CA2100882A1 - Method for measuring fibrinolytic capacity within whole human plasma - Google Patents
Method for measuring fibrinolytic capacity within whole human plasmaInfo
- Publication number
- CA2100882A1 CA2100882A1 CA 2100882 CA2100882A CA2100882A1 CA 2100882 A1 CA2100882 A1 CA 2100882A1 CA 2100882 CA2100882 CA 2100882 CA 2100882 A CA2100882 A CA 2100882A CA 2100882 A1 CA2100882 A1 CA 2100882A1
- Authority
- CA
- Canada
- Prior art keywords
- support
- lysine
- poly
- plasmin
- binding material
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Organic Chemistry (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Wood Science & Technology (AREA)
- Urology & Nephrology (AREA)
- Zoology (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Pathology (AREA)
- Neurosurgery (AREA)
- Biophysics (AREA)
- General Physics & Mathematics (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
This invention relates to a method for measuring the fibrinolytic capacity of whole plasma. This method comprises coating a solid support with a plasminogen binding material such as polylysine, fibrinogen or fibrin, adding a plasma sample or control and then measuring the amount of plasmin bound to the support. This method is particularly advantageous as the immobilized plasminogen binding material provides a surface which mimics the in vivo surface of a fibrin clot.
Description
W093/~1260 PCT/US92/09972 ~it~in ~hole_~um~n Pl a This i~ention relates to a ~ethod for measuring the fi~rinolytic GapaCity of wh~le plas~a. In particular t~is invention utilizes ~n i~Dobilized plasminogen ~inding ~aterial to provide ~ surface which mimics the i~ YiV~ urface of a fi~rin clot and thus ~llows for a better measure of plasma's fibrinolytic capacity.
2a~kqround ~ blo~d clot or thrombus is normally ~ormed in response to signals ~ssociated with a breaX or te~r in the blood vessel wall and t~us prevents excessi~e leakage of blood from the circulation. H~wever, ~xcessive growth of a thrombus can itsel~ fill and occlude the vessel, causing a lack of local ~lood ~low.
Thus, clotting ~ust be a dynzmic process of thrombus ~ormation ~nd breakdown, carefully balanced t~ ~eal thP
vessel wall without completely blocking its lumen.
A blood clot is formed by the process of coagulation, in which an ~bundant and very ~oluble circulating protein, fibrinogen, i5 converted into a ~uch less soluble protein, fibrin, by the action of the ~nzyme thrombin. Fi~rin polymerizes ~nd becomes covalently cros~linked to form the Jaatrix of a blood clot which entraps platel~ts, other cells and bl~od proteins within its meshw3rlc. On~ of these proteins, 30 pl~sminogen, later cAn be converl:ed into an ~cti~e enzyme, plasmin, through the action of other enzyr~es . ~nown as plasminogen activators. Plasmi~ catalyzes the proteolytie cleavage of ~ibrin st several ~iteC le~ding ~o formation of soluble ~i~rin degradation produGts ~nd thus can dissol~e thrombi. It is well ~nown ~hat soluble polylysine or its derivntives can aotivate :
WO 93/1~260 PCr/US92/09972 plasminogen. Allen, R.A.; Thromb. Haemostas 47(1):41-~5(1982); Petersen, L.A. and Swenson, E.; ~iochemica Elio~hvsic:a A~:ta, 883:313-325(1986~. ~
~ he plasma romponents which signal plasmin forma~ion are called the fibrinolytic system. When this system is not ~ufficiently active compared wit~ that of coagulation, a prothrombotic condition results that can lead to thrombo~ic disease including coronary thro~bosis, deep vein thrombosis, peripheral arterial occlusion, pulmonary embolism, occlusive stroke and others. Physirians attempting to correct such imbalance ~ay administer antit~rombotic agents ~eg., heparin or plasminogen activators).
In assessing th~ causes of clottiny, clinicians will routinely wish to measure both ~oagulation parameters (eg., thrombin time, prothrombin time) and fibrinolytic parameters. The latter, or fibrinolytic potential of the plasma is a complex function of the concentrations of plasminogen, plasmin inhibitors, plasminogen activators and plasminogen activator inhibitors. Ultimately it is reflected in the ability of plasma to produce sustained plasmin activity. Its assessment i~ an essential part of planning the treatment of coagulation disorders and thrombotic disease.
A number of tests have been developed to measure ..
t~e fibrinolytic potential in or fibrinolysis of w~ole plasma. For example, Wun and Capuano have described one such a method. J. Biol Chem., 260~8):5061-5066 (1985).
The Wun method detects the ~pontaneous fi~rinolysis of plasma clots by following the lysis of clots formed in an I125-fibrinogen-supplemented citrated plas~a. Using this method Wun determines the amount of lysis over a period of days by centrifuging a patient's plasma sample and plotting the radioactivity present in the supernatant of the centrifugal sample against the - ' , , 3 ~ 2 ~adioactivity S~c~und in the ~ample overall. This ~Dethod h~s ~he disadvant~ge of usi~g l~25-fibrinogerl, whioh is radioacti~e ~nd which als~ has a very hort ~helf life.
Moreover, it requires long incubation periods taking s~everal days to complete.
Another fibrinolytic assay commonly used to measure fibrinolysis is t~e fibrin plate lysis method, Astrup, T. and Darling, S., ~ hyg~ Scand., 4:45(1942). In this assay opaque plasma rlots are artificially ~rea~ed lo on plates or petri dishes. ~atient samples re then applied to the clots and clear lysis zones ~re f ormed and then examined. ~is assay ~as ~everal difficulties Associated with it. For example, clot lysis zones are di~ficult to qu~ntify And the ~ssay itself requires extremely long incubation ti~es -- someti~es ~p to 24 hours. A more rapid version of this test was developed by ~arsh and Gaf~ney. This version, however, requires ~ddition of plasminogen, thus altering the natural composition of the patient sample. Marsh, ~.A. ~nd Gaffney, P.J., Thrombos. Haemostas (Stuttg.), 38:
3545 (~977) -The euglobulin clot lysis time assay is ~lsocommonly used to test fibrinolytic potential. This ~ssay is conducted by formin~ a clot in the euglobulin 25 ~raction of the patient ~ample and then visually determining how long it takes or that clot to be dissolved by certain of the patient's own enzymes.
While this assay has the advantage of bei~g relatively ~ast, usually 60-~20 minutes, it has the di~dv~tage of being a ~u~jective t~st -- where the end point, ie., the clot lysis, is determined through ~isual ~nspec~ion of the reaction ~y the technician. In Addition, in preparing the euglo~ulin fraction, certain i~portant inhibito~s of the clot lysis process normally present in 35 the patient sample are ~emo~ed. Th~s, the ~uglobulin ' ~ ' . .
assay d~es not provide the clinici~n with a complete clinical profile of a patient.
Thus, a fibrinolytic assay is needed that will be fas~, reliable and representative of the in ~i~Q
fibrinolytic processes of a patient.
