# Patent

## Title
Methods and kits for determining von willebrand factor activity in the absence of ristocetin

## Abstract

Described herein are method(s), kit(s), reagent(s) and the like for determining von Willebrand factor (VWF) activity in a sample in the absence of ristocetin.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
The present application claims priority and benefit under 35 U.S.C. 120 and 365(c) as a continuation of International Application PCT/EP2008/005416, filed Jul. 3, 2008; which claims benefit under 35 U.S.C. 365(b) of German Patent Application No. DE 10 2007 031 708.7, filed Jul. 6, 2007. The entire contents of each of the above-referenced applications are hereby expressly incorporated herein by reference.


BACKGROUND
1. Field of the Discussed Inventive Concept(s)
The presently disclosed and claimed inventive concepts are generally within the field of coagulation diagnosis and relate to an in vitro method for determining von Willebrand factor (VWF) activity in a sample. A discussed method includes the use of a mutant gain-of-function variant of the GPIbα protein, thereby dispensing with the need to use ristocetin, botrocetin or another ristocetin- or botrocetin-equivalent substance. The presently disclosed and claimed inventive concept(s) further relate to a disclosed method for determining von Willebrand factor (VWF)-cleaving activity of ADAMTS-13 protease.
2. Relevant Background Information
Von Willebrand factor (VWF) is a high molecular weight, multimeric glycoprotein found in blood plasma, and functions in the process of primary hemostasis. VWF possesses, inter alia, binding sites for collagen and for glycoprotein Ib (GPIb) which is located on the surface of thrombocytes. GPIb is an integral membrane protein which, together with another integral membrane protein, glycoprotein IX (GPIX), forms the glycoprotein Ib-IX-receptor complex in the thrombocytic membrane. GPIb is a two-chain molecule comprising a heavy chain having an apparent molecular mass of about 145 kDa (synonyms: alpha-chain or GPIbα) and a light chain having an apparent molecular mass of about 22 kDa (synonyms: beta-chain or GPIbβ), which are linked to one another by disulfide bonds [Lopez, J. A. et al. (1987) Cloning of the α chain of human platelet glycoprotein Ib: A transmembrane protein with homology to leucine-rich α2-glycoprotein. Proc. Natl. Acad. Sci. USA 84: 5615-5619, the entire contents of which are hereby incorporated by reference].
In the case of a vessel injury, collagen surfaces are thereby exposed and to which VWF binds. Due to its binding to collagen and under the influence of increased shearing forces acting on the collagen-bound VWF, VWF is altered or activated in such a way that it can bind to the amino-terminal end of the GPIb heavy chain (GPIbα) in the GPIb-IX-receptor complex of the thrombocytic membrane. In this way, the activated VWF captures passing thrombocytes, resulting in the formation of a first agglomeration of VWF, collagen and thrombocytes at the site of the injury. Subsequently, the thrombocytes are activated, thereby also starting plasmatic coagulation which finally, after a plurality of amplifying cascades and attachment of further thrombocytes, results in the wound being closed.
Qualitative or quantitative VWF disorders are the cause of a “von Willebrand syndrome” (synonyms: von Willebrand disease, VWD), one of the most common hereditary hemorrhagic conditions. Various screening methods are available for diagnosing von Willebrand syndrome, such as, for example, but not by way of limitation, methods for determining bleeding time (BT), quantitative methods for determining the VWF antigen concentration (VWF:Ag), such as, for example, ELISA methods; and methods for determining the VWF activity, such as ristocetin-induced platelet agglutination (VWF:RCo).
The method of ristocetin-induced aggregation of stabilized thrombocytes, which is also referred to as ristocetin cofactor assay, also recognizes those functional defects of the VWF protein that are not detected by quantitative methods for determining the VWF antigen concentration. Complete diagnosis of a von Willebrand syndrome therefore requires carrying out a ristocetin cofactor assay for determining ristocetin cofactor activity. The ristocetin cofactor assay is usually carried out by mixing a patient's plasma sample with fixed thrombocytes and with ristocetin. Ristocetin induces binding of the VWF present in the sample to the GPIb receptor of the added thrombocytes, resulting in the latter aggregating. The extent of this aggregation reaction correlates with the amount of active VWF present in the patient's sample. Said aggregation reaction may be recorded optically, for example, by measuring the increase in transmission, thereby enabling the VWF:RCo activity to be quantified.
Compared to a VWF antigen assay, the ristocetin cofactor assay has the advantage of determining the activity of VWF and therefore enables functional VWF disorders and the classification of various subtypes of von Willebrand syndrome, only some of which are also accompanied by a reduced VWF antigen concentration, to be recognized. Said subtypes are often classified by forming the ratio of VWF ristocetin cofactor activity (VWF:RCo) and VWF antigen concentration (VWF:Ag). A VWF:RCo/VWF:Ag ratio of less than 1 is characteristic for the von Willebrand syndrome subtypes 2A, 2B and 2M. A frequently recommended threshold is a ratio of 0.7.
A disadvantage of the classical ristocetin cofactor assay is the fact that it is difficult to automate because the thrombocytes in the assay mix must constantly be stirred during the measurement. Another disadvantage is the relative imprecision of the assay and an insufficient determination of small VWF activities in the range of between 0 and 20% of standard variables.
In recent times, GPIbα-based VWF ELISAs have been developed which make use of recombinant GPIbα fragments (1-289 and 1-290, respectively) containing the amino-terminal VWF binding region [e.g., WO 01/02853 A2; Vanhoorelbeke, K. et al. (2002). A reliable von Willebrand factor: Ristocetin cofactor enzyme-linked immunosorbent assay to differentiate between type 1 and type 2 von Willebrand disease; and Semin Thromb Hemost. 28(2): 161-165 and Federici, A. B. et al. (2004) A sensitive ristocetin co-factor activity assay with recombinant glycoprotein Ibα for the diagnosis of patients with low von Willebrand factor levels. Haematologica 89(1): 77-85, the entire contents of each being hereby expressly incorporated by reference]. These assays involve binding a recombinant GPIbα fragment to an ELISA plate with the aid of a specific antibody. Ristocetin is added to the patient's sample in order for the VWF of the patient's sample to be able to bind to the recombinant GPIbα fragment. Finally, the bound VWF is quantitatively detected with the aid of an anti-VWF antibody. This kind of VWF ELISA has been shown to correlate very well with the results of the classical thrombocyte-based VWF ristocetin cofactor activity assay and to be even more sensitive and more precise.
Hui et al. (Abstract, ISTH 2007, the entire contents of which are expressly incorporated herein by reference) describe a GPIbα-based VWF ELISA which makes use of recombinant GPIbα fragments (1-483) having mutations in positions 233 and 239. These mutations are “gain-of-function” mutations which are known to have a higher affinity for VWF in the presence of low ristocetin concentrations and to interact with VWF more strongly than wild-type GPIbα protein (WO 93/16712, the entire contents of which are expressly incorporated herein by reference). The mutations mentioned are well known mutant variants of the GPIbα chain. Substitution of the glycine residue in position 233 of the GPIbα chain by a valine residue (G233V) has been described by Miller et al. (U.S. Pat. No. 5,317,097, the entire contents of which are expressly incorporated herein by reference). Said mutation is the cause of platelet-type von Willebrand syndrome (PT-VWD), an autosomally dominantly inherited hemorrhagic condition. Substitution of the methionine residue in position 239 of the GPIbα chain by a valine residue (M239V) has been described by Russell & Roth [Russell, S. D. & Roth, G. J. (1993) Pseudo-von Willebrand Disease: A mutation in the platelet glycoprotein Ibα gene associated with a hyperactive surface receptor. Blood 81(7), 1787-1791, the entire contents of which are expressly incorporated herein by reference]. This mutation also causes PT-VWD.
