Csl Behring Gmbh

Aventis Behring Gmbh

Zlb Behring Gmbh

Delta Biotechnology Limited

Annette Feussner

Juergen Roemisch

Hans-Arnold Stoehr

Wiegand Lang

Gudrun Muth-Naumann

Thomas Weimer

Claudia Nerlich

Margret Becker

J&uuml;rgen R&ouml;misch

Bernd Knoblauch

Jürgen Römisch

Hans-Arnold Stöhr

Stefan Kiechl

Johann Willeit

Christian Josef Wiedermann

Hans-Arnold St&ouml;hr

Joachim R&ouml;der

Volker Doersam

Stefan Schulte

Hubert Metzner

Klaus T Preissner

Uwe Liebing

Michael Moses

Uwe Kalina

Joerg Lemmer

Volker Gawantka

Christian Kannemeier

Jörg Weisse

Harald Stauss

Fumie Nakazawa

Christian Kannemaier

Jörg Lemmer

The present application relates to procedures for the determination of the activity of the protease which activates factor VII, also known as factor VII activating protease or FSAP. The application also relates to a method of detecting whether an individual has increased or lowered activity in the protease which activates factor VII compared to at least one standard sample, wherein the increased or lowered activity indicates an increased risk for disease or cardiovascular complications.

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

This application describes methods of treatment involving a protease for activating the blood clotting factor VII, which (a) activates blood clotting factor VII, (b) is inhibited by the presence of aprotinin, (c) is increased in its activity by the presence of at least one of the following: calcium ions, heparin, or heparin related substances, and (d) in SDS-PAGE, on subsequent staining in the non-reduced state, has one or more bands in the molecular weight range from 50 to 75 kDa, and in the reduced state has a band at 40 to 55 kDa and one or more bands in the molecular weight range from 10 to 35 kDa, and a band, which corresponds to a proenzyme, in the molecular weight range from 60 to 65 kDa.

Methods of treatment involving the protease for activating clotting factor VII

Mutants of the DNA sequence coding for the protease (FSAP) which activates blood clotting factor VII and single-chain plasminogen activators, the mutants comprising a G/C base exchange at nucleotide position 1177 and/or a G/A base exchange at nucleotide position 1601, are described. The corresponding protease has a Glu/Gln exchange at amino acid position 393 and/or a Gly/Glu exchange at amino acid position 534. Diagnostic methods which are used for detecting FSAP in body fluids or tissue cells and also for identifying patients with genetic heterozygous or homozygous FSAP expression are also described. In addition, antibodies against FSAP and its mutants are disclosed and diagnostic methods which can be used to detect antibodies against FSAP and its mutants are specified.

Mutants of the factor VII-activating protease and detection methods using specific antibodies

A monoclonal antibody which inhibits the blood clotting factor VII-activating protease and its proenzyme and a blood clotting factor VII-activating protease, stabilized by the addition of said monoclonal antibody, and its proenzyme are described. A suitable monoclonal antibody is produced by hybridoma cell line DSM ACC 2533. The application of the inhibitory, monoclonal antibody in the stabilization of blood clotting preparations and in preparations for reducing the coagulability of the blood is disclosed.

Inhibitory monoclonal antibody against blood clotting factor VII-activating protease

An arterial thrombosis risk factor comprising one or more of the identified mutants of coagulation factor VII activating protease (FSAP) is described. In addition, diagnostic determination methods for detecting these mutants which are identified as risk factors are described.

Marburg I mutant of factor VII activating protease (FSAP) as risk factor for arterial thrombosis and methods of detecting FSAP and FSAP mutations

The present invention relates to a process for purifying fibrinogen, which comprises one or more process steps in which one or more contaminating proteins are depleted by negative chromatography and/or negative adsorption using cation exchanger, hydrophobic gel and/or dye gel. In addition, the invention relates to the fibrinogen which is obtained by the process of the invention and which is notable for improved stability, and to the production and use of pharmaceutical preparations comprising this fibrinogen.

