Protease for activating clotting factor VII

A method of assaying prothrombin time comprising a protease or a mixture of the proenzyme of the protease and appropriate proenzyme activators, wherein the protease or the mixture is used instead of tissue factor or thromboplastin for assaying the prothrombin time;

• 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.

A pharmaceutical preparation, comprising a protease or a mixture of the protease and its proenzyme, in an amount adequate for dissolution of fibrin-containing thrombi;

• 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.

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.

A method for the detection of a protease, comprising:

• contacting a sample from an individual comprising the protease with a solution containing at least one plasminogen activator and measuring the activity of the at least one plasminogen activator, wherein the activity of the at least one plasminogen activator is dependent on the protease, and
• determining the antigen level of the protease in the sample;
• 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.

A method for the detection of a protease, comprising:

• contacting a sample from an individual comprising the protease with a solution containing factor VIII/VIIIa or factor V/Va and quantifying the activity of factor VIII/VIIIa or factor V/Va remaining after contact with the protease, wherein the remaining activity of factor VIII/VIIIa or factor V/Va is dependent on the protease, and
• determining the antigen level of the protease in the sample;
• 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.

A method for the detection of a protease, comprising:

• contacting a sample of an individual comprising the protease with blood, platelet-rich plasma, or plasma, quantifying a first clotting time, and measuring the difference between said first clotting time and a corresponding second clotting time obtained by contacting a solution, wherein the protease is inhibited or absent in the solution, with blood, platelet-rich plasma, or plasma, and
• determining the antigen level of the protease in the sample;
• 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.

A method for the detection of a protease, comprising:

• contacting a sample from an individual comprising the protease with an FVII-containing solution and measuring the resulting activity of FVII, wherein the resulting FVII activity is dependent on the protease, and
• determining the antigen level of the protease in the sample;
• 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.