Disadvantageously, the GPIbα-based VWF ELISAs as well as the ristocetin cofactor assay are based on using ristocetin or other exogenous, non-physiological modulators which mediate binding of VWF to the GPIbα chain. Ristocetin, an antibiotic glycopeptide of the bacterium Nocardia lurida, and botrocetin (synonym: co-agglutinin), a snake venom of the genus Bothrops, are known to be used for inducing binding of VWF to thrombocytes or to isolated GPIbα protein or fragments thereof in vitro. Using ristocetin has the disadvantage that it can bind not only to VWF but also to many other proteins, for example, but not by way of limitation, fibrinogen and anti-GPIbα antibodies (observed in some experiments). Using ristocetin in VWF assays therefore runs the risk of the occurrence of unspecific binding reactions or precipitation reactions, which may falsify the test result.
Accordingly, disclosed and claimed herein is at least one method for determining VWF activity, which can be carried out in the absence of ristocetin. One example of the disclosed and claimed method(s) is achieved by using a gain-of-function variant of the GPIbα chain, which has two point mutations, in position 233 and in position 239, as compared to the amino acid sequence of human GPIbα receptor. A gain-of-function variant of the GPIbα chain, as disclosed and claimed, has a significantly higher affinity for VWF and interacts with VWF more strongly than wild-type GPIbα protein in the presence of low ristocetin concentrations.
ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) is a metalloprotease which proteolytically cleaves von Willebrand factor (VWF), thereby reducing its activity. Congenital and acquired deficiencies in ADAMTS-13 enzyme activity are known, which cause various diseases such as, for example, thrombotic, thrombocytopenic purpura (TTP), a life-threatening thromboembolic disorder affecting microcirculation. Unduly large multimers of VWF are found in the plasma of TTP patients and are considered to be the cause of the formation of thrombi rich in VWF and thrombocytes. Determination of the VWF-cleaving protease activity of ADAMTS-13 protease in patients' samples is therefore of diagnostic importance.
Various methods for determining the VWF-cleaving activity of ADAMTS-13 protease and for diagnosing ADAMTS-13 deficiency have been disclosed. Furlan et al. (1996) describe a method, wherein a sample containing ADAMTS-13 protease is incubated with purified human VWF as substrate, with the proteolytic activity of the ADAMTS-13 protease being detected by subsequent analysis of VWF multimers by means of SDS agarose gel electrophoresis or by analyzing VWF fragments by means of SDS polyacrylamide gel electrophoresis (SDS PAGE) and subsequent immunoblotting [Furlan, M., Robles, R. and Lämmle, B. (1996) Partial purification and characterization of a protease from human plasma cleaving von Willebrand factor to fragments produced by in vivo proteolysis. Blood 87, 10: 4223-4234, the entire contents of which are hereby expressly incorporated by reference.] Multimer analyses by means of gel electrophoresis and immunoblotting require an extraordinary amount of work and time and are therefore not suitable for use in a clinical laboratory.
Other functional assay methods determine the proteolytic activity of ADAMTS-13 protease by incubating the sample containing said ADAMTS-13 protease with VWF as substrate and then determining the loss of activity of said VWF substrate. The more ADAMTS-13 protease present in the sample, the more VWF substrate is cleaved and the less VWF activity can then be detected in the reaction mixture. The advantage of these functional methods is that of simulating in vitro the in vivo-relevant function of ADAMTS-13 protease by using VWF substrate and measuring the loss of function of the large multimers. For example, WO 2004/005451 A2 (the entire contents of which are hereby expressly incorporated by reference) discloses a method, wherein a sample containing the ADAMTS-13 protease is incubated with purified human VWF substrate, with the proteolytic activity of said ADAMTS-13 protease being detected by subsequently determining the VWF activity remaining in the reaction mixture by means of a ristocetin cofactor assay. Alternatively, the VWF activity remaining in the reaction mixture may be assayed by means of a collagen binding assay (see, for example, WO 00/50904, the entire contents of which are hereby expressly incorporated by reference). Disadvantageously, the reaction mixture must be incubated for a very long time, sometimes overnight, in order to cause sufficient proteolytic degradation of VWF. An improved method with a reduced incubation time of 60 minutes is described in Kostousov et al. (Kostousov, V., Fehr, J., Bombeli, T. (2006) Novel, semi-automated, 60-min-assay to determine von Willebrand factor cleaving activity of ADAMTS-13. Thrombosis Res. 118: 723-731, the entire contents of which are hereby expressly incorporated by reference.)
US 2007/0065895 A1 and US 2005/0186646 A1 (the entire contents of both of which are hereby expressly incorporated by reference) disclose methods in which a sample containing ADAMTS-13 protease is incubated with peptides having a specific cleavage site for ADAMTS-13 protease or with VWF peptides (fragments) as substrate, with the proteolytic activity of ADAMTS-13 protease being detected by subsequently analyzing the resulting peptide fragments by means of SDS PAGE and optionally by subsequent immunoblotting.
The known assay systems are disadvantageous in that they either require complicated technologies, such as, for example, analysis of the substrate fragments resulting from proteolysis by means of SDS PAGE, or do not use the natural VWF substrate, thereby making it possible that some dysfunctionalities of ADAMTS-13 protease cannot be detected.
The presently disclosed and claimed inventive concept(s) provide a method for determining von Willebrand factor (VWF)-cleaving activity of ADAMTS-13 protease. The disclosed and claimed method(s) enable as many dysfunctionalities of ADAMTS-13 protease as possible to be detected. Furthermore, the presently disclosed and claimed method(s) can be carried out in a timesaving manner and automatically on conventional clinical analyzers. One such exemplary method comprises mixing a sample which is suspected to contain ADAMTS-13 protease with isolated VWF as substrate to give a reaction mix. The VWF substrate is a high molecular weight, multimeric VWF which includes VWF monomers having at least one amino acid sequence variation (polymorphism or mutation) resulting in each case in the VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a VWF substrate that is composed of wild-type VWF monomers.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE FIGURES
FIG. 1 is a graphical depiction of VWF binding as a function of ristocetin concentration. The design of the GPIbα-(aa1-285, G233V/M239V, SEQ ID NO: 24) and wild-type GPIbα ELISA assay systems shown in FIG. 1 are described in Example 4. Four different dilutions of the VWF/factor VIII concentrate HAEMATE® were used as samples. In the absence of ristocetin (0 mg/ml), strong and concentration-dependent binding of VWF occurs upon using the mutant GPIbα-(aa1-285, G233V/M239V, SEQ ID NO: 24) (top figure). Binding can be increased slightly by adding ristocetin, which is most likely due to unspecific binding taking place even without GPIbα. By contrast, similarly strong VWF binding can be achieved with wild-type GPIbα only by adding 1.2 mg/ml ristocetin. Without the addition of ristocetin, there is no binding observed upon use of wild-type GPIbα (bottom figure).
FIG. 2 is a calibration curve for determining VWF activity (10%-200% VWF activity) in human citrate plasma samples by means of a GPIbα latex particle agglutination assay without ristocetin (see Example 5). The rate of particle agglutination is depicted as a function of VWF activity.
FIG. 3 is a calibration curve for determining low VWF activities (≦3% VWF activity) in human citrate plasma samples by means of a GPIbα latex particle agglutination assay without ristocetin (see Example 5, 45 μl of sample). The rate of particle agglutination is depicted as a function of VWF activity.
FIG. 4 is a graphical representation of GPIbα latex particle agglutination assays without ristocetin in samples with different VWF concentrations (14.9% to 149.6%) and in the presence of different TWEEN® 20 concentrations in the assay mix (see Example 5, variation of TWEEN® 20 concentration, 15 μl of sample).
FIG. 5 is a graphical representation of GPIbα latex particle agglutination assays without ristocetin in samples with different VWF concentrations (8% to 40%) and in the presence of different THESIT® concentrations in the assay mix (see Example 5, but THESIT® is used instead of TWEEN® 20, 45 μl of sample).
FIG. 6 is a graphical representation of GPIbα latex particle agglutination assays without ristocetin in samples with different VWF concentrations, which samples were prediluted with MT-deficient plasma or with buffer, and in the presence of different THESIT® concentrations in the assay mix (see Example 5, but THESIT® is used instead of TWEEN® 20, 45 μl of sample).



