Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/56—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/86—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood coagulating time or factors, or their receptors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2337/00—N-linked chromogens for determinations of peptidases and proteinases
  • C12Q2337/10—Anilides
  • C12Q2337/12—Para-Nitroanilides p-NA
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
  • G01N2333/745—Assays involving non-enzymic blood coagulation factors
  • G01N2333/75—Fibrin; Fibrinogen
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
  • G01N2333/90—Enzymes; Proenzymes
  • G01N2333/914—Hydrolases (3)
  • G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
  • G01N2333/968—Plasmin, i.e. fibrinolysin

Definitions

• This invention relates to a method for measuring the fibrinolytic capacity of whole plasma.
• this invention utilizes an immobilized plasminogen binding material to provide a surface which mimics the in vivo surface of a fibrin clot and thus allows for a better measure of plasma's fibrinolytic capacity.
• a blood clot or thrombus is normally formed in response to signals associated with a break or tear in the blood vessel wall and thus prevents excessive leakage of blood from the circulation.
• excessive growth of a thrombus can itself fill and occlude the vessel, causing a lack of local blood flow.
• clotting must be a dynamic process of thrombus formation and breakdown, carefully balanced to seal the vessel wall without completely blocking its lumen.
• a blood clot is formed by the process of coagulation, in which an abundant and very soluble circulating protein, fibrinogen, is converted into a much less soluble protein, fibrin, by the action of the enzyme thrombin. Fibrin polymerizes and becomes covalently crosslinked to form the matrix of a blood clot which entraps platelets, other cells and blood proteins within its meshwork. One of these proteins, plasminogen, later can be converted into an active enzyme, plasmin, through the action of other enzymes known as plasminogen activators. Plasmin catalyzes the proteolytic cleavage of fibrin at several sites leading to formation of soluble fibrin degradation products and thus can dissolve thrombi.
• the plasma components which signal plasmin formation are called the fibrinolytic system.
• fibrinolytic system When this system is not sufficiently active compared with that of coagulation, a prothrombotic condition results that can lead to thrombotic disease including coronary thrombosis, deep vein thrombosis, peripheral arterial occlusion, pulmonary embolism, occlusive stroke and others. Physicians attempting to correct such imbalance may administer antithrombotic agents (eg. , heparin or plasminogen activators) .
• antithrombotic agents eg. , heparin or plasminogen activators
• coagulation parameters eg., thrombin time, prothrombin time
• fibrinolytic parameters eg., fibrinolytic potential of the plasma.
• fibrinolytic potential of the plasma is a complex function of the concentrations of plasminogen, plasmin inhibitors, plasminogen activators and plasminogen activator inhibitors.
• plasminogen activators e.g., plasminogen activators
• plasminogen activator inhibitors e.g., plasminogen activators
• fibrin plate lysis method A ⁇ trup, T. and Darling, S., Acta Physiol. Scand.. 4:45(1942).
• opaque plasma clots are artificially created on plates or petri dishes. Patient samples are then applied to the clots and clear lysis zones are formed and then examined.
• This assay has several difficulties associated with it. For example, clot lysis zones are difficult to quantify and the assay itself requires extremely long incubation times — sometimes up to 24 hours.
• a more rapid version of this test was developed by Marsh and Gaffney. This version, however, requires addition of plasminogen, thus altering the natural composition of the patient sample. Marsh, N.A. and Gaffney, P.J. , Thrombos. Haemostas. (Stuttg.), 38: 3545(1977) .
