Anticoagulant fusion protein anchored to cell membrane

Abstract

The invention relates to the inhibition of blood coagulation, especially during organ rejection, and in particular the inhibition of delayed vascular rejection. The invention provides anticoagulant proteins which are anchored to cell membranes. The anticoagulant function preferably provided by heparin, antithrombin, hirudin, TFPI, tick anticoagulant peptide, or a snake venom factor. These anticoagulant proteins are preferably prevented from being constitutively expressed at the cell surface. In particular, expression at the cell surface is regulated according to cell activation, for instance by targeting the protein to a suitable secretory granule. Expression of these proteins renders cells, tissues and organs less vulnerable to rejection after transplantation (e.g. after xenotransplantation).

1 . A protein comprising a region with anticoagulant activity and a region which can anchor said protein to a cell membrane.  
     
     
         2 . A protein according to  claim 1  further comprising a targeting sequence which prevents the protein from being constitutively expressed at the cell surface.  
     
     
         3 . A protein according to  claim 1  or  claim 2 , wherein the anticoagulant region comprises the sequence of a hirudin, a tissue factor pathway inhibitor, a tick anticoagulant peptide, or a protein C activator.  
     
     
         4 . A protein according to  claim 1  or  claim 2 , wherein the anchor region is a sequence capable of attaching the protein to a lipid bilayer.  
     
     
         5 . A protein according to  claim 4 , wherein the anchor region comprises the transmembrane sequence from a membrane protein.  
     
     
         6 . A protein according to  claim 2 , wherein said targeting sequence is a sequence which can target a nascent polypeptide to a secretory granule.  
     
     
         7 . A protein according to  claim 6  wherein said secretory granule does not fuse with the cell's plasma membrane until the cell is suitably stimulated.  
     
     
         8 . A protein according to  claim 7 , wherein said secretory granule is a Weibel-Palade body.  
     
     
         9 . A protein according to  claim 6  wherein said targeting sequence is the cytoplasmic domain of P-selectin.  
     
     
         10 . A protein according to any one of the previous claims wherein said anchor sequence is that of P-selectin.  
     
     
         11 . A polynucleotide encoding a protein according to any one of the previous claims.  
     
     
         12 . A vector comprising a polynucleotide according to  claim 11 .  
     
     
         13 . A delivery system comprising a protein according to any one of  claims 1  to  10 , a polynucleotide according to  claim 11 , or a vector according to  claim 12 .  
     
     
         14 . A method of transfecting a cell with a vector according to  claim 12 .  
     
     
         15 . A cell transfected according to  claim 14 .  
     
     
         16 . Biological tissue comprising a cell according to  claim 15 .  
     
     
         17 . An animal comprising biological tissue according to  claim 16  and/or cell according to  claim 15 .  
     
     
         18 . An animal according to  claim 17 , wherein said animal is a transgenic pig or sheep.  
     
     
         19 . A method of rendering a tissue or organ suitable for transplantation, comprising expressing a protein according to any one of  claims 1  to  10  on the surface of endothelial cells in said tissue or organ.  
     
     
         20 . A method of transplantation comprising transplanting biological tissue according to  claim 16  from a donor animal into a recipient animal.