Therefore it i5 an object of the present invention to create a fas~ and reliable screening assay to ide~tify abnormalities in ~ibrin-dependent fibrinolytic activation. It is a ~urther object of the present invention to develop an assay that will assist clinicians in evaluting and managing patients at risk for post-operative thromboem~olic complications. It is a further object o~ ~he present invention to develop an assay which mimics the in vivo fibrinolytic processes of a patient.
Des~ri~tion of Fi~ure~
Fiqure l. Figure l demonstrates the effe~t of the concentration of poly-d-lysine and molecular weight on absorbance.
Figure 2. 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.
~ iqure 3. Figure 3 shows absorbance data for 2,4,6, trinitrobenzyl polyAd-lysine with plasma pools.
Fi~ure 4. 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 Absorban~e x Reference Plasma Roference Plasma Absorban~e Assigned Value Fiqure 5. Figure 5 shows the %FP of patient plasma samples versus time pre, during and post ~ardiopulmonary bypass surgery.
.. . . . .
-~
. , -~
-. ,,~ ..... :
. ~
W093tl~260 PCT/US92/09972 2~ ~g~2 Fiaure 6. Figure 6 shows t~e ~FP of patient plasma ~a~ples using the ~e~hsd of present i~vention Yersus ti~e pre, during and post livPr transplant ~urgery.
E15Y~ Fi~ure 7 ~hows the ~FP of samples r~m S patients with various diseases deter~ined using the method of the present invention.
Eig9~Q ~. ~igure 8 sh~ws the effect of "~EPSTRACT"
l~ tradename for heparinase, ~n enzyme that degrades heparin or ~eparinized ~amples~ on heparinized plasma lo samples.
~ummarY of ~h~nyent~on The present invention relates to a ~ibranolytic kSSay which utilizes an imm~bilized plasminogen binding material, preferrably poly-d-lysine or a deri~ative thereof, tD mimi~ t~e ~ YiVo fibrin ~lot surface. The plasminogen binding material is applied to a solid-phase matrix or support in such ~ way that the ~upport becomes coated with the plzsminogen binding ma~erial. Plasma, containing all fibrinolytic factors, or contrGl amples, are added to the coated support and incu~ated ~t about room te~perature ~or a period of time ~ufficient to convert a portion of the pl~sminogen in the ~ample ~o plasmin. This incub~tion period can vary fro~ about 2 to about S0 ~inutes but preferrz~ly will be ~etween about 5 and ~bout 30 ~inutes. ~t ~hould be noted that incubation for ~ore than about 30 minutes can actually decrease the ~ignal, probably due to autolysis by plas~in. The ass~y, however, ~2n ~till be perfor~ed after more than 30 ~inutes of ~n~ubation proYided that all ~amples are treated consistently. The amount of ~lasmin ~or~ed dire~tly ~orrelates to the fibrinolytic potenti~l within the ~ample. The plasmin presen~ can be guan~i~ ied ~y any number o~ ~tandard ~ethods, 6uch ~s ~se o~ a plasmin synthetie su~str~te wit~ a fluorometric, chromogenic or radioacti~e la~el or ~y immun~chemical measurement. Husby, R.M. and S~ith, WO93/11260 PCT/~S92/O~g72 R.E., Semi~ars in ~hrombosis and Hemostatis Vol.~I(3):173-314 (1980).
This assay is a significant im~rovement over the currently available fibrinolytic assays as it provides a s ~uch ~aster -- as littl~ as about 5-30 ~inutes -- and more reliable 6ereening assay ~or determining abnormalities in the fibrinolytic processes o~ pa~ients.
Particularly, the assay o~ the present invention is superior to other currsntly available assays because it more accura~ely reflects the physiological conditi~n of the patient. ~irst, it is more accurate because the sample used contains all the ~ibrinslytio ~actors present L~ vivo. Moreover, the addition of exogenous fibrinolytic factors is not r~guired in order to initiate the reaetion as is required with other fibrinolysis tests. Second, it utilizes an i~mobilized plasminogen binding ~aterial. The advantage of an immobilized plasminogen binding material over the commonly used soluble ~aterials such as soluble polylysine is that the immobilized plasminogen binding material provides an activation surface which mimics the surface of a blood clot as it exists ~n vivo thus permitting the fibrinolyti~ factors to assemble on the arti~icial surface much as they do on the surface of a blood clot. Moreover, the relative concentratio~s of the blood factors are increased by providiny this artificial surface thus enabling the reaction to proceed without the addition of any extraneous eyzymes.
Detaile~ Des~r~ption of t~e Invention In the assay of the present invention a solid phase matrix or support made of any material commonly used for immunoassays s~ch 2S latex, glass ~iber or nitrocellulose is ~oated with a plasminogen binding material. In the most proferred embodiment the plasminogen binding material is poly-d-lysine or a derivative of poly-d-lysine such as trinitro~enzoylated .
WO93/11260 PCr/US92/09972 72 ~ 2 poly-d-lysine or other comparable derivative. The ~e~od of the present invention can also utilize p~ly-l-lysine, however, the l-isomer is not as efficient ~s t~e d--isom~r because it is ~ore readily degraded by plasmi~.
S ~ibrin and denatu2ed ~ibrinogen can ~lso be used as the plasminogen binding ~aterial. The ~olecular weight of the polylysine used can vary widely as almost any length of polymer ~bove about 50,000 daltons will suffice, but in the ~ost preferred ~mbodiment the polymer ~hould have ~ molecular weig~t between about l40,00D to about 250,000 ~altons, as ~h~wn in ~igure l.
The plasmin~gen binding materi l oan ~e suspended in-a buffer solution ~ueh as sodium phosphate, sD~ium bornte, ~odium bio~rbonate buffer or any comparable buffer. The concentration ~ plasminogen binding material used to coat the solid s~ppor~ can a'so vary widely, however, in the most preferred embodiment the polylysine concentration will range between about 5 ~g/ml to ~bout ~00 ~g/ml~
Incubation of the plasminogen ~inding ~aterial with t~e solid ~upport is ~ccomplished by passive adsorption at temperatures ranging from about 2C to about 25C for a period of time ~anging from about ~ minutes to about 24 hours. Following incubation excess reagent is removed by washing with a buffer ~olution. In t~e preferred embodiment the solution i5 an isotonic buffer containing ~ ~urfactant at a neutral p~, such ~s imidazole buffered saline. Although hyp~r- or hypotonic buffers, with ~r without surfact~nt and ~t acidic or b3sic pH, are ~l~o suitable.
