Activated protein c variants with normal cytoprotective activity but reduced anticoagulant activity

Abstract

Variants (mutants) of recombinant activated protein C (APC) or recombinant protein C (prodrug, capable of being converted to APC) that have substantial reductions in anticoagulant activity but that retain normal levels of anti-apoptotic activity are provided. Two examples of such recombinant APC mutants are KKK191-193AAA-APC and RR229/230AA-APC. APC variants and prodrugs of the invention have the desirable property of being cytoprotective (anti-apoptotic effects), while having significantly reduced risk of bleeding. The invention also provides a method of using the APC variants or prodrugs of the invention to treat subjects who will benefit from APC's cytoprotective activities that are independent of APC's anticoagulant activity. These subjects include patients at risk of damage to blood vessels or tissue in various organs caused, at least in part, by apopotosis. At risk patients include, for example, those suffering (severe) sepsis, ischemia/reperfusion injury, ischemic stroke, acute myocardial infarction, acute or chronic neurodegenerative diseases, or those undergoing organ transplantation or chemotherapy, among other conditions. Methods of screening for variants of recombinant protein C or APC that are useful in accordance with the invention are also provided.

1 . A method of selecting potentially therapeutic cytoprotective variants of recombinant activated protein C, said activated protein C having a protease domain, comprising: mutating a recombinant activated protein C at a surface loop of said protease domain to make an activated protein C variant; measuring the anticoagulant activity of said activated protein C variant; measuring the anti-apoptotic activity of said activated protein C variant; calculating the ratio of anti-apoptotic activity to anti-coagulant activity; and identifying the activated protein C variant as potentially therapeutic if said ratio is greater than 1.0. 
   
   
       2 . The method of  claim 1 , wherein the ratio is greater than about 2. 
   
   
       3 . The method of  claim 1 , wherein the ratio is greater than about 4. 
   
   
       4 . The method of  claim 1 , wherein the ratio is greater than about 8. 
   
   
       5 . The method of  claim 1 , wherein said surface loop is selected from the group consisting of loop 37, calcium loop, and autolysis loop. 
   
   
       6 . A method of selecting potentially therapeutic cytoprotective variants of recombinant protein C, said protein C having a protease domain, comprising: mutating a recombinant protein C at a surface loop of said protease domain to make a protein C variant; converting said protein C variant to an activated protein C variant; measuring the anticoagulant activity of said activated protein C variant; measuring the anti-apoptotic activity of said activated protein C variant; calculating the ratio of anti-apoptotic activity to anti-coagulant activity; and identifying the protein C variant as potentially therapeutic if said ratio is greater than 1.0. 
   
   
       7 . The method of  claim 6 , wherein the ratio is greater than about 2. 
   
   
       8 . The method of  claim 6 , wherein the ratio is greater than about 4. 
   
   
       9 . The method of  claim 6 , wherein the ratio is greater than about 8. 
   
   
       10 . The method of  claim 6 , wherein said surface loop is selected from the group consisting of loop 37, calcium loop, and autolysis loop. 
   
   
       11 . A method of selecting potentially therapeutic cytoprotective variants of recombinant activated protein C, comprising:
 (a) providing a library of candidate agents which are protein C variants, wherein each of said protein C variants has a protease domain, wherein each of said protein C variants comprises at least one mutation at a residue in a surface loop of said protease domain;   (b) converting each of said protein C variants to activated protein C variants;   (c) determining anti-apoptotic activity of said activated protein C variants in one or more stressed or injured cells by exposing said cells to an apoptotic-inducing concentration of staurosporine in the presence of an amount of a candidate agent;   (d) determining anticoagulant activity of said candidate agents that are assayed in (c) by performing a dilute prothrombin time clotting assay;   (e) calculating the ratio of the anti-apoptotic activity determined in (c) to the anticoagulant activity of (d); and   (f) selecting candidate agents having an anti-apoptotic:anticoagulant activity ratio greater than 1.0.   
   
   
       12 . The method of  claim 11 , wherein said ratio is greater than about 2. 
   
   
       13 . The method of  claim 11 , wherein said ratio is greater than about 4. 
   
   
       14 . The method of  claim 11 , wherein said ratio is greater than about 8. 
   
   
       15 . The method of  claim 11 , wherein said surface loop is selected from the group consisting of loop 37, calcium loop, and autolysis loop. 
   
   
       16 . A method of selecting potentially therapeutic cytoprotective variants of recombinant activated protein C, comprising:
 (a) providing a library of candidate agents which are activated protein C variants, wherein each of said activated protein C variants has a protease domain, wherein each of said activated protein C variants comprises at least one mutation at a residue in a surface loop of said protease domain;   (b) determining anti-apoptotic activity of said activated protein C variants in one or more stressed or injured cells by exposing said cells to an apoptotic-inducing concentration of staurosporine in the presence of an amount of a candidate agent;   (c) determining anticoagulant activity of said candidate agents that are assayed in (b) by performing a dilute prothrombin time clotting assay;   (d) calculating the ratio of the anti-apoptotic activity determined in (b) to the anticoagulant activity of (c); and   (e) selecting candidate agents having an anti-apoptotic:anticoagulant activity ratio greater than 1.0.   
   
   
       17 . The method of  claim 16 , wherein said ratio is greater than about 2. 
   
   
       18 . The method of  claim 16 , wherein said ratio is greater than about 4. 
   
   
       19 . The method of  claim 16 , wherein said ratio is greater than about 8. 
   
   
       20 . The method of  claim 16 , wherein said surface loop is selected from the group consisting of loop 37, calcium loop, and autolysis loop 
   
   
       21 . An agent selected by the method of  claim 1 . 
   
   
       22 . An agent selected by the method of  claim 6 . 
   
   
       23 . An agent selected by the method of  claim 11 . 
   
   
       24 . An agent selected by the method of  claim 16 .