Controlled platelet activation to monitor therapy of ADP antagonists
View Patent ↗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 for measuring inhibition of platelet aggregation by a P2Y12 antagonist, comprising the steps of: (a) providing a platelet containing blood sample from an individual treated with a P2Y12 antagonist; (b) contacting the platelet containing blood sample with particles comprising an attached GPIIb/IIIa receptor ligand, adenosine diphosphate (ADP) and prostaglandin El (PGE1) under conditions suitable for agglutination of said particles mediated by the platelet in the blood sample, and (c) assessing the agglutination to determine the level of inhibition of platelet aggregation by the P2Y12 antagonist in the individual, wherein the level of agglutination indicates whether the individual has reduced ability to form platelet aggregation in response to the P2Y12 antagonist treatment.
2. The method of claim 1 , wherein the P2Y12 antagonist is a thienopyridine.
3. The method of claim 2 , wherein the thienopyridine is clopidogrel.
4. The method of claim 1 , wherein the platelet containing blood sample is from an individual treated with a P2Y12 antagonist and aspirin.
5. The method of claim 1 , wherein the particles comprise polystyrene or latex.
6. The method of claim 1 , wherein the particles comprise an infrared dye, the contact between the platelet containing blood sample and the particles forms an assay mixture, and the agglutination of the particles mediated by the platelet is assessed by irradiating the assay mixture with a light in the infrared spectrum, and assessing the transmission of infrared light from the assay mixture.
7. The method of claim 1 , wherein the GPIIb/IIIa receptor ligand comprises a substance selected from the group consisting of fibrinogen, monoclonal antibody 10E5, monoclonal antibody c7E3, von Willebrand factor, fibronectin, vitronectin, a ligand that has an arginine-glycine-aspartic acid (RGD) sequence, a peptide that mimics the RGD sequence, and a peptidomimetic that mimics RGD sequence.
8. The method of claim 1 , wherein the GPIIb/IIIa receptor ligand is fibrinogen.
9. The method of claim 1 , which is conducted at a temperature ranging from 30° C. to 40° C., and the total time of the readings from the time of the contact among the platelet containing blood sample, the particles comprising an attached GPIIb/IIIa receptor ligand, ADP and PGE1 ranges from about 10 seconds to about 10 minutes.
10. The method of claim 1 , wherein the platelet containing blood sample is a whole blood sample or a plasma sample.
11. The method of claim 10 , wherein the plasma sample is a platelet rich plasma (PRP) sample.
12. The method of claim 1 , wherein the particles, ADP and PGE1 are contained in an assay medium.
13. The method of claim 12 , wherein the particles, ADP and PGE1 are contained in an assay medium on a disposable cartridge.
14. The method of claim 12 , wherein the assay medium has an absorption peak at about 800 nm.
15. The method of claim 2 , wherein the thienopyridine is ticlopidine.