Appropriate quantities of plasma ~amples or controls are then applied to the deriv~tized ~olid ~upport and the nhtural fi~rinolytic process i5 permitted to proceed. This incubataon with sample can range between about 5 ~nd ~out 60 ~inutes but in the most pre~erred embodiment the incubataon time of sample W093/1l260 PCT/US92/09972 with derivatized ~upport is between about 5 and about 30 minutes and ~ost preferably about 15 minutes. ~xcess ~ample or control i6 removed by washlng with a buffer solut~on. In the preferred embodiment t~e solution is S an isotonic buffer containing a ~urfactant at ~ neutral pH, ~uch as imidazole buffered saline. Although hyper-or hypotonic buffers, with or without surfactant and at acidic or basic pH are also suitable. The plasmin formed during the incubatio~ remains bound to t~e c~ated support and is then quantitated using standard quantitation methods, such as methods that employ a plasmin speeific synthetic or natural substrate chromogenically, fluorometrically or radioactiYely labelled. The plasmin speoific substrate is applied to the activated solid support containing the samples and an indicator is released. This incubation with substrate can range bPtwDen about 30 ~inutes 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. ~useby, R.M. and Smith, R.E., Seminars in Thrombosis and Hemostasis, Vol. VI(3):173-31~ tl980~. Some examples of such substrates which can be used to quantitate the plasmin formed by the method of the present invention are set fort~ in Figure 2. Plasmin may also be quantitated immunochemically using a plasmin/ plasminogen antibody.
Harpel, C.H., J. Clin. Invest. 68:46-55 ~1981~.
Th~ method of the present invention can also be used in kit format. Components of such a kit would include 2 plasminogen binding material coated micro-well titer plates, ~ lyophilized calibrator of normal plasma, a first lyophilized control of nor~al pooled plasma samples, a second lyophilized control of no~mal pooled plasma samples, a substrate buffer, a lyoph~lized substrate, a solution to terminate the substrate-plasmin reaction and a wash buffer. An example of the ',:
.
WO 93/1 1260 PC~/VS9210997~
9 2~ ~0~,~2 cc~mponents whic~ a kit ~ig~t include are as f ollows:
poly-d-lysi~e ~-oated ~ic:rowells, 2x8 ~Dodules, 6 dules/frame, ~6 tests/kit; calibrator (lyophilized), 1~0 (+/-) 5~ of nor~al plasma, 1. 0 ~/vial; 1 ~ial/kit;
a first control (level ~) (lyophilized~ t ~80~c of normal plasma, l~ ial, 1 vial/kit; a ~econd control (level II~ (lyophilized), ~40% of ~ormal pla ma, 1.0 ~L/vial, 1 vi~l/lcit; ~ubstra~e buffer, 12 ~/Yi~l, 1 vial/Xit;
~bs~rate (lyophilized), ~ ~/Yial, 1 vial/kit; ~top iO ~olution or ~olution to terminate ~he plasmin/substrate reaction, 6 mL/vial, 1 vial/kit and wash bu~fer, 20X
~oncentrated solution, 20 mL/vial, 1 vial/Xa~.
~ he assay system of the present invention may also ~e modi~ied using specific an~i~odies to measure lipoprotein(a), tissue plasminogen ~ctivator (tPA), plasminogen ~ctivator I (PAI), alpha-2-antiplasmin/plasmin complex and tPAlPAI complex.
Functional assays to quantify tPA, ~AI or ~lpha-2~
antiplasmin a~tivity are also possible using synthetic substrates ~nd immobilized polylysine.
E~M~ES
The method of the present invention can be further demonstrated ~y the ~ollowing examples~
E ~ I ~ L
Microwell titer pl~tes w~re coated with 10 ~g/ml of poly-d-lysine (molecular.weig~t r~nging ~rom 140,000-250,000 daltonsj in 100 mM ~odium bicarbonate buf~er, pH
9.5. Coating with poly-d-lysine was performed ~y passive adsorption at 4-C for 24 hours. Exces~ p~lymer ~ was remo~ed by w~shing with i~id~zole ~uffered ~aline.
Approximately 100 ul of eaeh plasma ~mple or contr~l was then applied to t~e cDated ~upport. The ~mples were incubated ~or 15 minutes. Excess reagents were removed ~y washing three times with ~bout 200 ul of imidazole buffered saline each ti~. ~he plasmin formed during the incubation w~s guantitated with a plasmin-. ~
~ -WO93/11260 pcT/us92/os972 specific synthetic substrate, H-D-n~rleucyl-hexa-hydrotyr~syl-lysine-p-~itroanilide d~acetat2 ~alt (but any lysine or arginine terminal peptide with any c~lorimetric or fluori~etric tag could be used~, that S contained a ~hromophoric leaving group, p~nitroaniline.
About lO0 ~l of the substrate was added to each well and incubated at room temperature for 60 minutes. The released chromophoEe, p-nitroanilide, was quantified in a spectrophotom~ter capable of ~easuring absorbance at 405 nm. Conveniently, the assay was performed in a 96-well microwell system and a miorowell 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.
EXPMPLE 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 O.lM
sodium bicar~onate buffer, pH 9Ø Each well was filled with llO ul of the poly-d-lysine solution and incubated for 4 hours at ambient or room temperature.
Approximately llO 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 ~Qr purposes of this ~xample, 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 mM 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 27-35C.).
2a~kqround ~ blo~d clot or thrombus is normally ~ormed in response to signals ~ssociated with a breaX or te~r in the blood vessel wall and t~us prevents excessi~e leakage of blood from the circulation. H~wever, ~xcessive growth of a thrombus can itsel~ fill and occlude the vessel, causing a lack of local ~lood ~low.
Thus, clotting ~ust be a dynzmic process of thrombus ~ormation ~nd breakdown, carefully balanced t~ ~eal thP
vessel wall without completely blocking its lumen.
A blood clot is formed by the process of coagulation, in which an ~bundant and very ~oluble circulating protein, fibrinogen, i5 converted into a ~uch less soluble protein, fibrin, by the action of the ~nzyme thrombin. Fi~rin polymerizes ~nd becomes covalently cros~linked to form the Jaatrix of a blood clot which entraps platel~ts, other cells and bl~od proteins within its meshw3rlc. On~ of these proteins, 30 pl~sminogen, later cAn be converl:ed into an ~cti~e enzyme, plasmin, through the action of other enzyr~es . ~nown as plasminogen activators. Plasmi~ catalyzes the proteolytie cleavage of ~ibrin st several ~iteC le~ding ~o formation of soluble ~i~rin degradation produGts ~nd thus can dissol~e thrombi. It is well ~nown ~hat soluble polylysine or its derivntives can aotivate :
WO 93/1~260 PCr/US92/09972 plasminogen. Allen, R.A.; Thromb. Haemostas 47(1):41-~5(1982); Petersen, L.A. and Swenson, E.; ~iochemica Elio~hvsic:a A~:ta, 883:313-325(1986~. ~
~ he plasma romponents which signal plasmin forma~ion are called the fibrinolytic system. When this system is not ~ufficiently active compared wit~ that of coagulation, a prothrombotic condition results that can lead to thrombo~ic disease including coronary thro~bosis, deep vein thrombosis, peripheral arterial occlusion, pulmonary embolism, occlusive stroke and others. Physirians attempting to correct such imbalance ~ay administer antit~rombotic agents ~eg., heparin or plasminogen activators).
In assessing th~ causes of clottiny, clinicians will routinely wish to measure both ~oagulation parameters (eg., thrombin time, prothrombin time) and fibrinolytic parameters. The latter, or fibrinolytic potential of the plasma is a complex function of the concentrations of plasminogen, plasmin inhibitors, plasminogen activators and plasminogen activator inhibitors. Ultimately it is reflected in the ability of plasma to produce sustained plasmin activity. Its assessment i~ an essential part of planning the treatment of coagulation disorders and thrombotic disease.
