Recombinant platelet collagen receptor glycoprotein vi and its pharmaceutical use

Abstract

The invention relates to Glycoprotein VI (GPVI), its isolation, purification, and methods for recombinant production. Especially, the invention relates to the use of GPVI, preferably recombinant GPVI, in the treatment of disorders and pathological events correlated directly or indirectly to blood coagulation disorders such as thrombotic and cardiovascular diseases. The extracellular recombinant protein can also be used for establishing screening assays to find potential inhibitors of the membrane bound GPVI in order to inhibit binding of thrombocytes and platelets, respectively, to collagen. Changes in GPVI can be used to monitor platelet age and exposure to thrombotic and cardiovascular diseases.

1 . A DNA coding for Glycoprotein VI which comprises the sequence of SEQ ID NO:1, or a biologically active fragment thereof. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . (canceled) 
     
     
         5 . A recombinant human Glycoprotein VI protein comprising the amino acid sequence of  FIG. 1 b    (SEQ ID NO:3) which is not glycosylated. 
     
     
         6 . A pharmaceutical composition comprising the protein of  claim 5  and a pharmaceutically acceptable diluent, carrier or excipient therefor. 
     
     
         7 . (canceled) 
     
     
         8 . A method of screening for an inhibitor of glycoprotein VI (GPVI)—collagen interaction, comprising binding recombinant glycoprotein VI polypeptide (GPVI) comprising a polypeptide sequence encoded by the nucleic acid sequence of SEQ ID NO: 1 or a fragment thereof comprising the extracellular domain of said polypeptide sequence and collagen in the presence of a potential inhibitor in a binding assay, wherein reduction of said binding in the presence of said potential inhibitor as compared to said binding in the absence of said potential inhibitor indicates the presence of an inhibitor. 
     
     
         9 . A method of treating thrombotic and cardiovascular events and disorders related to platelet-collagen interactions, comprising administering an effective amount of recombinant GPVI to a patient in need of such treatment. 
     
     
         10 . A method for detecting platelet age and platelet exposure to thrombotic and cardiovascular conditions or events, comprising detecting changes in GPVI as a marker. 
     
     
         11 . A purified antibody that specifically binds to a human glycoprotein VI (GPVI) polypeptide consisting of the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 1, or a fragment thereof that binds to the GPVI polypeptide, wherein said antibody or fragment thereof is non-naturally occurring, non-human, monoclonal, is a recombinant IgG, or was derived from an antibody raised against human GPVI in a non-human species and subsequently humanized. 
     
     
         12 . The purified antibody of  claim 11 , which is an immunoglobulin G (IgG) molecule, and which has been purified from polyclonal antisera raised in a rabbit against GPVI polypeptide consisting of the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 1 or an Fab fragment obtained by the digestion of said IgG molecule or an F(ab′) 2  fragment obtained by the digestion of said IgG molecule. 
     
     
         13 . The purified IgG molecule of  claim 12 , wherein the Fab fragment is obtained by the digestion of said the molecule with papain or the F(ab′) 2  fragment is obtained by the digestion of said IgG molecule with pepsin. 
     
     
         14 . The Fab fragment of  claim 13  obtained by the digestion of the IgG molecule with papain. 
     
     
         15 . The F(ab′) 2  fragment of  claim 13  obtained by the digestion of the IgG molecule with pepsin. 
     
     
         16 . A method for producing the purified IgG molecule of  claim 12 , or said Fab fragment thereof or said F(ab′) 2  fragment thereof, comprising
 (a) raising polyclonal antisera in a rabbit against human glycoprotein VI polypeptide consisting of the amino acid sequence encoded by the nucleic acid sequence set forth in SEQ ID NO: 1; 
 (b) purifying IgG from said polyclonal antisera; and 
 (c) optionally
 (i) digesting the IgG with papain to generate the Fab fragment thereof, or 
 (ii) digesting the IgG with pepsin to generate the F(ab′) 2  fragment. 
 
 
     
     
         17 . The method for producing the Fab fragment of  claim 16 , wherein the IgG is digested with papain to generate the Fab fragment. 
     
     
         18 . The method for producing the F(ab′) 2  fragment of  claim 16 , wherein the IgG is digested with pepsin to generate the F(ab′) 2  fragment. 
     
     
         19 . An Fab antibody fragment of the antibody of  claim 11 . 
     
     
         20 . An F(ab′) 2  antibody fragment of the antibody of  claim 11 . 
     
     
         21 . A method for measuring platelet binding profile of the purified antibody molecule of  claim 11 , comprising
 (a1) labelling the purified antibody molecule with a fluorescent marker or   (a2) adding a fluorescent-labelled second antibody or an affinity reagent specific for the antibody molecule; and   (b) detecting and quantitating the fluorescent marker, or the fluorescent-labelled second antibody or the affinity reagent specific for the antibody molecule, by flow cytometry.   
     
     
         22 . A method for measuring platelet binding profile of the purified IgG molecule of  claim 12 , comprising
 (a1) labelling the purified IgG molecule with a fluorescent marker or   (a2) adding a fluorescent-labelled second antibody or an affinity reagent specific for the IgG molecule; and   (b) detecting and quantitating the fluorescent marker, or the fluorescent-labelled second antibody or the affinity reagent specific for the IgG molecule, by flow cytometry.   
     
     
         23 . A method for measuring platelet binding profile of the purified IgG molecule of  claim 13 , comprising
 (a1) labelling the purified IgG molecule with a fluorescent marker or   (a2) adding a fluorescent-labelled second antibody or an affinity reagent specific for the IgG molecule; and   (b) detecting and quantitating the fluorescent marker, or the fluorescent-labelled second antibody or the affinity reagent specific for the IgG molecule, by flow cytometry.