DETAILED DESCRIPTION OF THE PRESENTLY DISCLOSED AND CLAIMED INVENTIVE CONCEPT(S)
Before explaining at least one embodiment of the presently disclosed and claimed inventive concept(s) in detail, it is to be understood that the presently disclosed and claimed inventive concept(s) is not limited in its application to the details of construction, experiments, exemplary data, and/or the arrangement of the components set forth in the following description or illustrated in the drawings. The presently disclosed and claimed inventive concept(s) is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for purpose of description and should not be regarded as limiting.
The presently disclosed and claimed inventive concept(s) include a method for determining von Willebrand factor (VWF) activity in a sample, wherein the sample is mixed with isolated GPIbα protein to give an assay mix, wherein neither ristocetin nor botrocetin are added to the assay mix. The GPIbα protein used includes at least an amino acid sequence which, when compared to the wild-type sequence of human GPIbα protein, includes at least the amino acid residues 1-268 of SEQ ID NO: 1 except that amino acid substitutions are found in positions 233 and 239: that is, any amino acid except glycine may be present at position 233, whereas any amino acid except methionine may be present at position 239 (i.e., SEQ ID NO: 2). In certain embodiments, the glycine residue in position 233 and the methionine residue in position 239 of the GPIbα chain are substituted with valine residues (G233V and M239V, respectively SEQ ID NO: 3) or serine residues (G233S and M239S, respectively SEQ ID NO: 4). Any combination of different amino acid substitutions in the two positions is also possible (see for example, but not by way of limitation, SEQ ID NO's: 5 and 6). The reference to GPIbα protein herein refers to a mutant variant thereof. Through use of such a gain-of-function variant of GPIbα, the required prior art step of applying shearing stress to the assay mix, for example by way of flow, stirring or shaking, is no longer required.
In an additional embodiment, “ristocetin- or botrocetin-equivalent substance” are also not added to the assay mix. The term “ristocetin- or botrocetin-equivalent substance” means an exogenous, (i.e., non-physiological) substance which is capable of inducing in vitro binding of dissolved VWF to the wild-type GPIbα chain or fragments thereof.
The term “sample” as used herein, refers to any material which is suspected to contain the substance to be detected, i.e. the VWF. The term “sample” comprises, for example but not by way of limitation, biological fluids, in particular of humans and animals, such as blood, plasma or serum, and also industrially produced products such as, for example, VWF calibrators or VWF controls or highly concentrated VWF concentrates, which are designed, for example, for substitution therapy of von Willebrand syndrome patients (e.g., HAEMATE P®, CSL Behring). Where appropriate, samples must be pretreated in order to make the analyte accessible to the detection method or to remove interfering sample components. Such a pretreatment of samples may include the removal and/or lysis of cells or centrifugation of samples. The term “sample” also comprises reaction mixtures comprising any of the above mentioned sample materials, to which isolated VWF has been added as substrate for determining the activity of a VWF-modifying factor and in which the residual VWF activity is to be determined after incubation of said VWF substrate with said VWF-modifying factor. Examples of such reaction mixtures include, for example but not by way of limitation, mixtures of a plasma sample with isolated high molecular weight VWF for determining the activity of VWF-cleaving ADAMTS-13 protease in the plasma sample. The ADAMTS-13 protease present in the plasma sample cleaves the added VWF substrate and thus reduces the VWF activity of the sample.
The GPIbα chain used according to the presently disclosed and claimed inventive concept(s) may be a recombinantly or synthetically produced GPIbα protein. Useful methods for producing recombinant GPIbα protein are known prokaryotic or eukaryotic expression systems such as, for example but not by way of limitation, expression in bacteria (e.g., E. coli), in yeasts (e.g. Saccharomyces cerevisiae, Pichia pastoris), and in plant, animal or human cell cultures. Suitable methods for producing synthetic GPIbα protein are known techniques for in vitro protein synthesis such as, for example but not by way of limitation, solid phase syntheses (e.g., Merrifield synthesis). The GPIbα protein is, in one embodiment, recombinantly produced GPIbα protein which has been produced in a culture of human cells, such as in a culture of human embryonic kidney cells (HEK cells), for example.
The GPIbα protein used may also be fused at the N terminus to the homologous, human GPIbα signal sequence MPLLLLLLLLPSPLHP (SEQ ID NO: 7, also referred to as “amino acid residues −16 to −1”). Alternatively, the GPIbα protein used may be fused at the N terminus to a heterologous signal sequence, i.e., to a polypeptide which normally is not present in the human GPIbα polypeptide but which has a beneficial influence on expression and/or secretion of the recombinantly expressed GPIbα protein in the chosen expression system. A suitable heterologous signal sequence is, for example but not by way of limitation, MPLQLLLLLILLGPGNSLQLWDTWADEAEKALGPLLARDRR (SEQ ID NO: 8).
Furthermore, the GPIbα protein may be fused at the C terminus to one or more affinity tags which enable the recombinantly expressed protein, for example, to bind to an affinity carrier, thereby enabling recombinantly expressed GPIbα protein to be purified. Additionally, the recombinant GPIbα protein may be bound to a solid phase in such a manner. Small affinity tags having a length of no more than 12 amino acids are particularly well-suited, for example. Known affinity tags from the group consisting of a His tag, a Flag tag, an Arg tag, a c-Myc tag, a Strep tag and combinations thereof are well suited for use. Examples of suitable affinity carriers which bind to an affinity tag with high affinity include, but are not limited to, specific antibodies, immobilized cations (e.g., Ni2+ with affinity for His tags) or other types of binding partners (e.g. streptavidin with affinity for Strep tags).
In one embodiment of the presently disclosed and claimed inventive concept(s), the GPIbα protein is associated with a solid phase. The term “associated” has a broad meaning and comprises, for example but not by way of limitation, covalent and noncovalent binding, direct and indirect binding, adsorption to a surface and inclusion in a depression. In covalent binding, the isolated GPIbα protein is bound via a chemical bond to the solid phase. A non-limiting example of noncovalent binding is surface adsorption. In addition to direct binding to the solid phase, the isolated GPIbα protein may also be bound indirectly via specific interaction with other specific binding partners to the solid phase, for example but not by way of limitation, via specific interaction with an antibody, preferably with an anti-GPIbα antibody or—if the isolated GPIbα protein has an affinity tag—with an anti-affinity tag antibody.
The term “solid phase” as used herein includes an object which comprises porous and/or nonporous, water-insoluble material and which may have very different shapes, such as, for example but not by way of limitation, vessel, tube, microtitration plate (i.e., ELISA plate), bead, microparticle, rod, strip, filter paper or chromatographic paper, and the like. The surface of the solid phase is normally hydrophilic or can be made hydrophilic. The solid phase may comprise very different materials such as, for example but not by way of limitation, inorganic and/or organic materials, synthetic materials, naturally occurring and/or modified naturally occurring materials. Examples of solid phase materials are polymers such as, for example but not by way of limitation, cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, crosslinked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate or nylon; latex; ceramics; glass; metals, in particular precious metals such as gold and silver; magnetite; and mixtures or combinations thereof.
The solid phase may have a coating of one or more layers, for example of proteins, carbohydrates, lipophilic substances, biopolymers, organic polymers or mixtures thereof, for example for suppressing or preventing unspecific binding of sample components to the solid phase, or for example for improving the suspension stability of particulate solid phases, the storage stability, the design stability or the resistance to UV light, microbes or other destructive agents.
One method of the presently disclosed and claimed inventive concept(s) for determining VWF activity comprises the use of a particle-associated GPIbα protein, such as, but not limited to, a latex particle-associated GPIbα protein, and determination of VWF activity. VWF activity may be determined on the basis of GPIbα-mediated agglutination of the particulate solid phase. In such a method, GPIbα protein may be associated with the particulate solid phase via an antibody. Suitable antibodies for this purpose include, but are not limited to, anti-GPIbα antibodies, in particular the monoclonal antibodies VM16d [Mazurov, A. V. et al. (1991) Characterization of an antiglycoprotein Ib monoclonal antibody that specifically inhibits platelet-thrombin interaction. Thromb Res. 62(6), 673-684; commercially available, for example, from Sanbio B. V., Uden, Netherlands: product number MON 1146] and SZ-2 [Ruan, C. et al. (1987) A murine antiglycoprotein Ib complex monoclonal antibody, SZ 2, inhibits platelet aggregation induced by both ristocetin and collagen. Blood 69(2), 570-577; commercially available, for example, from Beckman Coulter Inc., Fullerton, USA: product number IM0409]. If the GPIbα protein used is fused at the C terminus to one or more Flag tags, an anti-Flag antibody is equally suitable [see, for example, U.S. Pat. No. 5,011,912; commercially available, for example, from Sigma-Aldrich Chemie GmbH, Steinheim, Germany]. The agglutination reaction which correlates with the amount or activity of the VWF present in the sample can be determined quantitatively, for example but not by way of limitation, by utilizing the light scatter on the particle aggregates by way of measuring the intensity of the scattered light (nephelometry) or by way of measuring the turbidity of the medium (turbidimetry). The entire contents of all of the above references are hereby incorporated by reference in their entirety.