A number of tests have been developed to measure ..
t~e fibrinolytic potential in or fibrinolysis of w~ole plasma. For example, Wun and Capuano have described one such a method. J. Biol Chem., 260~8):5061-5066 (1985).
The Wun method detects the ~pontaneous fi~rinolysis of plasma clots by following the lysis of clots formed in an I125-fibrinogen-supplemented citrated plas~a. Using this method Wun determines the amount of lysis over a period of days by centrifuging a patient's plasma sample and plotting the radioactivity present in the supernatant of the centrifugal sample against the - ' , , 3 ~ 2 ~adioactivity S~c~und in the ~ample overall. This ~Dethod h~s ~he disadvant~ge of usi~g l~25-fibrinogerl, whioh is radioacti~e ~nd which als~ has a very hort ~helf life.
Moreover, it requires long incubation periods taking s~everal days to complete.
Another fibrinolytic assay commonly used to measure fibrinolysis is t~e fibrin plate lysis method, Astrup, T. and Darling, S., ~ hyg~ Scand., 4:45(1942). In this assay opaque plasma rlots are artificially ~rea~ed lo on plates or petri dishes. ~atient samples re then applied to the clots and clear lysis zones ~re f ormed and then examined. ~is assay ~as ~everal difficulties Associated with it. For example, clot lysis zones are di~ficult to qu~ntify And the ~ssay itself requires extremely long incubation ti~es -- someti~es ~p to 24 hours. A more rapid version of this test was developed by ~arsh and Gaf~ney. This version, however, requires ~ddition of plasminogen, thus altering the natural composition of the patient sample. Marsh, ~.A. ~nd Gaffney, P.J., Thrombos. Haemostas (Stuttg.), 38:
3545 (~977) -The euglobulin clot lysis time assay is ~lsocommonly used to test fibrinolytic potential. This ~ssay is conducted by formin~ a clot in the euglobulin 25 ~raction of the patient ~ample and then visually determining how long it takes or that clot to be dissolved by certain of the patient's own enzymes.
While this assay has the advantage of bei~g relatively ~ast, usually 60-~20 minutes, it has the di~dv~tage of being a ~u~jective t~st -- where the end point, ie., the clot lysis, is determined through ~isual ~nspec~ion of the reaction ~y the technician. In Addition, in preparing the euglo~ulin fraction, certain i~portant inhibito~s of the clot lysis process normally present in 35 the patient sample are ~emo~ed. Th~s, the ~uglobulin ' ~ ' . .
assay d~es not provide the clinici~n with a complete clinical profile of a patient.
Thus, a fibrinolytic assay is needed that will be fas~, reliable and representative of the in ~i~Q
fibrinolytic processes of a patient.
Therefore it i5 an object of the present invention to create a fas~ and reliable screening assay to ide~tify abnormalities in ~ibrin-dependent fibrinolytic activation. It is a ~urther object of the present invention to develop an assay that will assist clinicians in evaluting and managing patients at risk for post-operative thromboem~olic complications. It is a further object o~ ~he present invention to develop an assay which mimics the in vivo fibrinolytic processes of a patient.
Des~ri~tion of Fi~ure~
Fiqure l. Figure l demonstrates the effe~t of the concentration of poly-d-lysine and molecular weight on absorbance.
Figure 2. 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.
~ iqure 3. Figure 3 shows absorbance data for 2,4,6, trinitrobenzyl polyAd-lysine with plasma pools.
Fi~ure 4. 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 Absorban~e x Reference Plasma Roference Plasma Absorban~e Assigned Value Fiqure 5. Figure 5 shows the %FP of patient plasma samples versus time pre, during and post ~ardiopulmonary bypass surgery.
.. . . . .
-~
. , -~
-. ,,~ ..... :
. ~
W093tl~260 PCT/US92/09972 2~ ~g~2 Fiaure 6. Figure 6 shows t~e ~FP of patient plasma ~a~ples using the ~e~hsd of present i~vention Yersus ti~e pre, during and post livPr transplant ~urgery.
E15Y~ Fi~ure 7 ~hows the ~FP of samples r~m S patients with various diseases deter~ined using the method of the present invention.
Eig9~Q ~. ~igure 8 sh~ws the effect of "~EPSTRACT"
l~ tradename for heparinase, ~n enzyme that degrades heparin or ~eparinized ~amples~ on heparinized plasma lo samples.
~ummarY of ~h~nyent~on The present invention relates to a ~ibranolytic kSSay which utilizes an imm~bilized plasminogen binding material, preferrably poly-d-lysine or a deri~ative thereof, tD mimi~ t~e ~ YiVo fibrin ~lot surface. The plasminogen binding material is applied to a solid-phase matrix or support in such ~ way that the ~upport becomes coated with the plzsminogen binding ma~erial. Plasma, containing all fibrinolytic factors, or contrGl amples, are added to the coated support and incu~ated ~t about room te~perature ~or a period of time ~ufficient to convert a portion of the pl~sminogen in the ~ample ~o plasmin. This incub~tion period can vary fro~ about 2 to about S0 ~inutes but preferrz~ly will be ~etween about 5 and ~bout 30 ~inutes. ~t ~hould be noted that incubation for ~ore than about 30 minutes can actually decrease the ~ignal, probably due to autolysis by plas~in. The ass~y, however, ~2n ~till be perfor~ed after more than 30 ~inutes of ~n~ubation proYided that all ~amples are treated consistently. The amount of ~lasmin ~or~ed dire~tly ~orrelates to the fibrinolytic potenti~l within the ~ample. The plasmin presen~ can be guan~i~ ied ~y any number o~ ~tandard ~ethods, 6uch ~s ~se o~ a plasmin synthetie su~str~te wit~ a fluorometric, chromogenic or radioacti~e la~el or ~y immun~chemical measurement. Husby, R.M. and S~ith, WO93/11260 PCT/~S92/O~g72 R.E., Semi~ars in ~hrombosis and Hemostatis Vol.~I(3):173-314 (1980).
This assay is a significant im~rovement over the currently available fibrinolytic assays as it provides a s ~uch ~aster -- as littl~ as about 5-30 ~inutes -- and more reliable 6ereening assay ~or determining abnormalities in the fibrinolytic processes o~ pa~ients.
Particularly, the assay o~ the present invention is superior to other currsntly available assays because it more accura~ely reflects the physiological conditi~n of the patient. ~irst, it is more accurate because the sample used contains all the ~ibrinslytio ~actors present L~ vivo. Moreover, the addition of exogenous fibrinolytic factors is not r~guired in order to initiate the reaetion as is required with other fibrinolysis tests. Second, it utilizes an i~mobilized plasminogen binding ~aterial. The advantage of an immobilized plasminogen binding material over the commonly used soluble ~aterials such as soluble polylysine is that the immobilized plasminogen binding material provides an activation surface which mimics the surface of a blood clot as it exists ~n vivo thus permitting the fibrinolyti~ factors to assemble on the arti~icial surface much as they do on the surface of a blood clot. Moreover, the relative concentratio~s of the blood factors are increased by providiny this artificial surface thus enabling the reaction to proceed without the addition of any extraneous eyzymes.
