Nematode-extracted serine protease inhibitors and anticoagulant proteins

Abstract

Proteins which have activity as anticoagulants and/or serine protease inhibitors and have at least one NAP domain and are described. Certain of these proteins have factor Xa inhibitory activity and others have activity as inhibitors of factor VIIa/TF. These proteins can be isolated from natural sources as nematodes, chemically synthesized or made by recombinant methods using various DNA, expression systems.

1 . A method for treating abnormal thrombosis in a mammal in need thereof, the method comprising administering to said mammal a therapeutically effective amount of a protein having anticoagulant activity and having one or more Nematode Anticoagulant Protein (NAP) domains, wherein each NAP domain includes the sequence: Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7-Cys-A8-Cys-A9-Cys-A10 (FORMULA III), wherein
 (a) A1 is an amino acid sequence of 7 to 8 amino acid residues;   (b) A2 is an amino acid sequence;   (c) A3 is an amino acid sequence of 3 amino acid residues and has the sequence Asp-A3 a -A3 b  wherein A3 a  and A3 b  are independently selected amino acid residues;   (d) A4 is an amino acid sequence having a net anionic charge;   (e) A5 is an amino acid sequence of 4 amino acid residues and has the sequence A5 a -A5 b -A5 c -A5 d  (SEQ ID NO:85), wherein A5 a  through A5 d  are independently selected amino acid residues;   (f) A6 is an amino acid sequence;   (g) A7 is an amino acid residue selected from the group consisting of Val and Ile;   (h) A8 is an amino acid sequence of 11 to 12 amino acid residues;   (i) A9 is amino acid sequence of 5 to 7 amino acid residues;   (j) A10 is an amino acid sequence; and   each of A2, A4, A6 and A10 has an independently selected number of independently selected amino acid residues and each sequence is selected such that each NAP domain has in total less than 120 amino acid residues.   
     
     
         2 . The method of  claim 1 , wherein A3 has the sequence Asp-Lys-Lys; A5 a  is Leu, A5 c  is Arg, and A5 b  and A5 d  are independently selected amino acid residues (SEQ ID NO: 357); A7 is Val; and A8 comprises the amino acid sequence A8 a -A8 b -Gly-Phe-Tyr-Arg-Asn (SEQ ID NO: 79), wherein at least one of A8 a  and A8 b  is Glu or Asp. 
     
     
         3 . The method of  claim 1 , wherein the abnormal thrombosis occurs in the arterial vasculature. 
     
     
         4 . The method of  claim 2 , wherein the abnormal thrombosis occurs in the arterial vasculature. 
     
     
         5 . The method of  claim 1  wherein the abnormal thrombosis occurs in the venous vasculature. 
     
     
         6 . The method of  claim 2  wherein the abnormal thrombosis occurs in the venous vasculature. 
     
     
         7 . The method of  claim 5 , wherein the abnormal thrombosis occurs during surgery of the mammal in the lower extremities or abdominal area. 
     
     
         8 . The method of  claim 6 , wherein the abnormal thrombosis occurs during surgery of the mammal in the lower extremities or abdominal area. 
     
     
         9 . The method of  claim 1 , wherein the abnormal thrombosis is caused by rupture of an atherosclerotic plaque. 
     
     
         10 . The method of  claim 2 , wherein the abnormal thrombosis is caused by rupture of an atherosclerotic plaque. 
     
     
         11 . The method of  claim 1 , wherein the abnormal thrombosis is occlusive coronary thrombus. 
     
     
         12 . The method of  claim 2 , wherein the abnormal thrombosis is occlusive coronary thrombus. 
     
     
         13 . The method of  claim 11 , wherein the occlusive coronary thrombosis is caused by thrombolytic therapy. 
     
     
         14 . The method of  claim 12 , wherein the occlusive coronary thrombosis is caused by thrombolytic therapy. 
     
     
         15 . The method of  claim 11  wherein the occlusive coronary thrombosis is caused by percutaneous transluminal coronary angioplasty. 
     
     
         16 . The method of  claim 12  wherein the occlusive coronary thrombosis is caused by percutaneous transluminal coronary angioplasty. 
     
     
         17 . The method of  claim 1  wherein the abnormal thrombosis is disseminated intravascular thrombosis. 
     
     
         18 . The method of  claim 2  wherein the abnormal thrombosis is disseminated intravascular thrombosis. 
     
     
         19 . A method for inhibiting blood clot formation in a blood sample from a mammal, the method comprising mixing said blood sample in vitro with an effective amount of a protein having anticoagulant activity and having one or more Nematode Anticoagulant Protein (NAP) domains, wherein each NAP domain includes the sequence: 
       
         
           
                 
               
                   Cys-A1-Cys-A2-Cys-A3-Cys-A4-Cys-A5-Cys-A6-Cys-A7- 
                 
                     
                 
                   Cys-A8-Cys-A9-Cys-A10 (FORMULA III), 
                 
             
                
                
                
               
            
           
         
       
       wherein
 (a) A1 is an amino acid sequence of 7 to 8 amino acid residues; 
 (b) A2 is an amino acid sequence; 
 (c) A3 is an amino acid sequence of 3 amino acid residues and has the sequence Asp-A3 a -A3 b  wherein A3 a  and A3 b  are independently selected amino acid residues; 
 (d) A4 is an amino acid sequence having a net anionic charge; 
 (e) A5 is an amino acid sequence of 4 amino acid residues and has the sequence A5 a -A5 b -A5 c -A5 d  (SEQ ID NO:85), wherein A5 a  through A5 d  are independently selected amino acid residues; 
 (f) A6 is an amino acid sequence; 
 (g) A7 is an amino acid residue selected from the group consisting of Val and Ile; 
 (h) A8 is an amino acid sequence of 11 to 12 amino acid residues; 
 (i) A9 is amino acid sequence of 5 to 7 amino acid residues; 
 (j) A10 is an amino acid sequence; and 
 each of A2, A4, A6 and A 10 has an independently selected number of independently selected amino acid residues and each sequence is selected such that each NAP domain has in total less than 120 amino acid residues. 
 
     
     
         20 . The method of  claim 19 , wherein A3 has the sequence Asp-Lys-Lys; A5 a  is Leu, A5 c  is Arg, and A5 b  and A5 d  are independently selected amino acid residues (SEQ ID NO: 357); A7 is Val; and A8 comprises the amino acid sequence A8 a -A8 b -Gly-Phe-Tyr-Arg-Asn (SEQ ID NO: 79), wherein at least one of A8 a  and A8 b  is Glu or Asp.