blood flow. For this reason, blood clots cause damage to blood vessel walls. Carefully balance the blood to seal without completely blocking the cavity. It must be a dynamic process of plug formation and dissolution. Blood flow is abundant and non- Fibrinogen, a protein that is always soluble, is destroyed by the action of the enzyme thrombin. A protein that is more insoluble, formed by the clotting process and converted to fibrin will be accomplished. Fibrin polymerizes and binds platelets, other blood cells, and blood proteins. Covalently cross-linked to form a matrix of blood flow that traps within its meshwork It will be done. One of these proteins, plasminogen, is one of these proteins. It is converted to the active enzyme plasmin by the action of other enzymes known as activators. can be Plasmin is a protein in several parts of fibrin. Catalyzes the degradative cleavage of fibrin into soluble fibrin breakdown products and thus dissolves clots can. Soluble polylysine or its derivatives can activate plasminogen It is known. Allen, R, A; Thromb, Haemost as 47 (1): 41-45 (1982); Petersen.

L, A, and Swenson, E, ;BiochemicaBio h 5 ica Acta, 883:313-325 (19 signals plasmin production Plasma components are called fibrinolytic systems. When this system is not sufficiently active compared to blood clotting activity This includes coronary thrombosis, deep vein thrombosis, peripheral artery occlusion, pulmonary embolism, occlusive stroke, etc. resulting in a prothrombin state that can lead to plug disease. Qualify such an imbalance Clinicians who attempt to Genactivator) will be administered.

In evaluating the cause of clotting, the clinician should examine coagulation parameters (e.g., thrombin time, It is routinely desirable to measure both prothrombin time) and fibrinolytic parameters. It will be useless. The latter, or fibrinolytic capacity of plasma, is plasminogen, a plasmin inhibitor. , plasminogen activator, and plasminogen activator inhibitor. It is a complex function of the concentration of the harmful agent. Ultimately it is the plasma that produces long-lasting plasmin activity. reflected in the ability to produce. Its evaluation is an essential part of the coagulopathy and thrombosis treatment plan. It is.

A number of tests have been developed to measure fibrinolytic power in whole blood fibrinolysis. for example Wun and Capuano described one such method. J,Bi　o l, Chem, 260 (8): 5061-5066 (1985) Wu In the n method, 8 clumps/l are formed in the 1125 fibrinogen-enriched fraction, and then coagulated. Follow the lysis of clots formed in citrated plasma fed with some of the living person's own enzymes. Detect spontaneous fibrinolysis of plasma clots by tracing. Using this method, Wun centrifuges a patient's plasma sample and samples the radioactivity present in the supernatant of the centrifuged sample. Determine the solution over several days by plotting it against the radioactivity found throughout the pull. Detect the amount of solution. This method is radioactive and has a very short pot life112 5 has the disadvantage of using fibrinogen. Moreover, it will take several days to complete. Requires long incubation.

Other fibrinolytic assays commonly used to measure fibrinolysis include fibrin plates. Dissolution method, Astrup, T. and Darling, S., Acta Ph. 5iol 5cand, 4:45 (1942). In this assay An opaque plasma clot is artificially formed in a plate or vetri dish. then the patient A sample is passed through the mass, forming a clear lysis zone, which is then examined. this The assay has several difficulties associated with it. For example, the clot dissolution zone is difficult to quantify, and the assay itself requires extremely long incubations. A faster variation of this test, which requires hours, sometimes up to 24 hours, is Developed by S.H. and Gaffney. However, this variant Requires addition of lasminogen and thus alters natural sample of patient sample Marsh, N.A., and Gafney, P.J., Throm bos, Haemostas, (Stuttg, ), 38: True globulin clot Dissolution time assays are also commonly used to test fibrinolytic power. This assay is , patient sample true globulin S cover centro/gFp) left! Jushu Susu Check with the naked eye how long it takes for the lead to be dissolved by the culm stirrer. It will be implemented by making a decision. This assay is relatively fast, typically 60-1 There is a benefit in 20 minutes, but the endpoint, ie clot dissolution, is indicated. Twenty %FP is as follows.

Figure - Figure 5 shows patient plasma sample %FP versus time before, during and after cardiovascular bypass surgery. Indicates the interval.

■: Figure 6 shows patient plasma sample %FP vs. before liver transplant surgery using the method of the present invention. Indicates the middle and after times.

L: Figure 7 shows the %F of samples from patients with various diseases using the method of the present invention. Indicates P.