The determination of VWF activity on the basis of GPIbα-mediated agglutination of a particulate solid phase is also contemplated as being conducted in the presence of a detergent (e.g., in the presence of a detergent selected from the group consisting of TWEEN® 20 (CRODA INTERNATIONAL PLC), THESIT®, TRITON® detergent (e.g., TRITON® X-100 (UNION CARBIDE CORPORATION) or TRITON® X-405 (UNION CARBIDE CORPORATION)) and sodium dodecyl sulfate (SDS). The presence of a detergent influences the VWF-dependent rate of agglutination of the particulate solid phase, in particular of latex particles.
The rate of agglutination was determined at various VWF concentrations and at increasing TWEEN® 20 concentrations (see FIG. 4, determination in a manner analogous to Example 5). It is apparent that amplification of the reaction is particularly pronounced at relatively low TWEEN® 20 concentrations (0.02-0.1 g/l), which decreases again with increasing TWEEN® 20 concentration and attains a stable level. For 149.6% VWF, for example, the stable amplification level is reached at approximately 1 g/l TWEEN® 20, with the amplification then being 85% of the starting value without TWEEN® 20. Consequently, there are at least two methods contemplated when utilizing TWEEN® 20 as detergent within the presently disclosed and claimed inventive concept(s). First, TWEEN® 20 concentrations can be set low (0.02-0.1 g/l) in order to strongly amplify certain VWF concentrations up to 20%. For example, at 0.05 g/l TWEEN® 20 (in the assay mix), the rate of agglutination is amplified by 216%, with 30.4% VWF. Since low VWF concentrations are particularly difficult to determine, amplifying the reaction is very helpful. Second, a measuring system is generally more stable if it is operated in the saturation range of the detergent for all relevant VWF concentrations. This would be the case from 1 g/l TWEEN® 20 upward, for example. This variant is more suitable for a screening assay for a wide range of VWF concentrations. TWEEN® 20 concentrations of 0.6-20.0 g/l, such as 1 g/l, are advantageous if the agglutination reaction is to be amplified for determining VWF concentrations of more than 20%.
Other detergents such as THESIT® (synonym: Polidocanol, Schärer & Schläpfer Ltd., Rothrist, Switzerland), TRITON® detergent (e.g., TRITON® X-100 or TRITON® X-405) and sodium dodecyl sulfate (SDS) exhibit a steady concentration-dependent increase in the amplification of the agglutination reaction (see FIG. 5, determination in a manner analogous to Example 5: 45 μl of sample, but with THESIT® instead of TWEEN® 20).
The VWF activity of a plasma sample according to the presently disclosed and claimed inventive concept(s) is determined by diluting said sample beforehand, usually with a buffer or with VWF-deficient plasma. The same applies to normal plasma (e.g., standard human plasma) in order to generate a calibration curve. Dilution with VWF-deficient plasma and a customary detergent concentration (1 g/l in the assay mix) results in an undesired amplification of the GPIbα latex particle agglutination which can be suppressed by sufficient detergent. When, as FIG. 6 shows, the sample (in this case standard human plasma) in an assay mix containing 45 μl of sample is diluted with VWF-deficient plasma or with buffer, higher rates of agglutination occur at a particular VWF concentration in the sample in the presence of VWF-deficient plasma. In contrast, dispensing with detergent in the assay system altogether and diluting the standard human plasma sample with VWF-deficient plasma results in a lower rate of agglutination, as with dilution with a buffer. However, if sufficient detergent is added (e.g., ≧3 g/l THESIT®), the rates of agglutination for dilution with buffer or with deficient plasma become alike. This ensures that the dilution of a plasma sample in a buffer is proper. It is extremely advantageous for an assay to be able to use a simple buffer both for dilution of a normal plasma (as calibrator) and for possible dilution of the patient's sample, rather than having to use a VWF-deficient plasma. A buffer is inexpensive and ensures better storage stability, and many automated assay devices utilize a single buffer for multiple assays. Furthermore, VWF-deficient plasma is often not available in every laboratory. Measurements of VWF concentrates involve particularly high dilutions, with a proper dilution in buffer being likewise very advantageous.
VWF activity may also be determined according to the presently disclosed and claimed inventive concept(s) on the basis of GPIbα-mediated agglutination of a particulate solid phase in the presence of polyvinylpyrrolidone (PVP), such as at a polyvinylpyrrolidone concentration of from 0.1% to 1.0% in the assay mix, and/or in the presence of Dextran T500, preferably at a Dextran T500 concentration of from 0.1% to 3% in the assay mix, and/or in the presence of Alginate 500-600 cP, preferably at an Alginate 500-600 cP concentration of from 0.01% to 0.2% in the assay mix.
Another embodiment of a method of the presently disclosed and claimed inventive concept(s) for determining VWF activity is a competitive assay format. The competitive assay format includes the use of a particle-associated anti-GPIbα antibody, preferably VM16d, and particle-associated VWF in a reaction mix and the addition of GPIbα protein. Said particles agglutinate in the presence of GPIbα protein and in the absence of ristocetin, botrocetin or an equivalent substance. This agglutination reaction is inhibited by the addition of a sample containing VWF. Said inhibition of the agglutination reaction correlates with the amount or activity of the VWF present in said sample.
Another embodiment of a method of the presently disclosed and claimed inventive concept(s) for determining VWF activity includes the use of a GPIbα protein associated to a non-particulate solid phase, preferably associated to the surface of a microtiter plate, and determining said VWF activity on the basis of determining the amount of VWF bound to said GPIbα protein. A GPIbα protein associated via an antibody to the non-particulate solid phase may be used, for example. Suitable antibodies for this purpose include, but are not limited to, anti-GPIbα antibodies, in particular the monoclonal antibodies VM16d (see above) and MB45 [see, for example, Modderman, P. W. et al. (1992) Glycoproteins V and Ib-IX form a noncovalent complex in the platelet membrane. J. Biol. Chem. 267(1), 364-369, and reference 26 cited therein, the entire content of which are hereby expressly incorporated by reference in its entirety; commercially available, for example, from Sanguin, Amsterdam, Netherlands: PeliCluster CD42b, product number M9142]. If the GPIbα protein used is fused at the C terminus to one or more affinity tags such as, but not limited to, Flag tags, an anti-affinity tag antibody such as an anti-Flag antibody is equally suitable [see above]. The amount of VWF bound to the GPIbα protein can be determined, for example, by using an anti-VWF antibody which may be associated directly or indirectly with a component of a signal-producing system, thereby allowing the amount of GPIbα-bound VWF to be quantified. Since only “active” (functionally intact) VWF binds to the GPIbα receptor, the VWF antigen concentration calculated according to this principle correlates with the VWF activity.
The presently disclosed and claimed inventive concept(s) furthermore relate to a test kit for use in one or more of the methods described and claimed herein. For example, such a, test kit may comprise at least one reagent containing GPIbα protein which comprises the amino acid sequence of SEQ ID NO:2, and which GPIbα protein is associated with a particulate solid phase. In one embodiment, the test kit includes a reagent containing latex particle-associated GPIbα protein. The GPIbα protein may be associated with the particulate solid phase via an antibody. The reaction may be provided in a liquid or lyophilized form. If the reagent is a lyophilisate, the test kit may additionally include a solvent required for suspending said lyophilisate, such as distilled water or a suitable buffer, for example.
The presently disclosed and claimed inventive concept(s) furthermore relate to a test kit for use in one or more of the methods described and claimed herein. For example, such a test kit may comprise a non-particulate solid phase, preferably a microtitration plate, to which a GPIbα protein is associated, where the GPIbα protein comprises the amino acid sequence of SEQ ID NO: 2. In one embodiment, the GPIbα protein is associated to the non-particulate solid phase via an antibody. The test kit may additionally include a reagent which contains an anti-VWF antibody, preferably an anti-VWF antibody which is associated directly or indirectly with a component of a signal-producing system. The reagent containing the anti-VWF antibody may be provided in a liquid or lyophilized form. If the reagent is a lyophilisate, the test kit may additionally include a solvent required for suspending said lyophilisate, such as distilled water or a suitable buffer, for example.
The presently disclosed and claimed inventive concept(s) furthermore relate to the use of an isolated GPIbα protein having the amino acid sequence of SEQ ID NO: 2 in a method for determining von Willebrand (VWF) activity in vitro, wherein neither ristocetin nor botrocetin is used. In one embodiment, an isolated GPIbα protein in which at least one of the amino acid substitutions in positions 233 and 239 comprise a valine or a serine residue is used. Any combination of these different amino acid substitutions in positions 233 and 239 is contemplated herein.