• the euglobulin clot lysis time assay is also commonly used to test fibrinolytic potential. This assay is conducted by forming a clot in the euglobulin fraction of the patient sample and then visually determining how long it takes for that clot to be dissolved by certain of the patient's own enzymes. While this assay has the advantage of being relatively fast, usually 60-120 minutes, it has the disadvantage of being a subjective test — where the end point, ie.. the clot lysis, is determined through visual inspection of the reaction by the technician. In addition, in preparing the euglobulin fraction, certain important inhibitors of the clot lysis process normally present in the patient sample are removed. Thus, the euglobulin assay does not provide the clinician with a complete clinical profile of a patient.
• a fibrinolytic assay is needed that will be fast, reliable and representative of the jin vivo fibrinolytic processes of a patient.
• Figure 1 Figure l demonstrates the effect of the concentration of poly-d-lysine and molecular weight on absorbance.
• Figure 2 lists examples of various chromogenic substrates that can be used to measure or quantitate the plasmin formed using the method of the present invention.
• Figure 3 shows absorbance data for 2,4,6, trinitrobenzyl poly-d-lysine with plasma pools.
• Figure 4 shows the equation used to calculate the percent of Fibrinolytic Potential (%FP) of plasma samples tested using one of the methods of the present invention. This %FP equation is:
• %FP Sample Plasma Absorbance x Reference Plasma Reference Plasma Absorbance Assigned Value
• Figure 5 shows the %FP of patient plasma samples versus time pre, during and post cardiopulmonary bypass surgery.
• Figure 6 shows the %FP of patient plasma samples using the method of present invention versus time pre, during and post liver transplant surgery.
• Figure 7 shows the %FP of samples from patients with various diseases determined using the method of the present invention.
• Figure 8 shows the effect of "HEPSTRACT” (a tradename for heparinase, an enzyme that degrades heparin or heparinized samples) on heparinized plasma samples.
• HEPSTRACT a tradename for heparinase, an enzyme that degrades heparin or heparinized samples
• the present invention relates to a fibrinolytic assay which utilizes an immobilized plasminogen binding material, preferrably poly-d-lysine or a derivative thereof, to mimic the in vivo fibrin clot surface.
• the plasminogen binding material is applied to a solid-phase matrix or support in such a way that the support becomes coated with the plasminogen binding material.
• Plasma, containing all fibrinolytic factors, or control samples, are added to the coated support and incubated at about room temperature for a period of time sufficient to convert a portion of the plasminogen in the sample to plasmin. This incubation period can vary from about 2 to about 60 minutes but preferrably will be between about 5 and about 30 minutes.
• incubation for more than about 30 minutes can actually decrease the signal, probably due to autolysis by plasmin.
• the assay can still be performed after more than 30 minutes of incubation provided that all samples are treated consistently.
• the amount of plasmin formed directly correlates to the fibrinolytic potential within the sample.
• the plasmin present can be quantified by any number of standard methods, such as use of a plasmin synthetic substrate with a fluorometric, chromogenic or radioactive label or by immunochemical measurement. Husby, R.M. and Smith, R.E., Seminars in Thrombosis and He ostatis V ⁇ l.VI(3):173-314 (1980).
• This assay is a significant improvement over the currently available fibrinolytic assays as it provides a much faster — as little as about 5-30 minutes — and more reliable screening assay for determining abnormalities in the fibrinolytic processes of patients.
• the assay of the present invention is superior to other currently available assays because it more accurately reflects the physiological condition of the patient.
• the sample used contains all the fibrinolytic factors present jLn vivo.
• the addition of exogenous fibrinolytic factors is not required in order to initiate the reaction as is required with other fibrinolysis tests.
• Second, it utilizes an immobilized plasminogen binding material.
• immobilized plasminogen binding material provides an activation surface which mimics the surface of a blood clot as it exists m . vivo thus permitting the fibrinolytic factors to assemble on the artificial surface much as they do on the surface of a blood clot. Moreover, the relative concentrations of the blood factors are increased by providing this artificial surface thus enabling the reaction to proceed without the addition of any extraneous eyzymes.