Figure 8 shows “HEPSTRACT” for heparinized plasma samples. Trade name for hevarinase, an enzyme that degrades phosphorus or heparinized samples ) shows the effect of

Head office Ω base! The present invention uses immobilized plastic to mimic the in-vivo printer surface. A line that utilizes a minogen binding substance, preferably poly-α-lysine or a derivative thereof. Concerning dissolution assay. The plasminogen-binding substance has a support that binds plasminogen. applied to a solid phase matrix or support in such a manner that it is coated with a binding substance. It will be done. Plasma containing all fibrinolytic factors or control added to support coated. A portion of the plasminogen in the sample is converted to plasmin at approximately room temperature. Incubate for sufficient time. This incubation period is approximately 2 and can vary from about 60 minutes, but preferably from about 5 to about 30 minutes. Probably between. Incubation for about 30 minutes or longer is probably due to autologous reaction caused by plasmin. It should be noted that the signal may actually decrease due to lysis. However, this Assays can be performed in ink for 30 minutes or longer, as long as all samples are processed consistently. It can still be carried out after the evaluation. The generated plasmin is in the sample. is directly correlated to the fibrinolytic power of The plasmin present can be determined by fluorometry, panchromogenic or such as the use of radiolabeled plasmin synthetic substrates or immunochemical assays. It can be quantified by a number of standard methods. Husby, R.M. and S. mith, R, E, Sem1nar in Thrombosis and H emostasis Vol, VI(3):17 This assay Fast, about 5 to 30 minutes for determining process abnormalities is short and reliable Provides a screening assay that exceeds currently available fibrinolytic assays This is a significant improvement. In particular, the assays of the invention more accurately determine the physiological state of a patient. First, it is superior to other currently available assays because it reflects Samples containing all fibrinolytic factors present in the body are more accurate. . Additionally, exogenous fibrinolytic factors begin to react as other fibrinolytic tests require. You don't need it to do it. Second, this is an immobilized plasminogen-binding substance. Take advantage of. Immobilization that exceeds substances used for 1P such as soluble polylysine The advantage of immobilized plasminogen binding substances is that provides a blood coagulation surface as it exists in vivo, and for this reason fibrinolytic factors The idea is to allow as much of it to collect on the artificial surface as it collects on the surface of the mass. . Furthermore, the relative concentration of blood factors is increased by providing this artificial surface. , this book makes it possible to proceed with the reaction without adding any foreign enzymes. In the assay of the invention, latex, glass fiber or nitrocellulose solid phase matrices made of any material commonly used in immunoassays such as The substrate or support is coated with a plasminogen binding substance. Most preferred example In , the plasminogen binding substance is trinitrobenzoylated poly-d- The present invention is a derivative of poly d-lysine, such as lysine or other comparable derivatives. Polly invented the method! - Lysine can be used, but! -Isomers are The d-isomer is less efficient as it is more easily degraded by rasmin. centre Fibrin and denatured fibrinogen can also be used as plasminogen binding substances. can be used.

The molecular weight of the polylysine used is any length of polymer with a molecular weight of 50,000 daltons or more. may vary widely to be sufficient, but in preferred embodiments the point Rimmer ranges from about 140,000 to about 250,000 as shown in Figure 1. should have a molecular weight.

Plasminogen binding substances include sodium phosphate, sodium borate, and sodium bicarbonate. Can be suspended in a buffer such as thorium buffer or any comparable buffer Wear. A wide range of concentrations of plasminogen binding substances used to coat the solid phase However, in the most preferred embodiment the polylysine concentration is about 5 μg. /d to about 100 μg/d.

Incubation of the plasminogen binding substance with the solid phase is carried out at a temperature of about 2°C to about 25°C. This is accomplished by passive adsorption at a temperature of 0.degree. C. for a period of about 5 minutes to about 24 hours.

After incubation, excess reagent is removed by washing with buffer. preferred In some embodiments, the wash solution is at neutral pH, such as imidazole-buffered saline. It is an isotonic buffer containing a surfactant. However, it may contain surfactants. It is also permeable to hypertonic or hypotonic buffers that do not contain Ru.