The presently disclosed and claimed inventive concept(s) furthermore relate to a method for obtaining an isolated GPIbα protein having the amino acid sequence of SEQ ID NO: 2, which method comprises recombinantly expressing said GPIbα protein in a culture of prokaryotic or eukaryotic cells and isolating said GPIbα protein from the cell lysate or the cell culture supernatant in a manner such as, but not limited to, affinity chromatography using an affinity carrier. In one embodiment, the isolated GPIbα protein has a valine or serine residue present at the amino acid substitution(s) in positions 233 and/or 239. Any combination of these different amino acid substitutions in positions 233 and 239 is contemplated herein.
The presently disclosed and claimed inventive concept(s) further include at least one method for determining the von Willebrand factor (VWF)-cleaving activity of ADAMTS-13 protease.
The object is achieved by mixing a sample which is suspected to contain ADAMTS-13 protease with isolated VWF as substrate to give a reaction mix, said VWF substrate being high molecular weight, multimeric VWF which includes VWF monomers having at least one amino acid sequence variation (polymorphism or mutation) resulting in each case in said VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a VWF substrate that is high molecular weight, multimeric VWF composed of wild-type VWF monomers.
Human VWF monomer is synthesized in vivo initially as a 2813 amino acids precursor protein. Intracellular processing produces VWF multimers which can be more than 20,000 kDa in size. These multimers comprise linearly arranged 275 kDa, 2050 amino acids VWF monomers linked to one another via disulfide bonds. VWF circulates in the plasma in the form of globular multimers of different sizes from about 500 kDa (dimer) to over 15 000 kDa.
A “high molecular weight, multimeric VWF substrate” includes within its generally accepted definition a VWF protein which comprises more than 10 dimerized VWF molecules and which is more than 5000 kDa in size, as established by gel electrophoresis methods known to the skilled worker. Furthermore, high molecular weight, multimeric VWF may also include VWF monomers having at least one amino acid sequence variation resulting in each case in said VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a high molecular weight, multimeric VWF substrate that is composed of wild-type VWF monomers.
Amino acid sequence variations of the VWF protein which increase the sensitivity of VWF toward proteolytic cleavage by ADAMTS-13 protease, are known [See, for example, Hassenpflug, W. A., Budde, U., Obser, T., Angerhaus, D., Drewke, E., Schneppenheim, S., Schneppenheim, R. (2006) Impact of mutations in the von Willebrand factor A2 domain on ADAMTS 13-dependent proteolysis. Blood 107: 2339-2345; or Rayes, J., Hommais, A., Legendre, P., Tout, H., Veyradier, A., Obert, B., Ribba, A. S., Girma, J. P. (2006) Effect of von Willebrand disease type 2B and type 2M mutations on the susceptibility of von Willebrand factor to ADAMTS-13. J. Thromb Haemost. 5: 321-328, the entire contents of which are expressly incorporated herein by reference in their entirety]. A VWF substrate which is particularly suitable for the presently disclosed and claimed inventive concept(s) comprises a high molecular weight, multimeric VWF which contains VWF monomers having at least one amino acid sequence variation selected from the group consisting of P1648S (SEQ ID NO: 9), E1638K (SEQ ID NO: 10), G1505R (SEQ ID NO: 11), S1506L (SEQ ID NO: 12), M1528V (SEQ ID NO: 13), R1569del (SEQ ID NO: 14), R1597W (SEQ ID NO: 15), V1607D (SEQ ID NO: 16), G1609R (SEQ ID NO: 17), G1629E (SEQ ID NO: 18), G1631D (SEQ ID NO: 19), R1597Q (SEQ ID NO: 20), V1499E (SEQ ID NO: 21) and Y1584C (SEQ ID NO: 22). While this group of amino acid sequence variations is set forth, it is to be considered exemplary and not to be an exhaustive list of possible variations to be used with the presently disclosed and claimed inventive concepts.
The amino acid position indicated refers to the 2813 amino acid sequence of the VWF precursor protein (see, for example, NCBI Accession No. AAB594578, Version AA59458.1, GI: 340356, SEQ ID NO: 23). The abbreviation “del” means a deletion of the corresponding amino acid residue. For the nomenclature of mutations and polymorphisms of von Willebrand factor, (see also Goodeve et al., Goodeve, A. C., Eikenboom, J. C. J., Ginsburg, D., Hilbert, L., Mazurier, C., Peake, I. R., Sadler, J. E. and Rodeghiero, F. (2001), A standard nomenclature for von Willebrand factor gene mutations and polymorphisms. Thromb Haemost. 85: 929-931, the entire contents of which are hereby expressly incorporated by reference in its entirety).
Other naturally occurring or artificially generated amino acid sequence variations of VWF may likewise be suitable, as long as they result in each case in the resulting VWF substrate being broken down by ADAMTS-13 in an accelerated manner compared to a VWF substrate that is high molecular weight, multimeric VWF composed of wild-type VWF monomers, i.e., in the resulting VWF substrate having increased sensitivity to ADAMTS-13 proteolysis. Sensitivity to ADAMTS-13 proteolysis is increased if the high molecular weight, multimeric VWF substrate is proteolytically degraded in vitro by ADAMTS-13 more rapidly than a purely wild-type high molecular weight, multimeric VWF substrate, i.e., if greater fragmentation of the VWF substrate is caused under the same incubation conditions, which can be established, for example, by means of a gel electrophoresis or other methods known to the skilled worker.
An isolated high molecular weight, multimeric VWF substrate that can be used in at least one method of the presently disclosed and claimed inventive concept(s) may either be obtained from donor plasmas of heterozygous or homozygous carriers of a suitable VWF amino acid sequence variation or be recombinantly expressed with the aid of methods known to the skilled worker. Recombinantly produced VWF substrate, for example, is advantageous in that it has no ADAMTS-13 contamination whatsoever that could falsify the test results if it is introduced into the assay mix. In addition, recombinantly produced VWF substrate is fully multimerized and is not proteolyzed, which may be the case with VWF isolated from plasma.
In one embodiment of a method of the presently disclosed and claimed inventive concept(s) for determining von Willebrand factor (VWF)-cleaving activity of ADAMTS-13 protease, degradation of the VWF substrate may additionally be accelerated by the sample being mixed in addition with heparin and/or isolated native GPIbα protein and/or recombinant wild-type GPIbα protein and ristocetin or botrocetin. Similarly, the sample may also be mixed alternatively or in combination with recombinant or synthetic GPIbα protein comprising the amino acid sequence of SEQ ID NO: 2. The amino acid substitution in position 233 and/or position 239 of the GPIbα protein may, in one embodiment, comprise a valine or a serine residue. The VWF activity remaining in the reaction mix after incubating the sample with the VWF substrate may be determined in different ways, for example but not by way of limitation, by measuring the ristocetin-cofactor activity (VWF:RCo), or other known methods. In an additional embodiment, the VWF activity remaining in the reaction mix is determined by mixing the reaction mix or an aliquot thereof with isolated GPIbα protein comprising the amino acid sequence of SEQ ID NO: 2 and by adding neither ristocetin nor botrocetin. The GPIbα protein may also be coupled to a particulate solid phase which agglutinates as a function of the remaining VWF activity. The residual VWF activity in the reaction mix can be determined on the basis of the agglutination reaction, which activity in turn provides information about the ADAMTS-13 activity present in the sample.
A particular advantage of the method(s) of the presently claimed and disclosed inventive concept(s) for determining the von Willebrand factor (VWF)-cleaving activity of ADAMTS-13 protease in a sample is the fact that it is possible to dispense with treating the VWF substrate with urea, the application of which leads to a risk of determining VWF activities thereafter which are too low, if there is no strict adherence to the incubation times during pretreatment. Various known methods require a pretreatment of the VWF substrate with urea or similarly denaturing substances, in order to make VWF accessible to degradation by ADAMTS-13 protease in the first place (see, for example, WO 2004/005451 A2, the entire contents of which are hereby expressly incorporated by reference in its entirety).
Since each human plasma sample in which the VWF-cleaving activity of ADAMTS-13 protease is to be determined contains sample-intrinsic VWF, it is advantageous to carry out, in parallel to the actual assay mix, a measurement of a second assay mix, in which a second aliquot of the same sample is analyzed according to the present disclosed and claimed inventive concept(s), but without the sample mixed with the VWF substrate being incubated for the same period of time as the actual assay mix, but wherein the VWF activity in the second assay mix is measured immediately after mixing sample and VWF substrate. The difference between the VWF activity in the second assay mix (without incubation step) and the VWF activity in the actual assay mix (with incubation step for proteolytic VWF degradation) then constitutes the VWF activity degraded by ADAMTS-13 in the actual assay mix.
The examples described hereinbelow are used for illuminating by way of example individual aspects of the present invention and should not be construed as limiting to the presently disclosed and claimed inventive concept(s).
EXAMPLES
Example 1
Cloning of the GPIbα-(G233V/M239V)-(3×Flag)-(6×His) (SEQ ID NO: 24) Construct
The pIRES neo2 expression vector (BD Biosciences, Clontech, Art. No.: 6938-1) was modified so as to include a 3×Flag tag and a 6×His tag between the EcoRI and NotI restriction cleavage sites. A fragment coding for the signal peptide (SEQ ID NO: 7) and the amino acids 1-285 of the human GPIbα receptor (SEQ ID NO: 1) and containing a valine-encoding codon at the sites coding for amino acid positions 233 and 239 was inserted upstream (5′) of the tag sequences.
Example 2
Expression of the Recombinant GPIbα-(G233V/M239V)-3×Flag)-(6×His) (SEQ ID NO: 24) Fusion Protein in HEK Cells
HEK 293 cells (human embryonic kidney cells; ATCC number; CRL-1573; Cell Lines Service CLS, Heidelberg, Germany) were transformed with the construct described in Example 1.
The cells were cultured in:

