Antithrombotic thrombin variants

A physiologically acceptable composition comprising:

• (a) a variant thrombin, wherein the variant prothrombin has the amino acid substitution W215A and is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:
• 1;
• and
• (b) at least one pharmaceutically acceptable carrier.

A physiologically acceptable composition comprising:

• (a) a variant thrombin, wherein the variant prothrombin has the amino acid substitutions W215A and E217A, and is at least 80% identical to the amino acid sequence as set forth in SEQ ID NO:
• 3;
• and
• (b) at least one pharmaceutically acceptable carrier.

A method of inhibiting the formation of a thrombus, comprising the steps of:

• (a) delivering to the blood of an animal or human a physiologically acceptable composition comprising an effective amount of a variant thrombin with substantially reduced procoagulant activity, wherein the variant thrombin has the amino acid substitution W215A and comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:
• 1;
• and
• (b) allowing the variant thrombin to activate protein C, thereby inhibiting thrombus formation.

A method of inhibiting the formation of a thrombus, comprising the steps of:

• (a) delivering to the blood of an animal or human a physiologically acceptable composition comprising an effective amount of a variant thrombin with substantially reduced procoagulant activity, wherein the variant thrombin has the amino acid substitutions W215A and comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:
• 1;
• and
• (b) allowing the variant thrombin to activate protein C, thereby inhibiting thrombus formation.

A method of inhibiting the formation of a thrombus, comprising the steps of:

• (a) delivering to the blood of an animal or human a physiologically acceptable composition comprising an effective amount of a variant thrombin with substantially reduced procoagulant activity, wherein the variant thrombin has the amino acid substitutions W215A and E217A and comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:
• 3;
• and
• (b) allowing the variant thrombin to activate protein C, thereby inhibiting thrombus formation.

A method to determine the endogenous antithrombotic potential of the protein C system of an animal or patient, comprising the steps of:

• (a) administering to an animal or human an effective dose of a variant thrombin having a substantially reduced fibrinogen cleavage activity and capable of activating protein C;
• (b) obtaining from the animal or human a blood sample, and measuring the coagulation rate or APC amidolytic activity thereof;
• and
• (c) comparing the coagulation rate or APC amidolytic activity to the coagulation rate or APC amidolytic activity of a standard, thereby indicating the endogenous antithrombotic potential of the protein C system of the animal or human.

A method for producing activated protein C, comprising the steps of:

• (a) obtaining an sample of protein C;
• (b) incubating the sample of protein C with a variant thrombin with reduced procoagulant activity, wherein the variant thrombin has the amino acid substitution W215A and comprises an amino acid sequence at least 80% identical to the amino acid sequence set forth in SEQ ID NO:
• 1;
• and
• (c) cleaving the isolated protein C by the variant thrombin, thereby yielding activated protein C.

A method of determining the protein C activity in an individual, comprising the steps of:

• (a) obtaining a blood sample from an individual;
• (b) contacting the blood sample with a variant thrombin substantially free of fibrinogen cleavage activity and capable of protein C activation;
• and
• (c) measuring the level of activated protein C in the blood sample.