An appropriate amount of plasma sample or control is then passed through the derivatized solid support and the and the natural fibrinolytic process is allowed to proceed. Ink with this sample vation may range from about 5 to about 60 minutes, but in the most preferred embodiment The incubation time between the sample and the derivatized support is approximately 5 to 30 minutes, most preferably about 15 minutes. Excess sample or control Removed by washing with buffer. In a preferred embodiment, the cleaning solution is A neutral pH surfactant-containing isotonic buffer such as dazole buffered saline. hair However, hypertonic or hypotonic buffers with or without surfactants, and acidic Or even basic pH. Plasmin produced during incubation remains attached to the coated support and is then chromogenically, fluorescently or radioactively labeled. Standard methods such as recognized plasmin-specific synthesis or methods using natural substrates Quantitated using quantitative methods. Plasmin-specific substrate contains sample It is applied to an activated solid support and the incubation is released. this Incubation with the substrate can range from about 30 minutes to about 24 hours; In a preferred embodiment, the incubation time is about 30 minutes to 2 hours, up to Also preferably about 1 hour. Haseby, R.M., and Smith, R. E,, Sem1nar in Thrombosis and HemoSta sis, VoI, Vl (3): 173-314 (1980) How many such substrates can be used to quantify the plasmin produced? An example of this is described in FIG. Plasmin is also known as plasmin/plasminor. It can also be quantified immunochemically using genetic antibodies. Harppl, C0H , J. Cl1n, Invest, 68:46-55 (The method of the present invention also It can be used in a t-shape. The components of such kits include plasminogen binders. Compound-coated microwell titer plate, lyophilized normal calibration plasma, First lyophilization control of normal pooled plasma sample, second lyophilization of normal pooled plasma Control, substrate buffer, lyophilized substrate, solution to stop the group t/plasmin reaction, and wash buffer. An example of the ingredients included in the kit is as follows: It is. Poly d-lysine coated microwells, 2 x 8 modules, 6 modules le/frame, 96 tests/kit; calibration reagent (lyophilized), 100 (+/- ) 5% normal plasma, 1. Od/vial, 1 vial/kit; 1st control (level ■) (lyophilized), >80% normal plasma, 1.0d/vial, l vial/kit G; Second control (level II) (lyophilized), <40% normal plasma, 1.0IIL 1/vial, 1 vial/kit; I=1 in buffer, 12d/vial, 1 vial Al/Kit; Substrate (lyophilized), ljd! / vial, 1 vial / kit ; stop solution or solution to stop the plasmin/substrate reaction, 6af/vial, 1 Vial/quint; Washing solution, 20xl condensation solution, 20m/vial, 1 vial/ Kitsuto.

The assay system of the present invention uses lipoprotein(a)2 tissue plasminogen activator. ter (tPA), plasminogen activator-1 (PA, r), α-2- Specific to measure anti-plasmin/plasmin complexes and ten-PA/PAr complexes It can also be modified to use specific antibodies. tPA, PAI or α-2- Functional assays to quantify antiplasmin activity also utilize synthetic substrates and immobilized polylysine. It is possible using

! Give ■ The method of the invention can be further illustrated by the following examples.

Example 1 The microwell titer plate was filled with 100mM sodium bicarbonate buffer pH 9, Poly-alpha-lysine in 5 (molecular weight range 140,000-250,000 daltons) It was coated at 10 μg/d. Coating with poly d-lysine for 24 hours at 4°C. It was carried out by passive adsorption between. Excess polymer is imidapool-buffered saline. Removed by washing with water. Then approximately 100μ of each plasma sample or control! was applied to the coated support. Samples were incubated for 15 minutes. excess The reagents were washed three times with approximately 200 μ2 of imidazole-buffered saline each time. removed. The plasmin generated during the incubation has a chromophore leaving group p - Plasmin-specific synthetic substrate containing nitroaniline, HD-norleucyl- Hexahydrothyrosyl-lysine-P-nitroanilide diacetate salt (but optional Any lysine- or arginine-terminated peptide with a colorimetric or fluorometric label of interest. Chido can also be used. ) was quantified by Add 00 μl of substrate I to each well; Incubated for 60 minutes at room temperature. The released chromophore p-nitroaniline is Quantification was performed using a spectrophotometer capable of measuring absorbance at 5 nm. This app is convenient. The assay was performed in a 96-well microwell system, with one microwell absorbance readout. controller was used. Ratio of sample and control absorbance to normal reference plasma absorbance and reported as % of reference plasma.

