Novel platelet activation marker and method for determination thereof

Abstract

The present invention provides a convenient and highly sensitive method of determining sGPVI present in plasma; this is accomplished by establishing a plurality of mouse hybridomas that produce antibody against GPVI and combining the antibodies produced therefrom. Provided thereby are a novel platelet activation marker, a reagent and method for determining this novel platelet activation marker, and novel applications of this marker in, for example, the diagnosis of diseases associated with platelet activation/vascular endothelial injury.

1 . A method of diagnosing a disease, a susceptibility to the disease, or a risk of developing the disease using at least one anti-GPVI antibodies or a fragment thereof, comprising:
 1) obtaining from a subject in need thereof a sample that is a sample of a human body fluid or a sample prepared from a human body fluid, wherein said subject is suspected to be affected by a disease selected from angina pectoris, acute myocardial, infarction heart disease, cerebral infarction, atrial fibrillation, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation syndrome and/or dementia;   2) removing platelets from the sample, if the sample is a platelet containing sample;   3) contacting the sample with an anti-GPVI antibody or fragment thereof;   4) detecting, determining, or quantitating a value of sGPVI in said sample based on   said contacting with the anti-GPVI antibody; and   5) comparing the determined value with a standard sGPVI value of normal or healthy individuals, or a reference sGPVI value or a range of sGPVI values for patients of the disease to diagnose the disease, the susceptibility to the disease or the risk of developing the disease.   
     
     
         2 . The method of  claim 1 , wherein the disease is dementia. 
     
     
         3 . The method of  claim 1 , wherein a sensitivity of said detecting, determining, or quantitating is 30 pg/mL or less. 
     
     
         4 . The method of  claim 1 , wherein the first anti-GPVI antibody is an antibody that specifically binds to loop 2 or loop 9 of GPVI. 
     
     
         5 . The method of  claim 1 , wherein said contacting comprises contacting the sample with at least two anti-GPVI antibodies and wherein said detecting, determining, or quantitating comprises detecting, determining, or quantitating the value of sGPVI in said sample based on said contacting with the at least two anti-GPVI antibodies. 
     
     
         6 . The method of  claim 5 , wherein said at least two anti-GPVI antibodies comprise a) an anti-GPVI antibody that specifically binds to domain 1 of GPVI and b) an anti-GPVI antibody that specifically binds to domain 2 of GPVI. 
     
     
         7 . The method of  claim 5 , wherein said at least two anti-GPVI antibodies comprise a) an antibody that specifically binds to loop 2 of GPVI and b) an antibody that specifically binds to loop 9 of GPVI. 
     
     
         8 . A method of diagnosing a disease, a susceptibility to the disease or a risk of developing the disease using anti-GPVI antibodies or fragments thereof, comprising:
 1) obtaining from a subject in need thereof a sample that is a sample of a human body fluid or a sample prepared from a human body fluid;   2) removing platelets from the sample, if the sample is a platelet containing sample;   3) contacting the sample with a first anti-GPVI antibody or fragment thereof;   4) contacting said sample with a second anti-GPVI antibody or fragment thereof, wherein the second antibody or fragment thereof has a GPVI binding site different from that of the first antibody or fragment thereof;   5) detecting, determining, or quantitating a value of sGPVI in said sample based on said contacting with the first anti-GPVI antibody or fragment thereof and said contacting with the second anti-GPVI antibody or fragment thereof; and   6) comparing the determined value with a standard sGPVI value of normal or healthy individuals, or a reference sGPVI value or a range of sGPVI values for patients of the disease to diagnose the disease, the susceptibility to the disease or the risk of developing the disease;   wherein the disease is selected from the group consisting of angina pectoris, acute myocardial infarction, heart disease, cerebral infarction, atrial fibrillation, thrombotic thrombocytopenic purpura, disseminated intravascular coagulation syndrome and dementia.   
     
     
         9 . The method of  claim 8 , wherein the disease is dementia. 
     
     
         10 . The method of  claim 8 , wherein a sensitivity of said detecting, determining, or quantitating is 30 pg/mL or less. 
     
     
         11 . The method of  claim 8 , wherein each of the first anti-GPVI antibody and the second anti-GPVI antibody is an antibody that specifically binds to loop 2 or loop 9 of GPVI. 
     
     
         12 . The method of  claim 8 , wherein said first anti-GPVI antibody is an anti-GPVI antibody that specifically binds to domain 1 of GPVI and the second anti-GPVI antibody is an anti-GPVI antibody that specifically binds to domain 2 of GPVI. 
     
     
         13 . The method of  claim 8 , wherein said first anti-GPVI antibody is an antibody that specifically binds to loop 2 of GPVI and the second anti-GPVI antibody is an antibody that specifically binds to loop 9 of GPVI.