Substance with antithrombotic activity and method for detecting glycokallidin

Abstract

A method for conveniently detecting binding between the von Willebrand factor and glycoprotein Ib and a means to be used therein. The von Willebrand factor fixed in a reactor immobilized in a reaction vessel in the presence of bottrocetin is bound to a chimeric protein constructed by fusing the carboxyl terminal of a partial protein containing the von Willebrand factor-binding site of glycoprotein Ib with the amino terminal of the Fc region of an immunoglobulin molecule. Then the Fc region of the above immunoglobulin molecule is detected to thereby detect the binding between the von Willebrand factor and the glycoprotein Ib or inhibition of this binding.

1 . A method for detecting binding of von Willebrand factor and glycoprotein Ib or inhibition of the binding, comprising the steps of:
 immobilizing von Willebrand factor in a reaction vessel in the presence of a substance inducing the binding of von Willebrand factor and glycoprotein Ib, and,   reacting the immobilized von Willebrand factor with glycoprotein Ib.   
     
     
         2 . The method according to  claim 1 , wherein the substance that induces the binding of von Willebrand factor and glycoprotein Ib is botrocetin, ristocetin or the both substances. 
     
     
         3 . The method according to  claim 1 , wherein glycocalicin is measured by adding a sample containing glycocalicin to the reaction vessel during the reaction of von Willebrand factor and glycoprotein Ib, or prior to the reaction, and detecting inhibition of the binding of von Willebrand factor and glycoprotein Ib. 
     
     
         4 . The method according to  claim 1 , wherein a substance that inhibits the binding of von Willebrand factor and glycoprotein Ib is detected by adding a sample containing a substance to be detected to the reaction vessel during the reaction of von Willebrand factor and glycoprotein Ib, or prior to the reaction, and detecting inhibition of the binding of von Willebrand factor and glycoprotein Ib. 
     
     
         5 . A method for detecting binding of von Willebrand factor and glycoprotein Ib or inhibition of the binding, comprising the steps of:
 binding a chimeric protein that consists of an Fc region of immunoglobulin molecule fused at its amino terminus to a partial protein comprising a von Willebrand factor binding site of glycoprotein Ibα chain at its carboxyl terminus or the chimeric protein labeled with a labeling substance to von Willebrand factor immobilized in a reaction vessel, and   detecting the Fc region of the immunoglobulin molecule or the labeling substance.   
     
     
         6 . The method according to  claim 5 , wherein, when the chimeric protein is allowed to bind to von Willebrand factor, or prior to the binding, a substance that induces the binding of von Willebrand factor and glycoprotein Ib is added to the reaction vessel. 
     
     
         7 . The method according to  claim 6 , wherein the substance that induces the binding of von Willebrand factor and glycoprotein Ib is botrocetin, ristocetin or the both substances. 
     
     
         8 . The method according to  claim 6 , wherein von Willebrand factor is immobilized in the reaction vessel in the presence of a substance that induces the binding of von Willebrand factor and glycoprotein Ib. 
     
     
         9 . The method according to  claim 5 , wherein glycocalicin is measured by adding a sample containing glycocalicin to the reaction vessel when the chimeric protein is allowed to bind to von Willebrand factor, or prior to the binding, and detecting inhibition of the binding of von Willebrand factor and the chimeric protein. 
     
     
         10 . The method according to  claim 5 , wherein a substance that inhibits the binding of von Willebrand factor and glycoprotein Ib is detected by adding a sample containing a substance to be detected to the reaction vessel when the chimeric protein is allowed to bind to von Willebrand factor, or prior to the binding, and detecting inhibition of the binding of von Willebrand factor and the chimeric protein. 
     
     
         11 . A method for detecting binding of von Willebrand factor and glycoprotein Ib or inhibition of the binding, comprising the steps of:
 immobilizing a chimeric protein that consists of an Fc region of immunoglobulin molecule fused at its amino terminus to a partial protein comprising a von Willebrand factor binding site of glycoprotein Ibα chain at its carboxyl terminus in a reaction vessel,   binding von Willebrand factor or labeled von Willebrand factor to the chimeric protein, and   detecting bound von Willebrand factor or the labeling substance.   
     
     
         12 . The method according to  claim 11 , wherein, when the chimeric protein is allowed to bind to von Willebrand factor, or prior to the binding, a substance that induces the binding of von Willebrand factor and glycoprotein Ib is added to the reaction vessel. 
     
     
         13 . The method according to  claim 12 , wherein the substance that induces the binding of von Willebrand factor and glycoprotein Ib is botrocetin, ristocetin or the both substances. 
     
     
         14 . The method according to  claim 12 , wherein glycocalicin is measured by adding a sample containing glycocalicin to the reaction vessel when von Willebrand factor is allowed to bind to the chimeric protein, or prior to the binding, and detecting inhibition of the binding of von Willebrand factor and the chimeric protein. 
     
     
         15 . The method according to  claim 12 , wherein a substance that inhibits the binding of von Willebrand factor and glycoprotein Ib is detected by adding a sample containing a substance to be detected to the reaction vessel when von Willebrand factor is allowed to bind to the chimeric protein, or prior to the binding, and detecting inhibition of the binding of von Willebrand factor and glycoprotein Ib. 
     
     
         16 . A chimeric protein, which consists of an Fc region of immunoglobulin molecule fused at its amino terminus to a partial protein comprising a von Willebrand factor binding site of glycoprotein Ibα chain at its carboxyl terminus. 
     
     
         17 . The chimeric protein according to  claim 16 , wherein the immunoglobulin molecule is derived from mouse. 
     
     
         18 . A kit for measuring glycocalicin based on inhibition of a reaction of von Willebrand factor and glycoprotein Ib, comprising von Willebrand factor and a chimeric protein that consists of an Fc region of immunoglobulin molecule fused at its amino terminus to a partial protein comprising a von Willebrand factor binding site of glycoprotein Ibα chain at its carboxyl terminus. 
     
     
         19 . A compound which is detected by the method according to any one of  claims 1 ,  5  and  12 , which has an activity for specifically inhibiting platelet aggregation involving glycoprotein Ib and von Willebrand factor in blood plasma and a molecular weight of not more than 2000. 
     
     
         20 . The compound according to  claim 19 , which has a structure represented by the following formula:
 wherein R 1  and R 2  independently represent H or Cl, and R 3  represents CH 3  or H.