Detaile~ Des~r~ption of t~e Invention In the assay of the present invention a solid phase matrix or support made of any material commonly used for immunoassays s~ch 2S latex, glass ~iber or nitrocellulose is ~oated with a plasminogen binding material. In the most proferred embodiment the plasminogen binding material is poly-d-lysine or a derivative of poly-d-lysine such as trinitro~enzoylated .
WO93/11260 PCr/US92/09972 72 ~ 2 poly-d-lysine or other comparable derivative. The ~e~od of the present invention can also utilize p~ly-l-lysine, however, the l-isomer is not as efficient ~s t~e d--isom~r because it is ~ore readily degraded by plasmi~.
S ~ibrin and denatu2ed ~ibrinogen can ~lso be used as the plasminogen binding ~aterial. The ~olecular weight of the polylysine used can vary widely as almost any length of polymer ~bove about 50,000 daltons will suffice, but in the ~ost preferred ~mbodiment the polymer ~hould have ~ molecular weig~t between about l40,00D to about 250,000 ~altons, as ~h~wn in ~igure l.
The plasmin~gen binding materi l oan ~e suspended in-a buffer solution ~ueh as sodium phosphate, sD~ium bornte, ~odium bio~rbonate buffer or any comparable buffer. The concentration ~ plasminogen binding material used to coat the solid s~ppor~ can a'so vary widely, however, in the most preferred embodiment the polylysine concentration will range between about 5 ~g/ml to ~bout ~00 ~g/ml~
Incubation of the plasminogen ~inding ~aterial with t~e solid ~upport is ~ccomplished by passive adsorption at temperatures ranging from about 2C to about 25C for a period of time ~anging from about ~ minutes to about 24 hours. Following incubation excess reagent is removed by washing with a buffer ~olution. In t~e preferred embodiment the solution i5 an isotonic buffer containing ~ ~urfactant at a neutral p~, such ~s imidazole buffered saline. Although hyp~r- or hypotonic buffers, with ~r without surfact~nt and ~t acidic or b3sic pH, are ~l~o suitable.
Appropriate quantities of plasma ~amples or controls are then applied to the deriv~tized ~olid ~upport and the nhtural fi~rinolytic process i5 permitted to proceed. This incubataon with sample can range between about 5 ~nd ~out 60 ~inutes but in the most pre~erred embodiment the incubataon time of sample W093/1l260 PCT/US92/09972 with derivatized ~upport is between about 5 and about 30 minutes and ~ost preferably about 15 minutes. ~xcess ~ample or control i6 removed by washlng with a buffer solut~on. In the preferred embodiment t~e solution is S an isotonic buffer containing a ~urfactant at ~ neutral pH, ~uch as imidazole buffered saline. Although hyper-or hypotonic buffers, with or without surfactant and at acidic or basic pH are also suitable. The plasmin formed during the incubatio~ remains bound to t~e c~ated support and is then quantitated using standard quantitation methods, such as methods that employ a plasmin speeific synthetic or natural substrate chromogenically, fluorometrically or radioactiYely labelled. The plasmin speoific substrate is applied to the activated solid support containing the samples and an indicator is released. This incubation with substrate can range bPtwDen about 30 ~inutes 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. ~useby, R.M. and Smith, R.E., Seminars in Thrombosis and Hemostasis, Vol. VI(3):173-31~ tl980~. Some examples of such substrates which can be used to quantitate the plasmin formed by the method of the present invention are set fort~ in Figure 2. Plasmin may also be quantitated immunochemically using a plasmin/ plasminogen antibody.
Harpel, C.H., J. Clin. Invest. 68:46-55 ~1981~.
Th~ method of the present invention can also be used in kit format. Components of such a kit would include 2 plasminogen binding material coated micro-well titer plates, ~ lyophilized calibrator of normal plasma, a first lyophilized control of nor~al pooled plasma samples, a second lyophilized control of no~mal pooled plasma samples, a substrate buffer, a lyoph~lized substrate, a solution to terminate the substrate-plasmin reaction and a wash buffer. An example of the ',:
.
WO 93/1 1260 PC~/VS9210997~
9 2~ ~0~,~2 cc~mponents whic~ a kit ~ig~t include are as f ollows:
poly-d-lysi~e ~-oated ~ic:rowells, 2x8 ~Dodules, 6 dules/frame, ~6 tests/kit; calibrator (lyophilized), 1~0 (+/-) 5~ of nor~al plasma, 1. 0 ~/vial; 1 ~ial/kit;
a first control (level ~) (lyophilized~ t ~80~c of normal plasma, l~ ial, 1 vial/kit; a ~econd control (level II~ (lyophilized), ~40% of ~ormal pla ma, 1.0 ~L/vial, 1 vi~l/lcit; ~ubstra~e buffer, 12 ~/Yi~l, 1 vial/Xit;
~bs~rate (lyophilized), ~ ~/Yial, 1 vial/kit; ~top iO ~olution or ~olution to terminate ~he plasmin/substrate reaction, 6 mL/vial, 1 vial/kit and wash bu~fer, 20X
~oncentrated solution, 20 mL/vial, 1 vial/Xa~.
~ he assay system of the present invention may also ~e modi~ied using specific an~i~odies to measure lipoprotein(a), tissue plasminogen ~ctivator (tPA), plasminogen ~ctivator I (PAI), alpha-2-antiplasmin/plasmin complex and tPAlPAI complex.
Functional assays to quantify tPA, ~AI or ~lpha-2~
antiplasmin a~tivity are also possible using synthetic substrates ~nd immobilized polylysine.
E~M~ES
The method of the present invention can be further demonstrated ~y the ~ollowing examples~
E ~ I ~ L
Microwell titer pl~tes w~re coated with 10 ~g/ml of poly-d-lysine (molecular.weig~t r~nging ~rom 140,000-250,000 daltonsj in 100 mM ~odium bicarbonate buf~er, pH
9.5. Coating with poly-d-lysine was performed ~y passive adsorption at 4-C for 24 hours. Exces~ p~lymer ~ was remo~ed by w~shing with i~id~zole ~uffered ~aline.
Approximately 100 ul of eaeh plasma ~mple or contr~l was then applied to t~e cDated ~upport. The ~mples were incubated ~or 15 minutes. Excess reagents were removed ~y washing three times with ~bout 200 ul of imidazole buffered saline each ti~. ~he plasmin formed during the incubation w~s guantitated with a plasmin-. ~
~ -WO93/11260 pcT/us92/os972 specific synthetic substrate, H-D-n~rleucyl-hexa-hydrotyr~syl-lysine-p-~itroanilide d~acetat2 ~alt (but any lysine or arginine terminal peptide with any c~lorimetric or fluori~etric tag could be used~, that S contained a ~hromophoric leaving group, p~nitroaniline.
About lO0 ~l of the substrate was added to each well and incubated at room temperature for 60 minutes. The released chromophoEe, p-nitroanilide, was quantified in a spectrophotom~ter capable of ~easuring absorbance at 405 nm. Conveniently, the assay was performed in a 96-well microwell system and a miorowell 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.
EXPMPLE 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 O.lM
sodium bicar~onate buffer, pH 9Ø Each well was filled with llO ul of the poly-d-lysine solution and incubated for 4 hours at ambient or room temperature.
Approximately llO 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 ~Qr purposes of this ~xample, 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 mM 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 27-35C.).
3~ Approximately lO0 ul ea~h of plasma sample or control was then applied to the eoated support. The samples !
WO93/11260 PCT/VS92~09972 , . 1~ 2 ~ 2 were incu~ated for about ~5 minutes. ~xcess reagents were removed by washing t~ree times with ab~ut 20D ul of - imidazole buffered saline each time. The plasmin formed during the incubation was quantitated with a pla5min-~pecific ~ynthetic su~strate, H-D-norleueyl-hexahydrotyrosyl-lysine-p-nitr~anilide diace~ate salt tbut any lysine or ~rginine ter~inal peptide With any c~l~rimetric or fluorimetri~ tag could be used), that contained a chromophoric leaving grQup~ p-ni~roanilide.
About 100 ~1 o~ the substrate was added to each well and incubatPd at room temperature for 60 minutes. The released chromophore, p-nitroaniline, was qua~tified in a spectrophotometer capable of measuring absorbance at 405 nm. Convenien~ly the assay was performed in a microwell system and a ~icrowell a~sorbance reader w~s used. The absorbance of samples and controls were compared with th~ absorbance of a normal reference plasma and reported in terms of a perce~t of the reference plasma.
~XAMPL~ III
A 2,4,6-trinitrobenzyl (TNB) poly-d-lysine was prepared by incubating 20 mgs of poly-d-lysine (mean ~olecular Weight of 160,000~ with 13.2 u~ole of 2,4,6 trinitrobenzyl sulphonic acid in 4 mls of distilled water for 10 minutes at ambient or room temp2rature.
Unreacted 2,4,6 trinitrob~nzyl sulphonic acid was removed by dialysis. The alkylation can ~e increased or de~reased to proYide fewer or more ly~lne residues respectively.
~ icr~well titer pl~tes were coated w~b 10 ~glml of poly-d-lysine (mean ~olerul?r weight 143,700) in 0.1 ~odium bic~rbon~te bu~fer, p~ 9.5 or 10 ~ l of TNB-~oly-d-lysine (mean ~olecular weight 160,000) i~ 0.1 M
sodium bicarbonate buffer, pH 9.5. Coating w~s performed by passiYe adsorptio~ at 2-8C ~or 24 ho~rs.
Excess polymer was removed ~y washing wi~h 2a mM Tris wos3~ll26~ PCT/US92/09972 buffer containing 80 ~M sodium chloride and a surfactant (.02% Tween-20~. Approximately lOo ~l o~ ~ normal plasma pool sample was applied t~ the coated ~upport.
The samples were incubated for 15 minutes at room temperature. Excess reagents were removed by washing three times with about 200 ul of phosphate buffered ~aline. The plasmin formed durin~ the incubation was quantitated with a plasmin spe~ific substrate that contains a chromophoric leaving group, H-D-norleucyl-CHA-arginine-nitroanilide diacetate salt (but could be any lysine or arginine terminal peptide with any colorimetric or fluorimetric tag)~ Abou~ lO0 uL of the substrate was added t~ ~ach well and incubated at room temperature. Readings of the released chromophore, p-nitroaniline, were quantified in a spectrophotometer capable of measuring the absorbance at 405 nm. The readings were taken after 15~ 30, 60 and 120 minutes of incubation time. Conveniently the assay is performed in a 96-well microwell system and a microwell absorbance reader is used. The absorbance of samples using the TNB
poly-d-lysine coated wells was compared to the absorbance of the same samples using the poly-d-lysine coated wells. Data is shown in Table I of Figure 3.
EXAMPLE IV
Example III was repeated, except that the plasma pool samples were incubated with the coated wells for 30 mi~utes. Data is shown in ~able II of Figure 3.
EXAMPLE V
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.
When using a chromogenically or ~luorometrically labelled substrate as in the above Examples, the Fibrinolytic Potential (FP) cf the sample or co~trol is measured using the following equation:
. .:
.;
.,. . ... . .
. . . . :: .. . ..
W093f11260 PCT/US92/09972 13 21 ~82 ~FP ~ ~ x Reference Plasma Refer~nce Plasma Absor~ance Assign~d Value ~n this eguation ~he ~Sample Plasma ~bsorbance".is t~e S absorbance value fcr ~he chromophoric or fluor~ge~ic ~ubstance released in ~he rea~tion used to dete~mine plas~in conc~ntration in ~esting ~ plasma sample a~cording t~ the method of ~e present in~ention. ThP
"Referen~e Plasma Absorbance~ is ~he abs~rbance value of the chromop~oric or Sluorogenic ~u~stance released in the reaction used to determine plasmin con~entration in testing a re~erence or con~rol sample zccording to the ~ethod of the present invention. The value assigned to the "Reference ~lasma7~ ran be determined from multiple ~ssays against a 30 donor p~ol o~ fr~sh, normal plasma.
~he mean absorbance value of the fresh donor pool is assumed to represent lO0% fibrinolytic potential. The fibrinolytic potential is generally calcul~ted from results of ~ mini~um of 96 determinations (sixteen determinrtions ~rom each of six randomly sele~ted five-vial pools). Figure 4 ~ows the application of t~is equation.to patient and reference samples.
Figures 5, 6 ~nd 7 ~how the %FP o~ patient plasma samples tested as compared wi~h reference samples or controls using the ~ethod ~et out in Example II. These samples were obtained from patients that had undergone cardiopulmonary bypass and liver transplant surgery and from patients Wit~ various other debilitating diseases.
The determination Of ~FP in patients is particularly importa~t as preliminary research studies ~how that the %FP is reduced (2.7-69.3%) ~n pati~nts with angina, ~yocardi~l infar~ti~n, deep venous thr~mbosis, ~troke, disseminated intravascular coagulation and congestive heart disease. ~he FP ~alue is al50 compromised during surgery (c40$) in patients undergoing liv~r transplantation or cardiovascular bypass; ~lthough FP
: . . ~
WO93/11260 PCT/US~2/U9972 recovered to normal levels within 10 days post-surgery, the rate of recovery varies with each patient. Finally, Figure 8 shows the effeot "Hepstract" ~a tradename for heparinase, an enzyme that degrades heparin or heparinized samples in heparinized plasma sa~ples.