    
    
        DMEM (Art. No.: 31966-021, Invitrogen)
        +10% FBS Origin EU (Art. No.: 10270-106, Invitrogen), heat-inactivated or else in 10% FBS Origin USA (Art. No.: A15-003, Lot No.: A01123-678, PAA)
        +1% Antibiotic Antimycotic Solution (100×) (Art. No.: P11-002, PAA)
        +0.1% Gentamycin Solution 50 mg/ml (Art. No.: P11-005, PAA)
        +500 μg/ml Geneticin (G418) Solution 50 mg/ml active Geneticin (Art. No. 10131-027, Invitrogen)
    
    


For the expression (production):

    
    
        OPTIPRO-SFM (Art. No.: 12309-019, Invitrogen)
        +0.5% Antibiotic Antimycotic Solution (100×) (Art. No.: P11-002, PAA)
        +0.05% Gentamycin Solution 50 mg/ml (Art. No.: P11-005, PAA)
        +2% Glutamax I (100×) (Art. No.: 35050-038, Invitrogen) and no Geneticin.
    
    


The procedure was as follows:

    
    
        1) Cells were thawed (1 “Kryo” containing 5-10×106 cells) and cultured in T175 in serum-containing medium for 96 h;
        2) Cells were split into 4× T175 and cultured for 72-96 h;
        3) Cells were split into 25× T175 and cultured for 72-96 h;
        4) One T175 was split and continued cultured as reserve. The remaining 24× T175 were split into 3× CellStack Corning (10 levels per CellStack, with 6360 cm2 in total) and cultured for 72 h;
        5) Medium was removed followed by 1-2× washes of monolayer with DMEM without FBS, and serum-free OPTIPRO-SFM was added (1.8 liter per CellStack) and cultured for 96 h;
        6) The medium was harvested. The supernatant was removed by centrifugation and the pellet of detached cells was resuspended and returned them to the CellStack with fresh OPTIPRO-SFM, followed by culturing for 96 h;
        7) as in 6);
        8) The medium was finally harvested and the culture stopped.
    