Example 2 'A/S Nunc Maxi 5orp' microwell titer plate, 2 x 8 modules or equivalent in 0.1M sodium bicarbonate buffer PH9 ,0 coated with poly-d-lysine (average molecular weight 248.400) 20μg/d. Ta. Each well contains 110μ of poly d-lysine solution! filled with ambient or room temperature and incubated for 4 hours. 6 w/v% charcoal gelatin aqueous solution approx. 110 uffi is then added to the wells and incubated for approximately 30 minutes at ambient or room temperature. It was done. A 6 w/v% solution was used in this example, but other variations may also be used. obtain. Next, aspirate the contents of the wells and add 20mM Tris and salt to each well as wash solution. Solution 25 containing 80mM sodium, 200.02% Tween, pH 7.4 0 μm/well was added.

This washing liquid was aspirated and the washing operation was repeated twice. The wells are then lowered for approximately 20 hours. Air-dried in a humidity chamber (temperature approx. 8-20%, temperature approx. 27-35°C) ). 100 μ2 of each plasma or control was passed to the coated support. The sample is approximately 1 Incubated for 5 minutes.

The excess reagent is about 200 ul of imidazole buffered saline each time! Wash 3 times using was removed by The plasmin produced during the incubation is H-D-Nor, a plasmin-specific synthetic substrate containing the chromophore group p-nitroaniline Leucyl-hexahydrotyrosyl-lysine ~ P-nitroanilide diacetate salt (but any lysine or arginine with any colorimetric or fluorometric label) Terminal peptides can also be used. ) was quantified. Add approximately 100μ of substrate to each well. and incubated for 60 minutes at room temperature. Released chromophore P-nitroaniline was placed in a spectrophotometer capable of measuring absorbance at 405 nm. Conveniently this Assays are performed in a microwell system and one microwell absorbance reader was used.

The absorbance of the sample and control is compared to that of normal reference plasma and is calculated as % of reference plasma. It was reported as follows.

Example 3 2.4.6-1-linitrobenzyl (TNB) poly d-lysine is poly d-lysine 20 μm of lysine (average molecular weight 160,000), 13.2 μmo in 4 Id of distilled water l of 2.4.6-dolinitrobenzyl/I/phonic acid for 10 min at ambient or room temperature. prepared by incubating. Unreacted 2,4.61linitrohen Dilsulfonic acid was removed by dialysis. This alkylation is less or It can be increased or decreased by providing more lysine residues, respectively.

Microwell titer plates were prepared with 0.1M sodium bicarbonate buffer pH 9, 5, poly-d-lysine (average molecular weight 143,700) 10 μg/d or 0 ．． TNB-poly d-lysine in 1M sodium bicarbonate buffer pH 9.5 (average min. Coated with molecular weight 160,000) 10 μg/ll11. The coating should be kept at 2-8°C. It was carried out by passive adsorption for 24 hours. Excess polymer is sodium chloride 80mM and 20mM containing surfactant (0.Q2% Tween2Q) Removed by washing with Tris. Normal plasma pool sample approximately 100uA was applied to the coated support. Samples were incubated for 15 minutes at room temperature. past Excess reagents are removed by washing three times with approximately 200 μl of phosphate buffered saline. removed. The plasmin generated during the incubation has a chromophore leaving group p -To plasmin-specific synthetic substrate HD-norleucyl-containing nitroaniline xahydrotyrosyl-lysine-p-nitroanilide diacetate (any Any lysine- or arginine-terminated peptide with a colorimetric or fluorometric label can also be used. ) was quantified by Add 100 μi of substrate to each well and leave at room temperature. Incubated with The released chromophore P-nitroaniline has a reading of 405n. It was quantified using a spectrophotometer capable of measuring absorbance at m. The reading is incubi The samples were taken after 15, 30, 60 and 120 minutes of session time. This assay is useful was performed in a 96-well microwell system with one microwell absorbance reader. was used. Absorption of samples using TNB-poly d-lysine coated wells The absorbance was compared to the absorbance of the same sample using poly d-lysine coated wells.

The data are shown in Table I of FIG.

Example 4 except that the plasma pool samples were incubated with coated wells for 30 min. Example 3 was repeated. The data are shown in Table 3 of FIG.

Example 5 except that the plasma pool samples were incubated with coated wells for 60 min. Example 3 was repeated. The data are shown in Table 3 of FIG.

When using a substrate labeled chromogenically, chromogen- or fluorometrically as in the examples above, The fibrinolytic power (FP) of a sample or control is measured using the following equation.