WO93/11260 PCT/VS92~09972 , . 1~ 2 ~ 2 were incu~ated for about ~5 minutes. ~xcess reagents were removed by washing t~ree times with ab~ut 20D ul of - imidazole buffered saline each time. The plasmin formed during the incubation was quantitated with a pla5min-~pecific ~ynthetic su~strate, H-D-norleueyl-hexahydrotyrosyl-lysine-p-nitr~anilide diace~ate salt tbut any lysine or ~rginine ter~inal peptide With any c~l~rimetric or fluorimetri~ tag could be used), that contained a chromophoric leaving grQup~ p-ni~roanilide.
About 100 ~1 o~ the substrate was added to each well and incubatPd at room temperature for 60 minutes. The released chromophore, p-nitroaniline, was qua~tified in a spectrophotometer capable of measuring absorbance at 405 nm. Convenien~ly the assay was performed in a microwell system and a ~icrowell a~sorbance reader w~s used. The absorbance of samples and controls were compared with th~ absorbance of a normal reference plasma and reported in terms of a perce~t of the reference plasma.
~XAMPL~ III
A 2,4,6-trinitrobenzyl (TNB) poly-d-lysine was prepared by incubating 20 mgs of poly-d-lysine (mean ~olecular Weight of 160,000~ with 13.2 u~ole of 2,4,6 trinitrobenzyl sulphonic acid in 4 mls of distilled water for 10 minutes at ambient or room temp2rature.
Unreacted 2,4,6 trinitrob~nzyl sulphonic acid was removed by dialysis. The alkylation can ~e increased or de~reased to proYide fewer or more ly~lne residues respectively.
~ icr~well titer pl~tes were coated w~b 10 ~glml of poly-d-lysine (mean ~olerul?r weight 143,700) in 0.1 ~odium bic~rbon~te bu~fer, p~ 9.5 or 10 ~ l of TNB-~oly-d-lysine (mean ~olecular weight 160,000) i~ 0.1 M
sodium bicarbonate buffer, pH 9.5. Coating w~s performed by passiYe adsorptio~ at 2-8C ~or 24 ho~rs.
Excess polymer was removed ~y washing wi~h 2a mM Tris wos3~ll26~ PCT/US92/09972 buffer containing 80 ~M sodium chloride and a surfactant (.02% Tween-20~. Approximately lOo ~l o~ ~ normal plasma pool sample was applied t~ the coated ~upport.
The samples were incubated for 15 minutes at room temperature. Excess reagents were removed by washing three times with about 200 ul of phosphate buffered ~aline. The plasmin formed durin~ the incubation was quantitated with a plasmin spe~ific substrate that contains a chromophoric leaving group, H-D-norleucyl-CHA-arginine-nitroanilide diacetate salt (but could be any lysine or arginine terminal peptide with any colorimetric or fluorimetric tag)~ Abou~ lO0 uL of the substrate was added t~ ~ach well and incubated at room temperature. Readings of the released chromophore, p-nitroaniline, were quantified in a spectrophotometer capable of measuring the absorbance at 405 nm. The readings were taken after 15~ 30, 60 and 120 minutes of incubation time. Conveniently the assay is performed in a 96-well microwell system and a microwell absorbance reader is used. The absorbance of samples using the TNB
poly-d-lysine coated wells was compared to the absorbance of the same samples using the poly-d-lysine coated wells. Data is shown in Table I of Figure 3.
EXAMPLE IV
Example III was repeated, except that the plasma pool samples were incubated with the coated wells for 30 mi~utes. Data is shown in ~able II of Figure 3.
EXAMPLE V
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.
When using a chromogenically or ~luorometrically labelled substrate as in the above Examples, the Fibrinolytic Potential (FP) cf the sample or co~trol is measured using the following equation:
. .:
.;
.,. . ... . .
. . . . :: .. . ..
W093f11260 PCT/US92/09972 13 21 ~82 ~FP ~ ~ x Reference Plasma Refer~nce Plasma Absor~ance Assign~d Value ~n this eguation ~he ~Sample Plasma ~bsorbance".is t~e S absorbance value fcr ~he chromophoric or fluor~ge~ic ~ubstance released in ~he rea~tion used to dete~mine plas~in conc~ntration in ~esting ~ plasma sample a~cording t~ the method of ~e present in~ention. ThP
"Referen~e Plasma Absorbance~ is ~he abs~rbance value of the chromop~oric or Sluorogenic ~u~stance released in the reaction used to determine plasmin con~entration in testing a re~erence or con~rol sample zccording to the ~ethod of the present invention. The value assigned to the "Reference ~lasma7~ ran be determined from multiple ~ssays against a 30 donor p~ol o~ fr~sh, normal plasma.
~he mean absorbance value of the fresh donor pool is assumed to represent lO0% fibrinolytic potential. The fibrinolytic potential is generally calcul~ted from results of ~ mini~um of 96 determinations (sixteen determinrtions ~rom each of six randomly sele~ted five-vial pools). Figure 4 ~ows the application of t~is equation.to patient and reference samples.
Figures 5, 6 ~nd 7 ~how the %FP o~ patient plasma samples tested as compared wi~h reference samples or controls using the ~ethod ~et out in Example II. These samples were obtained from patients that had undergone cardiopulmonary bypass and liver transplant surgery and from patients Wit~ various other debilitating diseases.
The determination Of ~FP in patients is particularly importa~t as preliminary research studies ~how that the %FP is reduced (2.7-69.3%) ~n pati~nts with angina, ~yocardi~l infar~ti~n, deep venous thr~mbosis, ~troke, disseminated intravascular coagulation and congestive heart disease. ~he FP ~alue is al50 compromised during surgery (c40$) in patients undergoing liv~r transplantation or cardiovascular bypass; ~lthough FP
: . . ~
WO93/11260 PCT/US~2/U9972 recovered to normal levels within 10 days post-surgery, the rate of recovery varies with each patient. Finally, Figure 8 shows the effeot "Hepstract" ~a tradename for heparinase, an enzyme that degrades heparin or heparinized samples in heparinized plasma sa~ples.
Claims (8)
1. A method for determining or detecting the fibrinolytic capacity within whole plasma comprising:
(a) incubating a plasminogen binding material with a solid support for a sufficient period of time that the support becomes coated with the plasminogen binding material;
(b) removing excess plasminogen binding material;
(c) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(d) removing excess sample or control;
(e) measuring the concentration of plasmin bound to the support.
(a) incubating a plasminogen binding material with a solid support for a sufficient period of time that the support becomes coated with the plasminogen binding material;
(b) removing excess plasminogen binding material;
(c) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(d) removing excess sample or control;
(e) measuring the concentration of plasmin bound to the support.
2. A method for determining or detecting the fibrinolytic capacity within whole plasma comprising:
(a) incubating a poly-d-lysine or a derivative of poly-d-lysine with a solid support for a sufficient period of time that the support becomes coated with the poly-d-lysine or a derivative of poly-d-lysine;
(b) removing excess poly-d-lysine or a derivative of poly-d-lysine;
(c) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(d) removing excess sample or control;
(e) measuring the concentration of plasmin bound to the support.
(a) incubating a poly-d-lysine or a derivative of poly-d-lysine with a solid support for a sufficient period of time that the support becomes coated with the poly-d-lysine or a derivative of poly-d-lysine;
(b) removing excess poly-d-lysine or a derivative of poly-d-lysine;
(c) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(d) removing excess sample or control;
(e) measuring the concentration of plasmin bound to the support.