    


Example 3
Isolation of the Recombinant GPIbα-(G233V/M239V)-(3×Flag)-(6×His) (SEQ ID NO: 24) Fusion Protein by Affinity Chromatography
The cells or cell debris still remaining in the GPIbα-(G233V/M239V)-3×Flag)-(6×His)-containing medium obtained according to Example 2 were removed by centrifugation (35 min, 10 000 rpm, Beckman J2-21, Beckman Coulter GmbH, Germany). The cell-free supernatant thus obtained was concentrated to 1/10 of the starting volume by means of tangential flow ultrafiltration using an ultrafiltration cassette with a cut-off of 10 kDa (PES 10, Schleicher & Schüll, Germany).
The purification was carried out by means of affinity chromatography using Ni2+-Sepharose (His Prep FF16/10, GE Healthcare, Sweden) according to the manufacturer's instructions. GPIbα-(G233V/M239V)-(3×Flag)-(6×His) was bound by adding to the concentrated supernatant 500 mmol of NaCl, 20 mmol of Na2HPO4 and 5 mM imidazole and adjusting the pH to pH 7.4 by adding 5M HCl. Unbound components were washed off by rinsing the column with a buffer of 500 mmol of NaCl, 20 mmol of Na2HPO4 and 5 mM imidazole, pH 7.4. The bound GPIbα-(G233V/M239V)-(3×Flag)-(6×His) was eluted with a buffer of 20 mmol of Na2HPO4, 500 mmol of NaCl, and 500 mmol of imidazole, pH 7.4. The eluate thus obtained was concentrated to 1/10 of the starting volume in a stirred ultrafiltration cell with an ultrafiltration membrane, 10 kDa cut-off (OMEGA 10 K, Pall Life Sciences, USA). Further purification and removal of contaminations were carried out by means of gel filtration using a SUPERDEX® 200 prep grade 35/600 (GE Healthcare, Sweden) chromatography column according to the manufacturer's instructions. The chromatography was carried out with a flow rate of 5.0 ml/min using a buffer of 0.048 mol/l Na2HPO4, 0.02 mol/l KH2PO4, 0.145 mol/l NaCl, and 0.015 mol/l NaN3, pH 7.2. After the sample was loaded, GPIbα-(G233V/M239V)-(3×Flag)-(6×His) eluted from the chromatography column in a peak after an elution volume of approx. 300 ml.
Example 4
Method for Determining VWF Activity in an Anti-FLAG/GPIbα ELISA without Ristocetin 
ELISA plates which had been coated previously with an antibody to the Flag tag (Sigma, Saint Louis, USA, ANTI-FLAG HS, M2 coated 96-well Plates (clear), Product Number P 2983) were utilized in Example 4. Each well of the ELISA plate was charged with 100 μl of a phosphate buffer solution containing recombinant wild-type GPIbα-(3×Flag)-(6×His) fusion protein (SEQ ID NO: 25) (1:10 diluted supernatant of the culture medium after cell sedimentation) or isolated recombinant GPIbα-(aa1-285, G233V/M239V)-(3×Flag)-(6×His) fusion protein (SEQ ID NO: 24) (see Example 3) in a concentration of 2.4 μg/ml, followed by incubation at 2-8° C. overnight. After washing 4 times with phosphate buffer (+0.01% TWEEN® 20), 50 μl of a dilution of HAEMATE® (CSL Behring, Marburg, Germany) in phosphate buffer+0.1% bovine serum albumin and 50 μl of a ristocetin dilution in phosphate buffer+0.1% bovine serum albumin were added. This was followed by incubation at room temperature for 1 hour. After washing 4 times as above, 100 μl of a horse radish peroxidase-coupled anti-VWF antibody (rabbit anti-human VWF/HRP, DAKO, Ref. P0226) were added, followed by incubation at room temperature for 1 hour and subsequent washing as above. Thereafter, 100 μl of tetramethylbenzidine solution as substrate (TMB substrate, Dade Behring Marburg GmbH, Marburg, Germany, Ref. 450684) were added, followed by incubation at room temperature for 4 minutes. The reaction was stopped by adding 100 μl of 0.5N sulfuric acid. Extinction at 504 nm was then measured in a spectrophotometer.
In the absence of ristocetin, strong and concentration-dependent binding of VWF occurred upon usage of the mutant GPIbα prepared according to Examples 1-3. Addition of ristocetin slightly increased said binding, which can be attributed probably to unspecific binding that takes place even without GPIbα. In contrast, similarly strong VWF binding was achieved with wild-type GPIbα only by adding 1.2 mg/ml ristocetin. Without the addition of ristocetin, there was no binding at all upon usage of wild-type GPIbα (FIG. 1).
Example 5
Method for Determining VWF Activity in a GPIbα Latex Particle Agglutination Assay without Ristocetin 
Three reagents were prepared: (1) Reagent 1 comprised the monoclonal anti-GPIbα antibody VM16d (Sanbio B. V., Uden, Netherlands: product number MON 1146) was bound to the surface of latex particles, and the latter were resuspended in a buffer for long term storage (0.6 g/l TRIS, 1.1% leucine, 12.5% sucrose, 0.05% HSA, 6.25 mg/l gentamycin and 0.625 mg/l amphotericin, pH 8.25, latex concentration 0.22%); (2) Reagent 2 comprised VWF-deficient plasma; and (3) for Reagent 3, the following components were mixed resulting in a total volume of 30 ml: (i) 2.5 ml of a 7.5% strength solution of polyvinylpyrrolidone (PVP) (Fluka, Germany) in phosphate buffer (pH 7.1), (ii) 12.9 ml (0.625 mg/ml) Heterophilic Blocking Reagent 1 (Scantibodies Laboratory Inc, Santee, Calif. 92071, USA) in Tris buffer (pH 7.1), (iii) 3.4 ml of a solution containing 21 g/l TWEEN® 20 (Sigma, St. Louis, Mo., USA) in phosphate buffer (pH 7.2), (iv) 0.086 ml of a solution of 5.59 mg/ml recombinant GPIbα-(G233V/M239V)-(3×Flag)-(6×His) (see Example 3) in phosphate buffer (pH 7.1), and (v) 11.2 ml of phosphate buffer (pH 7.1).
The assay was carried out on the BCS® automatic coagulation analyzer (Behring Coagulation System, Dade Behring Marburg GmbH, Marburg, Germany). 15 μl of a human citrate plasma sample, 30 μl of Reagent 2 and 70 μl of Reagent 3 were mixed. The reaction was started by adding 40 μl of Reagent 1 with mixing. The extinction of the reaction mixture was measured continuously at 575 nm. The rate of latex particle agglutination was a function of the activity of VWF in the sample.
The VWF activity was calculated by generating a calibration curve using standard human plasma (Dade Behring Marburg GmbH, Marburg, Germany). The VWF target value used was the declared ristocetin cofactor value for the BCS® system. VWF activities of up to 200% were generated by increasing the volume of standard human plasma and simultaneously reducing the corresponding amount of Reagent 2. Lower VWF activities were generated by diluting standard human plasma with Reagent 2. FIG. 2 depicts a typical calibration curve. The VWF activity of a sample can be read from the calibration curve with the aid of the reaction rate measured.
To measure particularly low VWF activities in a sample, 45 μl of sample rather than 15 or 30 μl of sample (see above) were used in an assay mix. For calibration, standard human plasma was diluted with Reagent 2 in such a way that low VWF activities of up to 3% were generated (see FIG. 3). This approach enables extremely low VWF activities to be determined accurately in the assay, which is particularly important for calculating VWF activity/antigen ratios in order to classify subtypes of von Willebrand syndrome. VWF-dependent agglutination can additionally be amplified by increasing the PVP concentration to 0.35% in the reaction mix and the sample volume to 60 μl thus enabling even 1% VWF and 0% VWF to be differentiated. As a result, a type 3 von Willebrand disease (0% VWF) can very reliably be distinguished from extremely large deficiencies of other types (e.g. 1-5%).
Example 6
Method for Determining the VWF-Cleaving Activity of ADAMTS-13 Protease Using Urea-Pretreated, High Molecular Weight, Multimeric VWF (P1648S) (SEQ ID NO: 9) as VWF Substrate
First, a reaction buffer containing 1 mmol/l Pefabloc SC (Roche Diagnostics GmbH, Mannheim, Germany), 12.5 mmol/l BaCl2, and 5 mmol/l TRIS, pH 8.0 was prepared. The substrate buffer was prepared and incubated at room temperature for exactly 5 minutes: 8.3 U/ml (830%) high molecular weight, multimeric, recombinantly produced VWF-P1648S, 4.6 mol/l urea, and 4.6 mmol/l TRIS, pH 8.0. Next, 15 μl of human plasma sample were admixed with 300 μl of reaction buffer and then mixed with 150 μl of substrate buffer. The assay mix was then incubated at 37° C. for 4 to 8 hours. Measurement of VWF activity was measured using this reaction mixture as sample as described in Example 5.
Example 7
Method for Determining the VWF-Cleaving Activity of ADAMTS-13 Protease Using Urea-Pretreated, Recombinant High Molecular Weight, Multimeric VWF (P1648S) (SEQ ID NO: 9) as VWF Substrate
In this example, 6 μl of human plasma sample (diluted 1:6 with reaction buffer, see Example 6) were mixed with 20 μl of reaction buffer and incubated at 37° C. for 5 minutes, to activate ADAMTS-13 protease. Next, 12 μl of substrate buffer (5 U/ml (500%) recombinant high molecular weight, multimeric VWF-P1648S, 5 mol/l urea, 5 mmol/l Tris-HCl, pH 8.0) was added, and the assay mix was incubated at 37° C. for 20 minutes. VWF activity was measured using this reaction mixture as sample as described in Example 5.
Example 8
Method for Determining the VWF-Cleaving Activity of ADAMTS-13 Protease Using High Molecular Weight, Multimeric VWF (P1648S) as VWF Substrate and Calculating the Difference with/without ADAMTS-13 Degradation
Assay 1:
10 μl of a human plasma sample were mixed with 20 μl of ADAMTS-13 activating buffer (18.75 mmol/l BaCl2, pH 8.0, 5 mmol/l Tris-HCl, and 1.5 mmol/l Pefabloc SC (Roche Diagnostics GmbH, Mannheim, Germany)) and incubated at 37° C. for 5 minutes. 20 μl containing 1.25 U/ml (125%) recombinant VWF substrate (P1648S) in 5 mmol/l Tris-HCl, pH 8.0 was added and the assay mix was incubated at 37° C. for 20 minutes. VWF activity was then measured using this reaction mixture as sample as described in Example 5.
Assay 2:
Another 10 μl of the same human plasma sample were treated as in assay 1, but with the difference that the incubation step of 20 minutes at 37° C. was not carried out, but the VWF activity was measured using this reaction mixture as sample as in Example 5 immediately after addition of the VWF substrate (P1648S). The difference in VWF activity of assay 1 and assay 2 provided the amount of VWF activity degraded and therefore the activity of ADAMTS-13. The activity of ADAMTS-13 in the plasma sample was determined therefrom on the basis of a calibration.
Thus, in accordance with the presently disclosed and claimed inventive concept(s), there have been provided compositions and methods that fully satisfy the objectives and advantages set forth hereinabove. Although the presently disclosed and claimed inventive concept(s) has been described in conjunction with the specific drawings, experimentation, results and language set forth hereinabove, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the presently disclosed and claimed inventive concept(s).