In this equation, "Sample Plasma Absorbance" is defined as "Sample Plasma Absorbance" Released during the reaction used to determine plasmin concentration in pull tests Absorbance value for a chromogen or fluorogen. “The reference plasma absorbance parameter is Determining the plasmin concentration in a reference or control sample test according to the invention absorbance for the chromogen or fluorogen released during the reaction used to determine It is a value. The values assigned to “reference plasma” are based on fresh normal plasma from 30 donors. It can be determined from multiple assays on the pool. fresh doner boo The average absorbance value for the sample is considered to be representative of 100% fibrinolytic power. Line molten power is general a minimum of 96 measurements (16 from each of 6 randomly selected 5-vial pools) (times measurements). Figure 4 shows the application of this equation to patient and reference samples. shows.

Figures 5.6 and 7 show reference samples or samples using the method described in Example 2. %FP of tested patient plasma compared to control.

These samples were collected from patients who underwent cardiovascular bypass and liver transplant surgery; Obtained from patients with various other debilitating diseases. Determination of a patient's %FP is based on angina, cardiac have myocardial infarction, deep vein thrombosis, stroke, contagious vascular procoagulation and congestive heart disease. Preliminary studies have shown that %FP is decreased (2.7-69.3%) in patients with This is especially important because FP values also determine whether liver transplant or cardiovascular bypass surgery It decreases during surgery in patients undergoing surgery (<40%), but it returns to normal within 10 days after surgery. The rate of recovery to normal values varies for each patient. Finally, Figure 8 shows a heparinized plasma sample. “’Heptract” degrades heparin or heparinized samples in samples. shows the effect of

Table I Poly d-lysine well TNB-poly d-lysine well table ■ Poly d-lysine well TNB-poly d-lysine well table ■ Poly d-lysine well TNB-poly d-lysine well quality incubation time A405 A405 (minutes) Sample 3 0.529 0.515 68.780.500 Time (days) Time (days) FIG.8 International search report OrT/II<9’109972orTt1<Q7109Q 77

Claims (1)

[Claims] 1. (a) A plasminogen binding substance is attached to a solid phase support, and the support is attached to a plasminogen. incubate for sufficient time to coat with the binding substance, (b) remove excess plasminogen binding material; (c) remove plasma sample or Incubate the light-coated support for a sufficient time to generate plasmin. , (d) remove excess sample or control, and (e) remove plasmid bound to support. A method for determining or detecting fibrinolytic capacity in whole plasma comprising measuring the concentration of 2. (a) poly-d-lysine or a derivative of poly-d-lysine as a solid support; sufficient for the support to be coated with poly-d-lysine or a derivative of poly-d-lysine. (b) excess poly-d-lysine or poly-d-lysine; remove the gin derivative, (c) Support coated with plasma sample or control and plasmin generated. (d) remove excess sample or control; (e) incubate for sufficient time; (d) remove excess sample or control; ) to determine the fibrinolytic capacity in whole plasma, including measuring the concentration of plasmin bound to the support. How to determine or detect. 3. (a) A plasminogen binding substance is attached to a solid phase support, and the support is attached to a plasminogen. incubate for sufficient time to coat with the binding substance, (b) Add fish gelatin/water solution to the incubation mixture (c) Remove excess plastic. removes sminogen binding substances and fish gelatin/aqueous solution; (d) Support coated with plasma sample or control and plasmin generated. incubate for sufficient time, (e) remove excess sample or control, and (f ) to determine the fibrinolytic capacity in whole plasma, including measuring the concentration of plasmin bound to the support. How to determine and also detect. 4(a) A plasminogen-binding substance is attached to a solid support, and the support is a plasminogen-binding substance. incubate for sufficient time to coat with compound; (b) Add fish gelatin/water solution to the incubation mixture (c) Remove excess plastic. removes sminogen binding substances and fish gelatin/aqueous solution; (d) washing the support with a buffer; (e) removing the buffer from the support; (f) Support coated with plasma sample or control and plasmin generated. Incubate for sufficient time, (g) remove excess sample or control, (h ) Adding a plasmin-specific synthetic substrate to the support, (i) measuring the concentration of the generated indicator; A method for determining or detecting fibrinolytic capacity in whole plasma. 5. The amount of poly-d-lysine or poly-d-lysine derivative is about 5 μg/ml. 3. The method of claim 2, wherein the amount is about 100 μg/ml. 6. The amount of poly-d-lysine or poly-d-lysine derivative added to the support is about 1 The method of claim 2 having a molecular weight of 40,000 to about 250,000 Daltons. . 7. The concentration of plasmin bound to the support is determined by the synthetic substrate containing the chromophore leaving group. 3. The method of claim 2, wherein the method is measured using: