US20030124622A1 - Procedure for the determination of the activity of the protease which activates factor VII from protein solutions - Google Patents

Procedure for the determination of the activity of the protease which activates factor VII from protein solutions Download PDF

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US20030124622A1
US20030124622A1 US10/287,047 US28704702A US2003124622A1 US 20030124622 A1 US20030124622 A1 US 20030124622A1 US 28704702 A US28704702 A US 28704702A US 2003124622 A1 US2003124622 A1 US 2003124622A1
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protease
activity
proenzyme
level
solid phase
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Juergen Roemisch
Annette Feussner
Hans-Arnold Stoehr
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CSL BEHRING GmbH
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Assigned to CSL BEHRING GMBH reassignment CSL BEHRING GMBH CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: ZLB BEHRING GMBH
Priority to US12/155,619 priority patent/US7892842B2/en
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    • 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
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • 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)
    • G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
    • G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
    • G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
    • G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
    • G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
    • G01N2333/96433—Serine endopeptidases (3.4.21)
    • G01N2333/96441—Serine endopeptidases (3.4.21) with definite EC number
    • G01N2333/96447—Factor VII (3.4.21.21)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/324—Coronary artery diseases, e.g. angina pectoris, myocardial infarction
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/368—Pregnancy complicated by disease or abnormalities of pregnancy, e.g. preeclampsia, preterm labour
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/966—Chemistry: molecular biology and microbiology involving an enzyme system with high turnover rate or complement magnified assay, e.g. multi-enzyme systems
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S436/00—Chemistry: analytical and immunological testing
    • Y10S436/811—Test for named disease, body condition or organ function

Definitions

  • the invention relates to a procedure for the qualitative and quantitative determination of the protease which activates factor VII in complex protein solutions such as plasma.
  • the blood clotting system comprises two different cascade-like pathways for activating clotting factors which are present in the plasma.
  • the intrinsic or the extrinsic pathway is used for initiating clotting, depending on the triggering mechanism.
  • thromboplastin tissue factor, TF with phospholipids
  • the membrane located thromboplastin can bind both clotting factor VII (FVII) and circulating activated FVII (FVIIa).
  • FVII clotting factor VII
  • FVIIa circulating activated FVII
  • FXa activated form
  • FVIIa or FVIIa-containing concentrates is used in certain clinical situations.
  • FVIII-bypassing activity of FVIIa is used in patients who are suffering, for example, from hemophilia A and have developed antibodies against FVIII as a consequence of the administration of FVIII.
  • FVIIa is well tolerated in this context and, while it does not lead to any tendency to thrombosis, it is suitable for ensuring that clotting takes place to a limited but adequate extent.
  • Recombinant FVIIa is already being used therapeutically and prophylactically.
  • FVII which has been isolated from blood plasma can also be activated and then used.
  • Proteases such as thrombin can be used for this activation, however, these proteases strongly activate clotting and lead to the risk of a thrombosis. For this reason, subsequent removal or inactivation of thrombin is necessary and leads to yield losses. As a result of the risk of thrombosis which is associated with it, the use of FXa or FIIa (thrombin) is frequently contraindicated and only indicated in emergencies, e.g., in association with extreme loss of blood and unstoppable hemorrhages.
  • FVIIa is found in very low concentrations in the plasma of healthy subjects. Very little is known about the formation and origin of FVIIa which is circulating in the blood. Traces of thromboplastin which has been expressed or released in association with cell destruction might play a role in this context. Although it is known that factor XIIa, for example, can lead to FVII activation under certain conditions, the physiological relevance of this reaction has not yet been clarified.
  • novel protease for activating the blood clotting factor VII is:
  • protease the activated form of the protease
  • protezyme the non-activated form
  • the solution is adjusted to a pH of between 4 and 7.2, preferably to a pH of between 5.0 and 7.0.
  • stabilizers apart from citrate, such as glutamate, amino acids, such as arginine, glycine or lysine, calcium ions and sugars such as glucose, arabinose or mannose in quantities of 1-200 mmol/l, preferably in quantities of 5-100 mmol/l.
  • Efficient stabilization was also achieved by adding glycols such as ethylene glycol or glycerol, with quantities of 5-80% by weight, preferably of 10-60% by weight, being used.
  • the pH of the stabilized solution should then be between 4 and 9.
  • novel protease and also the proenzyme
  • PPSB prothrombin complex
  • the starting material is first subjected to anion exchange chromatography, which is followed by an affinity chromatography of the elute.
  • Heparin which is immobilized on a matrix, or a heparin-related substance such as heparan sulfate or dextran sulfate, is particularly suitable for the affinity chromatography.
  • novel protease and/or the proenzyme can be selectively bound and then eluted once again using known methods.
  • the use of a spacer is advisable for coupling the ligand to the matrix.
  • a heparin-lysine matrix has been found to be particularly suitable for isolating the novel protease.
  • the protease which has been isolated by this method exhibits, in the non-reduced state, one to several bands which lie closely together in the molecular weight range of 55-75 kDa. Following reduction, one to several bands were observed in the molecular weight range of 15-35 kDa and one band was observed at 40-55 kDa. A further band between 60 and 65 kDa, which, after scanning and quantitative evaluation, constituted 5-10% of the total protein, showed that non-activated proenzyme was also present. This result was supported by appropriate investigations using monoclonal antibodies against this protease.
  • proenzyme of this protease can also be prepared, pasteurized and used by the method according to the invention.
  • the proportion of the proenzyme to the weight of the total protein is indicated by the band between 60 and 65 kDa.
  • amino acid sequence which constitutes the activation region of the proenzyme, thrombin, kallikrein or FXIa are, in accordance with their substrate specificities, examples of suitable physiological activators of the proenzyme.
  • the preparation was essentially achieved using an aprotinin matrix.
  • the activity was described as being a thrombin-like activity. Hunfeld et al. did not find any influence on global clotting parameters such as prothrombin time, Quick or platelet aggregation.
  • novel protease can be used diagnostically and therapeutically, it is desirable to qualitatively and quantitatively detect the protease is complex protein solutions such as plasma.
  • German Patent Application 199 03 693, and its corresponding Canadian Patent Application 2,269,109 already discloses test systems and procedures for the qualitative and quantitative detection of the protease which activates blood clotting factor VII. These include chromogenic test procedures, which are based on the cleavage of labeled, low molecular weight peptide substrates and the photometric determination of the extinction occurring in this case, and test procedures in which the biological properties of the protease mentioned are utilized. In these procedures, the protease or its proenzyme can be detected in that it has
  • the determination of the activity of the protease only leads to reliable results if the protease is present in a purified or enriched state and no interfering effects of impurities distort the measurement result.
  • Very complex protein mixtures such as plasma or tissue fluids contain a large number of proteins which can prevent or at least hinder a specific qualitative and quantitative determination of the protease.
  • the protease is present in the plasma especially as a proenzyme, such that activation to give the active protease is necessary for the purpose of the subsequent activity determination.
  • the object of the present invention is to develop a procedure which makes possible the qualitative and quantitative determination, in a manner which is as simple and specific as possible, of one or more biological activities of the factor VII-activating protease.
  • the protein solution comprising the protease and/or its proenzyme is incubated with a solid phase to which an antibody directed against the protease and/or its proenzyme has been coupled beforehand, and
  • the protease and/or its proenzyme fixed thereto are incubated with reagents which allow for the determination of their activity.
  • this determination procedure can be used not only on the protease in its activated form but also with its non-activated proenzyme.
  • the protease circulates in plasma mainly as a proenzyme and, therefore, in order then to be able to display its biological activities, the proenzyme must be activated to the protease after being bound to the solid phase, it has now surprisingly been found that such an activation is not necessary.
  • the proenzyme bound to the solid phase displays its biological activity in immobilized form in the same manner as the protease. A separate activation step is therefore unnecessary, allowing for more rapid and interference-free determination.
  • Activation of the proenzyme can be done in order to ensure that the proenzyme bound to the solid phase has been completely activated.
  • Suitable solid phases are matrices known to the person skilled in the art, such as activated Sepharose® or Fraktogel®.
  • Microtiter plates are preferably coated with antibodies directed against the protease or its proenzyme, which can be of polyclonal or monoclonal origin.
  • Antibody fragments such as F(ab) or F(ab) 2 can also be used.
  • the present invention can utilize chromogenic substrates which allow for the determination of the activity of the protease.
  • a particularly preferred chromogenic substrate is S2288 from Chromogenix AB (H-D isoleucyl-L-prolyl-L-arginine-pNA ⁇ 2 HCl), which, like similar compounds, shows significant concentration and time dependent increase in the absorption due to amidolysis of the substrate.
  • the protease retains its biological activities and properties even after binding to the antibody, namely the capability to activate FVII and plasminogen activators. The specific determination of the functionality of the protease from a complex protein solution is thereby possible.
  • the other substrates mentioned in Canadian Patent Application 2,269,109 such as S2765 (N-a-Cbo-D-Arg-Gly-Arg-pNA), also offer themselves for the activity determination, i.e., the inactivation of the blood clotting factors VIII/VIIIa or V/Va and also the activation of the FVII and the plasminogen activators.
  • the proportion of activated factor VII can be determined by direct amidolysis of a chromogenic substrate which is specific for the FVII or by a coupled reaction such as the so-called FVIIa-rTF test.
  • single-chain plasminogen activators scuPA, single chain urokinase plasminogen activator or sctPA, single chain tissue plasminogen activator
  • substrate reaction for example, S2444 (pyroGlu-Gly-Arg-pNA ⁇ HCl).
  • substances can also be added for detection which stimulate the activity of the protease, for example, soluble calcium salts and/or heparin or substances related to heparin such as dextran sulfate.
  • the process is moreover suitable for the determination of the activity of the FVII activating protease in extracts of tissues or cells. Such a determination gives an indication about the presence of the protease activity or about potential pathological conditions in the case of over or underexpression of this protein.
  • a particular interest applies to the detection of the protease activities in body fluids, such as blood and plasma, seminal plasma, urine, cerebrospinal fluid, bronchioalveolar lavage, amniotic fluid, saliva or lacrimal fluid.
  • body fluids such as blood and plasma, seminal plasma, urine, cerebrospinal fluid, bronchioalveolar lavage, amniotic fluid, saliva or lacrimal fluid.
  • a valuable supplement to present invention is the antigen determination system (e.g. ELISA) mentioned in Canadian Patent Application 2,269,109. With the aid of both determination systems, a more comprehensive picture can be obtained, for example, in the case of a disorder.
  • FIG. 1 shows protease activities (pPEU/ml) of healthy men (A) and women (B).
  • FIG. 2 shows protease activities (pPEU/ml) from patients with acute myocardial infarction (AMI) (A) and from healthy donors (B).
  • this parameter can lead to early detection and can be used as a criterion of a change in the syndrome. This includes the diagnosis of further cardiovascular-associated complications.
  • the determination system described can also be used for diagnosis and therapeutic monitoring in the case of malignant diseases, inflammation, autoimmune diseases, vasculitis, respiratory defects or for the diagnosis of hemostasis (clotting and fibrinolysis), and also in the case of sepsis and associated reactions, such as disseminated intravasal clotting.
  • Further application areas include the diagnosis of organ defects, such as cerebral, respiratory and kidney diseases. In patients with cirrhosis of the liver, we found significantly decreased activities of the protease, which in most cases were accompanied by decreased antigen levels.
  • Microtiter plates (96 wells) were coated with a monoclonal antibody against the protease by pipetting 150 ⁇ l of a solution comprising 10 ⁇ g/ml of the monoclonal antibody into each hollow. After incubation at room temperature for 16 hours, the plates were washed several times. 100 ⁇ l of increasing concentrations of purified protease or various dilutions of a standard human plasma (SHPL) were in each case pipetted into the hollows. After incubation at 37° C., the solutions were removed by washing several times and the activities were determined.
  • SHPL standard human plasma
  • a prourokinase solution (10 ⁇ g/ml, American Diagnostica, US) were pipetted into each hollow, as were 50 ⁇ l of buffer, which contained 30 mM CaCl 2 and 100/ml of heparin. Two minutes later, a further 100 ⁇ l of buffer and 25 ⁇ l of the substrate S2444 (3 mM) were added. The increase in the absorption at 405 nm per minute was determined.
  • FIG. ( 1 ) shows the protease activities of the investigated healthy men (A) and women (B). It is clear that 5-10%, both men and women, show a markedly decreased activity compared with the average.
  • protease activities and the antigen levels of the corresponding people (x-axis) belonging to them are shown in the figure.
  • the arbitrary ‘normal ranges’ of the antigen and activity levels are in each case shown by horizontal and vertical lines as upper and lower limits of the parameters.
  • the rectangles resulting therefrom (in each case in the center of the figure) accordingly represent the ‘normal ranges’ of healthy donors. It is again particularly clear here that the majority of the samples having decreased activity were not accompanied by a corresponding reduction of the antigen levels. This could indicate a heterozygotic mutation (or several), i.e., for example about 50% of the protease molecules could be modified by one or more mutations such that a reaction with biological substrates is no longer guaranteed.
  • the detection of the protease activity also in association with antigen determination, can be seen as a parameter for early recognition and prophylaxis/therapeutic control.
  • Citrate plasma of pregnant women was tested as described in Example 3. Samples were obtained at various times during pregnancy and then investigated.
  • Plasma from 54 patients with acute myocardial infarct was obtained on admission (before intensive treatment) to the emergency ward and used for routine analysis. Later, plasma residues (unthawed aliquots) were used for the quantification of the protease activities (and antigen contents).
  • FIG. ( 2 ) summarizes the results of the investigation. Compared with a group of healthy donors (B), significantly higher protease activities (and also the antigen contents) can be measured in the plasma of patients with acute myocardial infarct (A).
  • these parameters can be used for the early detection of an infarct, i.e., even in the case of stable and unstable angina pectoris. In patients with these coronary heart disorder we also found significantly increased activities on average. The height of the measured values can make possible evaluation of the degree of severity of the disease or give valuable indications about the condition of the patient in the course of infarct and angina pectoris prophylaxis and therapy. Moreover, these parameters can be used for the assessment of other complications associated with the cardiovascular system.

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US10/287,047 US20030124622A1 (en) 1999-06-10 2002-11-04 Procedure for the determination of the activity of the protease which activates factor VII from protein solutions
US12/155,619 US7892842B2 (en) 1998-04-24 2008-06-06 Procedure for the determination of the activity of the protease which activates factor VII from protein solutions

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DE19926531.3 1999-06-10
US59133800A 2000-06-09 2000-06-09
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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020142316A1 (en) * 2000-07-26 2002-10-03 Juergen Roemisch Mutants of the factor VII-activating protease and detection methods using specific antibodies
US20030077271A1 (en) * 2001-09-28 2003-04-24 Christian Kannemeier Use of coagulation factor VII-activating protease for the prophylaxis and therapy of vasoproliferative disorders
US20030215447A1 (en) * 2002-02-08 2003-11-20 Juergen Roemisch Inhibitory monoclonal antibody against blood clotting factor VII-activating protease
US20040009543A1 (en) * 2000-07-26 2004-01-15 Stefan Kiechl Marburg I mutant of factor VII activating protease (FSAP) as risk factor for arterial thrombosis
US20050202002A1 (en) * 1998-04-24 2005-09-15 Zlb Behring Gmbh Protease for activating clotting factor VII
US20060045879A1 (en) * 2004-08-24 2006-03-02 Harald Althaus Antibodies which are directed against the Marburg I polymorphism of factor VII-activating protease (FSAP), and their preparation and use
US20070190574A1 (en) * 2005-12-22 2007-08-16 Dade Behring Marburg Gmbh Diagnostic method for identifying carriers of the Marburg I variant of factor VII-activating protease (FSAP) on the basis of differential modulation of FSAP activity
WO2008046125A1 (de) 2006-10-19 2008-04-24 Apeiron Biologics Forschungs- Und Entwicklungsgesellschaft M.B.H. Verfahren zur bestimmung der aktivität von ace2
US20090087864A1 (en) * 1998-04-24 2009-04-02 Csl Behring Gmbh Procedure for the determination of the activity of the protease which activates factor VII from protein solutions
US20100316624A1 (en) * 2007-12-21 2010-12-16 Hans Loibner Treatment of fibroses and liver disorders
US20110091918A1 (en) * 2009-09-12 2011-04-21 Gerlinde Christ Heterogeneous coagulation test
US20120046642A1 (en) * 2003-04-25 2012-02-23 Medtronic, Inc. Optical detector for use in therapy
EP2729809B1 (de) * 2011-07-07 2018-10-17 DuPont Nutrition Biosciences ApS Assay

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10036641A1 (de) * 2000-07-26 2002-02-14 Aventis Behring Gmbh Monoklonale Antikörper für die den Blutgerinnungsfaktor VII aktivierende Protease (FSAP)und ihre Verwendung
DE10238429A1 (de) * 2002-03-19 2003-10-30 Aventis Behring Gmbh Intellect Marburg I Mutante der Faktor VII aktivierenden Protease (FSAP) als Risikofaktor für Atherosklerose
EP2386652A1 (de) 2010-05-12 2011-11-16 Siemens Healthcare Diagnostics Products GmbH Homogenes Verfahren zur Bestimmung der Plasminogenaktivator-aktivierenden Aktivität der FSAP

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4784950A (en) * 1985-04-17 1988-11-15 Zymogenetics, Inc. Expression of factor VII activity in mammalian cells
US5175087A (en) * 1987-07-06 1992-12-29 Biopool International, Inc. Method of performing tissue plasminogen activator assay
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