• a solid phase matrix or support made of any material commonly used for immunoassays such as latex, glass fiber or nitrocellulose is coated with a plasminogen binding material.
• the plasminogen binding material is poly-d-lysine or a derivative of poly-d-lysine such as trinitrobenzoylated poly-d-lysine or other comparable derivative.
• the method of the present invention can also utilize poly-1- lysine, however, the 1-isomer is not as efficient as the d-isomer because it is more readily degraded by plasmin. Fibrin and denatured fibrinogen can also be used as the plasminogen binding material.
• the molecular weight of the polylysine used can vary widely as almost any length of polymer above about 50,000 daltons will suffice, but in the most preferred embodiment the polymer should have a molecular weight between about 140,000 to about 250,000 daltons, as shown in Figure 1.
• the plasminogen binding material can be suspended in a buffer solution such as sodium phosphate, sodium borate, sodium bicarbonate buffer or any comparable buffer.
• concentration of plasminogen binding material used to coat the solid support can also vary widely, however, in the most preferred embodiment the polylysine concentration will range between about 5 ⁇ g/ l to about 100 ⁇ g/ml.
• Incubation of the plasminogen binding material with the solid support is accomplished by passive adsorption at temperatures ranging from about 2°C to about 25°C for a period of time ranging from about 5 minutes to about 24 hours. Following incubation excess reagent is removed by washing with a buffer solution.
• the solution is an isotonic buffer containing a surfactant at a neutral pH, such as imidazole buffered saline.
• a surfactant at a neutral pH such as imidazole buffered saline.
• hyper- or hypotonic buffers with or without surfactant and at acidic or basic pH, are also suitable.
• Appropriate quantities of plasma samples or controls are then applied to the derivatized solid support and the natural fibrinolytic process is permitted to proceed.
• This incubation with sample can range between about 5 and about 60 minutes but in the most preferred embodiment the incubation time of sample with derivatized support is between about 5 and about 30 minutes and most preferably about 15 minutes.
• Excess sample or control is removed by washing with a buffer solution.
• the solution is an isotonic buffer containing a surfactant at a neutral pH, such as imidazole buffered saline.
• hyper- or hypotonic buffers with or without surfactant and at acidic or basic pH are also suitable.
• the plasmin formed during the incubation remains bound to the coated support and is then quantitated using standard quantitation methods, such as methods that employ a plasmin specific synthetic or natural substrate chro ogenically, fluorometrically or radioactively labelled.
• the plasmin specific substrate is applied to the activated solid support containing the samples and an indicator is released.
• This incubation with substrate can range between about 30 minutes to up to about 24 hours but in the preferred embodiment the incubation time is between about 30 minutes to 2 hours and most preferably about one hour. Huseby, R.M. and Smith, R.E., Seminars in Thrombosis and Hemostasis. Vol. VI(3) :173-314 (1980).
• Plasmin may also be quantitated immunochemically using a plasmin/ plasminogen antibody. Harpel, C.H. , i. Clin. Invest. 68:46-55 (1981).
• the method of the present invention can also be used in kit format.
• Components of such a kit would include a plasminogen binding material coated micro-well titer plates, a lyophilized calibrator of normal plasma, a first lyophilized control of normal pooled plasma samples, a second lyophilized control of normal pooled plasma samples, a substrate buffer, a lyophilized substrate, a solution to terminate the substrate-plasmin reaction and a wash buffer.
• kits An example of the components which a kit might include are as follows: poly-d-lysine coated microwells, 2x8 modules, 6 modules/frame, 96 tests/kit; calibrator (lyophilized) , 100 (+/-) 5% of normal plasma, 1.0 mL/vial; 1 vial/kit; a first control (level I) (lyophilized) , >80% of normal plasma, 1.0 mL/vial, 1 vial/kit; a second control (level II) (lyophilized), ⁇ 40% of normal plasma, 1.0 mL/vial, l vial/kit; substrate buffer, 12 mL/vial, 1 vial/kit; substrate (lyophilized) , 1 mL/vial, 1 vial/kit; stop solution or solution to terminate the plasmin/substrate reaction, 6 mL/vial, 1 vial/kit and wash buffer, 20X concentrated solution, 20 mL/vial, 1 vial/kit.
