Methods and reagents for protease inhibition

Abstract

The invention discloses a protease inhibitor and a method of inhibiting a protease selected from the group consisting of thrombin, chymotrypsin and neuropsin, by contacting the protease with an effective amount of a member of the phosphoethanolamine binding protein (PEBP) family.

1 . A method of inhibiting a protease selected from the group consisting of thrombin, chymotrypsin and neuropsin, said method comprising contacting said protease with an effective amount of a phosphoethanolamine binding protein (PEBP) family member.  
     
     
         2 . The method of  claim 1 , wherein said protease is thrombin.  
     
     
         3 . The method of  claim 1 , wherein said protease is chymotrypsin.  
     
     
         4 . The method of  claim 1 , wherein said protease is neuropsin.  
     
     
         5 . The method of claims  1 - 4 , wherein said PEBP family member is a mammalian PEBP or a fragment thereof.  
     
     
         6 . The method of  claim 5 , wherein said PEBP family member is human PEBP.  
     
     
         7 . The method of  claim 5 , wherein said PEBP is encoded by the sequence provided in  FIG. 2 .  
     
     
         8 . The method of claims  1 - 7 , further comprising contacting said protease and protease inhibitor with a potential protease modulator and determining a change in the level of protease activity as compared to when said protease inhibitor is absent.  
     
     
         9 . A kit comprising a phosphoethanolamine binding protein (PEBP) family member for use in any of the methods 1 to 8.  
     
     
         10 . A method for identifying modulators of a biological activity of a PEBP family member, said method comprising: 
 contacting a protease susceptible to inhibition by a PEBP family member with a candidate compound in the presence of a partially inhibitory amount of said PEBP family member,    assaying proteolytic activity of said protease in the presence of a susceptible substrate, said candidate compound and partially inhibitory amount of said PEBP family member, and    comparing the proteolytic activity to a standard level of activity, the standard level of activity being assayed when contact is made between the protease and said substrate in the presence of the partially inhibitory amount of PEBP family member and the absence of said candidate compound.    
     
     
         11 . The method of  claim 10 , further comprising correlating an increase in inhibition of proteolytic activity over the standard with the presence of an agonist of PEBP inhibitory activity.  
     
     
         12 . The method of  claim 10 , further comprising correlating a decrease in inhibition of proteolytic activity compared to the standard with the presence of an antagonist of PEBP inhibitory activity.  
     
     
         13 . The method of  claim 10 , wherein said protease is provided in a sample of a body fluid or cell extract.  
     
     
         14 . A method for identifying modulators of a biological activity of a PEBP family member, said method comprising: 
 contacting a PEBP-susceptible protease with a PEBP polypeptide and a candidate compound, and    determining whether binding of said PEBP family member to said PEBP-susceptible protease is increased or decreased due to the presence of the candidate compound.    
     
     
         15 . The method of  claim 14 , wherein said protease is selected from the group consisting of thrombin, chymotrypsin and neuropsin.  
     
     
         16 . A method of inhibiting blood coagulation, said method comprising contacting blood with a phosphoethanolamine binding protein (PEBP) family member in an amount sufficient to inhibit blood coagulation.  
     
     
         17 . The method of  claim 16 , wherein said PEBP family member is a mammalian PEBP or fragment thereof.  
     
     
         18 . The method of  claim 16 , wherein said PEBP is encoded by the sequence provided in  FIG. 2 .  
     
     
         19 . The method of  claim 16 , wherein said PEBP family member is coated onto a solid surface prior to contacting said surface with blood.  
     
     
         20 . The method of  claim 16 , further comprising contacting blood with an additional anti-coagulant.  
     
     
         21 . The method of  claim 16 , further comprising contacting blood with a compound promoting blood coagulation.  
     
     
         22 . A receptacle coated with a PEBP family member.  
     
     
         23 . The receptacle of  claim 22  coated with a mammalian PEBP or fragment thereof.  
     
     
         24 . The receptacle of  claim 22  selected from the group consisting of: intravascular cannulas, vascular access shunts in hemodialysis patients, hemodialysis machines, and cardiopulmonary bypass machines.  
     
     
         25 . A method of treating a disorder or disease, or predisposition thereto, characterized by an increase in the activity of a protease susceptible to inhibition by a PEBP family member, said method comprising administering an effective-amount of a PEBP family member to an individual.  
     
     
         26 . The method of  claim 25 , wherein said disorder is a neurodegenerative disorder.  
     
     
         27 . A composition comprising a PEBP family member and a pharmaceutically appropriate carrier.  
     
     
         28 . The composition of  claim 27 , wherein said pharmaceutically appropriate carrier is a liposome.  
     
     
         29 . The use of a PEBP family member as a medicament.  
     
     
         30 . A kit comprising a PEBP family member and a catheter.  
     
     
         31 . A PEBP family member comprising the sequence provided in  FIG. 2 .  
     
     
         32 . A method of diagnosing a disorder or disease characterized by an increase in the activity of a protease susceptible to inhibition by a PEBP family member, said method comprising determining whether the presence of said PEBP family member is reduced in a sample relative to a standard level, said standard level being from a non-afflicted individual.