Fibrinogen purification

A stabilized liquid preparation of a protease which activates blood coagulation Factor VII or of its proenzyme is herein described. The preparation also contains one or more compounds selected from the group consisting of ornithine, diaminopimelic acid, agmatine, creatine, guanidinoacetic acid, acetylornithine, citrulline, arginoinosuccinic acid, tranexamic acid and epsilon-aminocaproic acid or their salts and has a pH of 2&#x2013;8.

Stabilized liquid preparation of the protease which activates blood coagulation factor VII, or of its proenzyme

A novel arterial thrombosis risk factor comprising one or more of the identified mutants of coagulation factor VII-activating protease (FSAP) is described. In addition, diagnostic determination methods for detecting these mutants which are identified as risk factors are described.

Marburg I mutant of factor VII activating protease (FSAP) as risk factor for arterial thrombosis

A protease for activating the blood clotting factor VII is described, wherein the protease is inhibited by the presence of aprotinin and is increased in its activity by calcium ions and/or heparin or heparin-related substances, and wherein in SDS-PAGE, on subsequent staining in the non-reduced state, the protease has one or more brands in the molecular weight range from 50 to 75 kDa, and in the reduced state, the protease has a band at 40 to 55 kDa and one or more bands in the molecular weight range from 10 to 35 kDa. The proenzyme of this protease is also characterized. Further, a process for obtaining this protease and its use in hemorrhage prophylaxis or hemostasis is described. Moreover, a test system for the qualitative and quantitative detection of a protease which activates the blood clotting factor VII is described.

Protease for activating clotting factor VII

A process for the partial or complete separation of glycosylated and nonglycosylated proteins is described, in which: a) a triazine dye immobilized on a matrix is incubated with a mixture of glycosylated and nonglycosylated proteins, b) the matrix is then washed to remove the unbound proteins, and c) the proteins are eluted by means of a stepwise or continuous increase in the ionic strength or in the pH, nonglycosylated proteins and proteins having an increasing degree of glycosylation being collected separately from one another in the eluate fractions obtained. By means of the use of this process it is possible, for example, to prepare a human albumin which is free of glycoside bonds and is expressed in yeast cells.

Process for the separation of glycosylated and nonglycosylated proteins

A monoclonal antibody has been developed which only specifically binds activated factor VII, and not factor VII, and which does not bind to an activated factor VII which is complexed with antithrombin III. This monoclonal antibody is isolated from the hybridoma cell line DSM ACC 2332. It can be used for qualitatively and quantitatively detecting factor VIIa in body fluids, blood coagulation preparations or the intermediate stages in the production of these preparations, on cell surfaces or in tissues, and can also be used as a humanized monoclonal antibody in therapeutic preparations.

Monoclonal antibody which is specific for activated coagulation factor VII, and its use

A process for the preparation of a pure form of protein, wherein the protein is protease activating blood clotting factor VII, the proenzyme for the protease, or a mixture of both the-protease and the proenzyme, is described. The protein is obtained from biological fluids or through genetic engineering and is isolated by anion- and/or cation-exchange chromatography at a pH below the isoelectric point of the protein to be isolated, or by a combination of anion- or cation-exchange chromatography with a chromatography process and/or fractional precipitation at pH between 2.5 and 9.0.

Process for the preparation in pure form of the protease activating blood clotting factor VII, its proenzyme or a mixture of both proteins by means of ion-exchange chromatography

A process for the preparation in pure form of the protease activating blood clotting factor VII and/or its proenzyme by the use of a chromatography separation processes and/or fractional precipitation is described. The process used may include adsorption on calcium phosphate/hydroxyapatite, a hydrophobic matrix, a chelate matrix, a matrix on which heparin or a substance related to heparin, such as heparin sulfate or dextran sulfate, is immobilized, or a matrix that is coated with an immobilized monoclonal or polyclonal antibody directed against the protein to be isolated, or F(ab) or F(ab) fragments of antibodies directed against the protein to be isolated. A pharmaceutical preparation and a reagent are described which contain the said protease and/or its proenzyme.

Process for the preparation in pure form of the protease activating blood clotting VII, its proenzyme or a mixture of both proteins by means of affinity chromatography

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Marburg, Germany

Annette Feussner