Controlled platelet activation to monitor therapy of adp antagonists

Abstract

A method is provided of determining whether an individual has reduced ability to form platelet thrombi due to inhibition of platelet activation initiation, signal transduction and/or GPIIb/IIIa blockade. A blood sample is obtained from the individual being assessed. The blood sample is mixed in combination with 1) an anticoagulant; 2) sufficient buffer to maintain the pH and salt concentration of the anticoagulated blood within a range suitable for platelet aggregation; 3) a platelet GPIIb/IIIa receptor ligand immobilized on a solid surface; 4) one or more agents to enhance a signal transduction pathway and 5) a receptor activator. The combination is incubated under conditions for agglutinating particles. Platelet-mediated agglutination is assessed in the agitated mixture. The absence of agglutination indicates that the individual has a reduced ability to form platelet thrombi.

1 . A method of determining whether an individual has reduced ability to form platelet thrombi due to inhibition of platelet activation initiation, signal transduction and/or GPIIb/IIIa blockade, comprising the steps of:
 a) obtaining a blood sample from the individual being assessed;   b) mixing the blood sample in combination with 1) an anticoagulant; 2) sufficient buffer to maintain the pH and salt concentration of the anticoagulated blood within a range suitable for platelet aggregation; 3) a platelet GPIIb/IIIa receptor ligand immobilized on a solid surface; 4) one or more agents to enhance a signal transduction pathway and 5) a receptor activator;   c) incubating said combination under conditions for agglutinating particles; and   d) assessing platelet-mediated agglutination in the agitated mixture, wherein the absence of agglutination indicates that the individual has reduced ability to form platelet thrombi.   
     
     
         2 . The method of  claim 1 , wherein the receptor activator is a platelet activator and the agent is a platelet inhibitor. 
     
     
         3 . The method of  claim 2 , wherein the platelet activator is ADP. 
     
     
         4 . The method of  claim 2 , wherein the platelet inhibitor is Prostaglandin E1 (PGE1). 
     
     
         5 . The method of  claim 1 , wherein the conditions comprise; agitating the mixture for a period of time sufficient for activated platelet GPIIb/IIIa receptors to bind with the platelet GPIIb/IIIa receptor ligands on said solid surface. 
     
     
         6 . The method of  claim 1 , wherein the GPIIb/IIIa receptor ligand is selected from the group consisting of fibrinogen, monoclonal antibody 10E5, monoclonal antibody c7E3, von Willebrand factor, fibronectin, vitronectin, and synthetic ligands having an arginine-glycine-aspartic acid (RGD) GPIIb/IIIa binding sequence. 
     
     
         7 . The method of  claim 1  wherein the solid surface comprises glass microparticles or small polymeric beads, the surface of said microparticles or beads modified with a GPIIb/IIIa receptor ligand. 
     
     
         8 . The method of  claim 5  wherein the small polymeric beads have diameters from about 1 to about 6 microns. 
     
     
         9 - 15 . (canceled) 
     
     
         16 . A method of determining whether an individual has reduced ability to form platelet thrombi due to inhibition of platelet activation initiation, signal transduction and/or GPIIb/IIIa blockade using controlled activation of the platelet, comprising:
 providing a platelet activator;   providing one or platelet inhibitors; and   producing an alternate signal transduction pathway.   
     
     
         17 . The method of  claim 16 , wherein the platelet activator is ADP. 
     
     
         18 . The method of  claim 16 , wherein the platelet inhibitor is Prostaglandin E1 (PGE1). 
     
     
         19 . A kit comprising:
 a platelet GPIIb/IIIa receptor ligand immobilized on a solid surface;   one or more agents to enhance a signal transduction pathway; and   a receptor activator.   
     
     
         20 . The kit of  claim 19 , wherein receptor activator is a platelet activator and the agent is a platelet inhibitor. 
     
     
         21 . The kit of  claim 20 , wherein the platelet activator is ADP. 
     
     
         22 . The kit of  claim 20 , wherein the platelet inhibitor is Prostaglandin E1 (PGE1). 
     
     
         23 . The kit of  claim 19 , wherein the solid surface is selected from glass microparticles or small polymeric beads. 
     
     
         24 . The kit of  claim 19 , wherein the small polymeric beads have diameters from about 1 to about 6 microns. 
     
     
         25 . The kit of  claim 19 , wherein the GPIIb/IIIa receptor ligand is selected from the group consisting of fibrinogen, monoclonal antibody 10ES, monoclonal antibody c7E3, von Willebrand factor; fibronectin, vitronectin and synthetic ligands having an arginine-glycine-aspartic acid (RGD) GPIIb/IIIa binding sequence.