• the assay system of the present invention may also be modified using specific antibodies to measure lipoprotein(a) , tissue plasminogen activator (tPA) , plasminogen activator I (PAI) , alpha-2- antiplasmin/plasmin complex and tPA/PAI complex.
• tPA tissue plasminogen activator
• PAI plasminogen activator I
• alpha-2- antiplasmin/plasmin complex and tPA/PAI complex.
• Functional assays to quantify tPA, PAI or alpha-2- antiplasmin activity are also possible using synthetic substrates and immobilized polylysine.
• EXAMPLE I Microwell titer plates were coated with 10 ⁇ g/ml of poly-d-lysine (molecular weight ranging from 140,000- 250,000 daltons) in 100 mM sodium bicarbonate buffer, pH 9.5. Coating with poly-d-lysine was performed by passive adsorption at 4°C for 24 hours. Excess polymer was removed by washing with imidazole buffered saline. Approximately 100 ul of each plasma sample or control was then applied to the coated support. The samples were incubated for 15 minutes. Excess reagents were removed by washing three times with about 200 ul of imidazole buffered saline each time.
• the plasmin formed during the incubation was quantitated with a plasmin-specific synthetic substrate, H-D-norleucyl-hexa- hydrotyrosyl-lysine-p-nitroanilide diacetate salt (but any lysine or arginine terminal peptide with any colorimetric or fluorimetric tag could be used) , that ' contained a chromophoric leaving group, p-nitroaniline.
• About 100 ⁇ l of the substrate was added to each well and incubated at room temperature for 60 minutes.
• the released chromophore, p-nitroanilide was quantified in a ⁇ pectrophotometer capable of measuring absorbance at 405 nm.
• the assay was performed in a 96- well microwell system and a microwell absorbance reader was used. The absorbance of samples and controls were compared with the absorbance of a normal reference plasma and reported in terms of a percent of the reference plasma. EXAMPLE II.
• A/S Nunc Maxi Sorp" microwell titer plates 2x8 modules or equivalent, were coated with 20 ⁇ g/ml of poly-d-lysine (mean Molecular Weight of 248,400) in 0.1M sodium bicarbonate buffer, pH 9.0. Each well was filled with 110 ul of the poly-d-lysine solution and incubated for 4 hours at ambient or room temperature. Approximately 110 ul of a 6% w/v fish gelatin/water solution was then added to the wells and incubated at ambient or room temperature for about 30 minutes. A 6% w/v solution was used for purposes of this Example, however, other variations may be used.
• the contents of the well were then aspirated and 250 ul/well of a solution containing 20 M Tris, 80 M Sodium Chloride and 0.02% "Tween 20" pH 7.4, was added to each well as a wash.
• the wash solution was then aspirated and the wash procedure repeated twice.
• the wells were then air dried in a low humidity chamber for approximately 20 hours (humidity about 8-20%, temperature about 27-35°C). Approximately 100 ul each of plasma sample or control was then applied to the coated support. The samples were incubated for about 15 minutes. Excess reagents were removed by washing three times with about 200 ul of imidazole buffered saline each time.
• the plasmin formed during the incubation was guantitated with a pla ⁇ min- specific synthetic substrate, H-D-norleucyl- hexahydrotyrosyl-lysine-p-nitroanilide diacetate salt (but any lysine or arginine terminal peptide with any colorimetric or fluorimetric tag could be used) , that contained a chromophoric leaving group, p-nitroanilide.
• About 100 ⁇ l of the substrate was added to each well and incubated at room temperature for 60 minutes.
• the released chromophore, p-nitroaniline was quantified in a spectrophotometer capable of measuring absorbance at 405 nm.
• the assay was performed in a microwell system and a microwell absorbance reader was used.
• the absorbance of samples and controls were compared with the absorbance of a normal reference plasma and reported in terms of a percent of the reference plasma.
• a 2,4,6-trinitrobenzyl (TNB) poly-d-lysine was prepared by incubating 20 mgs of poly-d-lysine (mean Molecular Weight of 160,000) with 13.2 umole of 2,4,6 trinitrobenzyl sulphonic acid in 4 mis of distilled water for 10 minutes at ambient or room temperature. Unreacted 2,4,6 trinitrobenzyl sulphonic acid was removed by dialysis. The alkylation can be increased or decreased to provide fewer or more lysine residues respectively.
