Protease for activating clotting factor VII

Abstract

A protease for activating the blood clotting factor VII is described, which (a) is inhibited by the presence of aprotinin, (b) is increased in its activity by calcium ions and/or heparin or heparin-related substances, and (c) in SDS-PAGE, on subsequent staining in the non-reduced state, has one or more brands in the molecular weight range from 50 to 75 kDa and kDa 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. 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.

1 .- 27 . (canceled)  
     
     
         28 . A reagent for the immunological detection of a protease or its proenzyme, wherein the protease 
 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,    comprises one or more bands in the molecular weight range from 50 to 75 kDa; and in SDS-PAGE, on subsequent staining in the reduced state, comprises a band at 40 to 55 kDa, 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,    and wherein the reagent comprises a polyclonal or monoclonal antibody against the protease or its proenzyme.    
     
     
         29 . (canceled)  
     
     
         30 . An assay system comprising the protease of  claim 28  or a mixture of its proenzyme and appropriate proenzyme activators, wherein the protease or the mixture substitute for tissue factor or thromboplastin in assaying prothrombin time.  
     
     
         31 . A pharmaceutical preparation, comprising the protease of  claim 28  or a mixture of the protease and its proenzyme, in an amount adequate for dissolution of fibrin-containing thrombi.  
     
     
         32 . The pharmaceutical preparation as claimed in  claim 31 , optionally further comprising single-chain or two-chain plasminogen activators.  
     
     
         33 . The pharmaceutical preparation as claimed in  claim 31 , optionally further comprising anticoagulants.  
     
     
         34 . The pharmaceutical preparation as claimed in  claim 31 , optionally further comprising soluble calcium salts.  
     
     
         35 . The pharmaceutical preparation as claimed in  claim 31 , optionally further comprising heparin or heparin-like substances.  
     
     
         36 . A pharmaceutical preparation, comprising an inhibitor of the protease of  claim 28 , wherein the protease inhibitor is aprotinin, C1-inhibitor, α 2 -antiplasmin, inter-α-trypsin-inhibitor, or ATIII/heparin.  
     
     
         37 . A process for preparing a pharmaceutical preparation comprising a protease, wherein the protease 
 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, comprises one or more bands in the molecular weight range from 50 to 75 kDa; and in SDS-PAGE, on subsequent staining in the reduced state, comprises a band at 40 to 55 kDa, 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 between 60 and 65 kDa,    and wherein the process comprises 
 1) preparing the pharmaceutical preparation in a pH range from 3.5 to 8.0;  
 2) adding one or more amino acids in an amount greater than 0.01 mol/L;  
   3 ) optionally, adding a sugar or a combination of different sugars in an amount greater than 0.05 g/ml; and  
   4 ) optionally, adding one or more substances which are able to complex calcium ions.  
   
     
     
         38 . (canceled)  
     
     
         39 . (canceled)  
     
     
         40 . A pharmaceutical preparation obtainable by the process as claimed in  claim 37 .  
     
     
         41 . A method for promoting wound healing and hemostasis in a patient comprising administering to the patient the protease or proenzyme of  claim 28 , optionally together with proenzyme activators, wherein the protease or proenzyme is prepared from blood plasma or prothrombin complex (PPSB) concentrates or expressed recombinantly or transgenically.  
     
     
         42 . A method for rapid wound closure comprising 
 a) adding to at least one of a fibrin adhesive, fleece, or other release system the protease or proenzyme of  claim 28 , optionally together with proenzyme activators, wherein the protease or proenzyme is prepared from blood plasma or prothrombin complex (PPSB) concentrates or expressed recombinantly or transgenically: and    b) applying said fibrin adhesive, fleece, or other release system to a wound.    
     
     
         43 . A method for treating inborn or acquired deficiencies in a protease or proenzyme of a patient, comprising administering to the patient the protease or proenzyme of  claim 28 , optionally together with proenzyme activators, wherein the protease or proenzyme is prepared from blood plasma or prothrombin complex (PPSB) concentrates or expressed recombinantly or transgenically.