3. A method for determining or detecting the fibrinolytic capacity within whole plasma comprising:
(a) incubating a plasminogen binding material with a solid support for a sufficient period of time that the support becomes coated with the plasminogen binding material;
(b) adding a fish gelatin/water solution to the incubation mixture;
(c) removing the excess plasminogen binding material and the fish gelatin/water solution from the support;
(d) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(e) removing excess sample or control;
(f) measuring the concentration of plasmin bound to the support.
(a) incubating a plasminogen binding material with a solid support for a sufficient period of time that the support becomes coated with the plasminogen binding material;
(b) adding a fish gelatin/water solution to the incubation mixture;
(c) removing the excess plasminogen binding material and the fish gelatin/water solution from the support;
(d) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(e) removing excess sample or control;
(f) measuring the concentration of plasmin bound to the support.
4. A method for determining or detecting the fibrinolytic capacity within whole plasma comprising:
(a) incubating a plasminogen binding material with a solid support for a sufficient period of time that the support becomes coated with the plasminogen binding material;
(b) adding a fish gelatin/water solution to the incubation mixture;
(c) removing the excess plasminogen binding material and fish gelatin/water solution from the support;
(d) washing the support with a buffer solution;
(e) removing the buffer solution from the support;
(f) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(g) removing excess sample or control;
(h) adding a plasmin specific synthetic substrate to the support; and (i) measuring the concentration of indicator formed.
(a) incubating a plasminogen binding material with a solid support for a sufficient period of time that the support becomes coated with the plasminogen binding material;
(b) adding a fish gelatin/water solution to the incubation mixture;
(c) removing the excess plasminogen binding material and fish gelatin/water solution from the support;
(d) washing the support with a buffer solution;
(e) removing the buffer solution from the support;
(f) incubating a plasma sample or control with the coated support for a sufficient period of time for plasmin to be formed;
(g) removing excess sample or control;
(h) adding a plasmin specific synthetic substrate to the support; and (i) measuring the concentration of indicator formed.
5. The method of Claims 1, 3 and 4 wherein the plasminogen binding material is selected from a group consisting of poly-l-lysine, poly-d-lysine, a poly-d-lysine derivative, fibrinogen or fibrin.
6. The method of Claim 2 wherein the amount of poly-d-lysine or a derivative of poly-d-lysine added to said support is about 5 µg/ml to about 100 µg/ml
7. The method of Claim 2 wherein the poly-d-lysine or the poly-d-lysine derivative added to said support has a molecular weight of about 140,000 to about 250,000 daltons.
8. The method of Claims 1 or 2 wherein the concentration of plasmin bound to the support is measured using a synthetic substrate that contains a chromophoric leaving group.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US79756491A | 1991-11-25 | 1991-11-25 | |
| US7/797,564 | 1991-11-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2100882A1 true CA2100882A1 (en) | 1993-05-26 |
Family
ID=25171191
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2100882 Abandoned CA2100882A1 (en) | 1991-11-25 | 1992-11-20 | Method for measuring fibrinolytic capacity within whole human plasma |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0572605A1 (en) |
| JP (1) | JPH06506605A (en) |
| AU (1) | AU655862B2 (en) |
| CA (1) | CA2100882A1 (en) |
| WO (1) | WO1993011260A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5246666A (en) * | 1992-05-08 | 1993-09-21 | Becton, Dickinson And Company | Additive having dual surface chemistry for blood collection container and assembly containing same |
| WO2024091477A1 (en) * | 2022-10-24 | 2024-05-02 | The Johns Hopkins University | Mini-scale euglobulin clot lysis assays and methods of using the same |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE3502878A1 (en) * | 1985-01-29 | 1986-07-31 | Boehringer Mannheim Gmbh, 6800 Mannheim | METHOD FOR DETERMINING THE FIBRINOLYTIC STATE OF PLASMA |
| FI91777C (en) * | 1990-04-02 | 1994-08-10 | Elomit Oy | Method for determining plasmin activity |
-
1992
- 1992-11-20 JP JP5510158A patent/JPH06506605A/en active Pending
- 1992-11-20 EP EP19920925328 patent/EP0572605A1/en not_active Withdrawn
- 1992-11-20 CA CA 2100882 patent/CA2100882A1/en not_active Abandoned
- 1992-11-20 AU AU31424/93A patent/AU655862B2/en not_active Ceased
- 1992-11-20 WO PCT/US1992/009972 patent/WO1993011260A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| EP0572605A4 (en) | 1993-09-17 |
| WO1993011260A1 (en) | 1993-06-10 |
| EP0572605A1 (en) | 1993-12-08 |
| JPH06506605A (en) | 1994-07-28 |
| AU3142493A (en) | 1993-06-28 |
| AU655862B2 (en) | 1995-01-12 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Jespersen et al. | A study of the fibrin plate assay of fibrinolytic agents | |
| Dvilansky et al. | Factor XIII assay by an isotope method | |
| Lorand et al. | Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation | |
| EP0420332B1 (en) | Method for determining the endogenous thrombin potential of plasma and blood, and also a kit for use in said method | |
| Lorand et al. | Transamidating enzymes: II. A continuous fluorescent method suited for automating measurements of factor XIII in plasma | |
| US5721140A (en) | Stable coagulation controls | |
| JPS60230066A (en) | Reagent for measuring prothrombin time | |
| JP2003517610A (en) | Hematological assays and reagents | |
| AU9139798A (en) | Method for determining the anticoagulatory potential of a sample | |
| FI64466B (en) | REQUEST OF REAGENTS FOR THE PROTECTION OF THE PROTOCOL | |
| Hoffmann et al. | Automated nephelometry of fibrinogen: analytical performance and observations during thrombolytic therapy. | |
| WO2003100376A2 (en) | Hematological assay and kit | |
| JPS59187800A (en) | Measurement of activated partial thromboplastin time | |
| EP0216891B1 (en) | A solubilizable, fibrin based composition and its use | |
| CA1079166A (en) | Process for the quantitative determination of antithrombin iii | |
| JPS6356501B2 (en) | ||
| CA2100882A1 (en) | Method for measuring fibrinolytic capacity within whole human plasma | |
| Orthner et al. | A sensitive and facile assay for the measurement of activated protein C activity levels in vivo | |
| Ito et al. | Centrifugal analysis for plasma kallikrein activity, with use of the chromogenic substrate S-2302. | |
| US5939325A (en) | Stable whole blood coagulation controls | |
| EP0637633B1 (en) | Method of determining activity of tested substance | |
| JPH03503486A (en) | Methods of assaying enzymes and kits and materials suitable for the methods | |
| Heimburger et al. | Blood coagulation and fibrinolysis | |
| US5200322A (en) | Method for assaying protein C and measuring kit for the same | |
| US5298401A (en) | Process for the quantitative determination of the function and antigenic concentration of a substance contained in a biological liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| EEER | Examination request | ||
| FZDE | Dead |