• Microwell titer plates were coated with 10 ⁇ g/ml of poly-d-lysine (mean molecular weight 143,700) in 0.1 M sodium bicarbonate buffer, pH 9.5 or 10 ⁇ g/ml of TNB- poly-d-lysine (mean molecular weight 160,000) in 0.1 M sodium bicarbonate buffer, pH 9.5. Coating was performed by passive adsorption at 2-8°C for 24 hours. Excess polymer was removed by washing with 20 mM Tris buffer containing 80 M sodium chloride and a surfactant (.02% Tween-20) . Approximately 100 ul of a normal plasma pool sample was applied to the coated support. The samples were incubated for 15 minutes at room temperature.
• Example III was repeated, except that the plasma pool samples were incubated with the coated wells for 60 minutes. Data is shown in Table III of Figure 3.
• FP Fibrinolytic Potential
• the "Sample Plasma Absorbance” is the absorbance value for the chromophoric or fluorogenic substance released in the reaction used to determine plasmin concentration in testing a plasma sample according to the method of the present invention.
• the “Reference Plasma Absorbance” is the absorbance value of the chromophoric or fluorogenic substance released in the reaction used to determine plasmin concentration in testing a reference or control sample according to the method of the present invention.
• the value assigned to the "Reference Plasma” can be determined from multiple assays against a 30 donor pool of fresh, normal plasma. The mean absorbance value of the fresh donor pool is assumed to represent 100% fibrinolytic potential.
• the fibrinolytic potential is generally calculated from results of a minimum of 96 determinations (sixteen determinations from each of six randomly selected five- vial pools) .
• Figure 4 shows the application of this equation to patient and reference samples.
• Figures 5, 6 and 7 show the %FP of patient plasma samples tested as compared with reference samples or controls using the method set out in Example II. These samples were obtained from patients that had undergone cardiopulmonary bypass and liver transplant surgery and from patients with various other debilitating diseases. The determination of %FP in patients is particularly important as preliminary research studies show that the %FP is reduced (2.7-69.3%) in patients with angina, myocardial infarction, deep venous thrombosis, stroke, disseminated intravascular coagulation and congestive heart disease. The FP value is also compromised during surgery ( ⁇ 40%) in patients undergoing liver transplantation or cardiovascular bypass; although FP recovered to normal levels within 10 days post-surgery, the rate of recovery varies with each patient.
• Figure 8 shows the effect "Hepstract" (a tradename for heparinase, an enzyme that degrades heparin or heparinized samples in heparinized plasma samples.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Hematology (AREA)
• Organic Chemistry (AREA)
• Molecular Biology (AREA)
• Immunology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• General Health & Medical Sciences (AREA)
• Biotechnology (AREA)
• Analytical Chemistry (AREA)
• Microbiology (AREA)
• Physics & Mathematics (AREA)
• Urology & Nephrology (AREA)
• Wood Science & Technology (AREA)
• Zoology (AREA)
• Biochemistry (AREA)
• Biomedical Technology (AREA)
• Biophysics (AREA)
• General Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Cell Biology (AREA)
• Neurosurgery (AREA)
• Food Science & Technology (AREA)
• Medicinal Chemistry (AREA)
• General Physics & Mathematics (AREA)
• Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Investigating Or Analysing Biological Materials (AREA)

Applications Claiming Priority (2)

Publications (2)

Family

ID=25171191

Family Applications (1)

Country Status (5)

Families Citing this family (2)

Family Cites Families (2)

• 1992
  • 1992-11-20 WO PCT/US1992/009972 patent/WO1993011260A1/en not_active Application Discontinuation
  • 1992-11-20 EP EP19920925328 patent/EP0572605A1/de not_active Withdrawn
  • 1992-11-20 JP JP5510158A patent/JPH06506605A/ja active Pending
  • 1992-11-20 CA CA 2100882 patent/CA2100882A1/en not_active Abandoned
  • 1992-11-20 AU AU31424/93A patent/AU655862B2/en not_active Ceased

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events