## Claims

1. A method for determining von Willebrand factor (VWF) activity in a sample from a human or animal, comprising the steps of:
mixing the sample with an isolated GPIbα protein associated with a particulate solid phase, wherein the solid phase comprises a material selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, crosslinked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate, nylon, latex, ceramics, glass, a precious metal, and magnetite, and wherein the amino acid sequence of the isolated GPIbα protein comprises the wild-type sequence of human GPIbα protein (SEQ ID NO: 1), having at least the amino acid residues 1-268 of SEQ ID NO: 1, except with substitutions found in positions 233 and 239;
wherein the amino acid substitutions in each of positions 233 and 239 of the GPIbα protein comprise a valine or a serine residue;
wherein the sample is mixed absent added ristocetin or botrocetin; and
determining the VWF activity in the sample by detecting agglutination of the particulate solid phase in the mixed sample.

2. The method of claim 1, wherein no ristocetin or botrocetin equivalent substance is added to the assay mix.

3. The method of claim 1, wherein the GPIbα protein comprises SEQ ID NO: 3.

4. The method of claim 1, wherein the GPIbα protein comprises SEQ ID NO: 4.

5. The method of claim 1, wherein the GPIbα protein comprises SEQ ID NO: 5.

6. The method of claim 1, wherein wherein the GPIbα protein comprises SEQ ID NO: 6.

7. The method of claim 1, wherein the GPIbα protein is a recombinant or synthetic protein.

8. The method of claim 1, wherein the GPIbα protein is fused at the N terminus to a compound selected from the group consisting of a homologous, human GPIbα signal sequence (SEQ ID NO: 7) and a heterologous signal sequence.

9. The method of claim 1, wherein the GPIbα protein is fused at the C terminus to at least one affinity tag.

10. The method of claim 9, wherein the at least one affinity tag is selected from the group consisting of a His tag, a Flag tag, an Arg tag, a c-Myc tag and a Strep tag.

11. The method of claim 10, wherein the GPIbα protein is associated with the particulate solid phase via an antibody.

12. The method of claim 11, wherein the GPIbα protein is associated with the particulate solid phase via an anti-GPIbα antibody.

13. The method of claim 11, wherein the GPIbα protein is associated with the particulate solid phase via an anti-affinity tag antibody.

14. The method of claim 1, wherein the VWF activity is determined by detecting GPIbα-mediated agglutination of the particulate solid phase.

15. The method of claim 14, wherein the GPIbα protein is associated with the particulate solid phase via a monoclonal anti-GPIbα antibody selected from the group consisting of VM16d and SZ 2.

16. The method of claim 14, wherein the GPIbα protein is fused at the C terminus to at least one Flag tag and is associated with the particulate solid phase via an anti-Flag antibody.

17. The method of claim 14, wherein the assay mix further includes a detergent.

18. The method of claim 17, wherein the detergent is selected from the group consisting of polysorbate 20, Polyethylene glycol dodecyl ether, octoxynol-9 and sodium dodecyl sulfate.

19. The method of claim 17, wherein the assay mix contains polysorbate 20 in a concentration of from 0.02 g/l to 20 g/l.

20. The method of claim 1, wherein the sample is a biological fluid of human or animal origin.

21. The method of claim 1, wherein the sample is a reaction mixture comprising a biological fluid to which isolated VWF has been added as a substrate.

22. A test kit for determining von Willebrand factor (VWF) activity in a sample from a human or animal, the test kit comprising:
a reagent containing an isolated GPIbα protein associated with a particulate solid phase comprising a material selected from the group consisting of cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, crosslinked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate, nylon, latex, ceramics, glass, a precious metal, and magnetite, wherein the amino acid sequence of the isolated GPIbα protein comprises the wild-type sequence of human GPIbα protein (SEQ ID NO: 1), including at least the amino acid residues 1-268 of SEQ ID NO: 1, except that amino acid substitutions are found in positions 233 and 239;
wherein the amino acid substitutions in each of positions 233 and 239 of the GPIbα protein comprise a valine or a serine residue.

23. The test kit of claim 22, wherein the reagent contains neither ristocetin, botrocetin nor ristocetin or botrocetin equivalent substances.

24. The test kit of claim 22, wherein the GPIbα protein comprises SEQ ID NO: 3.

25. The test kit of claim 22, wherein the GPIbα protein comprises SEQ ID NO: 4.

26. The test kit of claim 22, wherein the GPIbα protein comprises SEQ ID NO: 5.

27. The test kit of claim 22, wherein the GPIbα protein comprises SEQ ID NO: 6.