Methods for measuring platelet reactivity of individuals treated with drug eluting stents
View Patent ↗A method is provided for measuring inhibition of platelet reactivity in an individual treated with a drug-eluting stent (DES). First, a blood sample is obtained from an individual treated with a DES and a P2Y12 antagonist. The blood sample is then mixed with particles comprising an attached GPIIb/IIIa receptor ligand, adenosine diphosphate (ADP) and prostaglandin E1 (PGE1). The mixture is incubated under conditions suitable for agglutinating particles, and platelet-mediated agglutination is assessed in the mixture. The absence or reduction of agglutination indicates that the individual treated with a DES has reduced platelet reactivity. Also provided is a kit for measuring inhibition of platelet aggregation by a P2Y12 receptor antagonist that includes a GPIIb/IIIa receptor ligand immobilized on a particle, adenosine diphosphate (ADP), prostaglandin E1 (PGE1), an anticoagulant, and a buffer to maintain the anticoagulated blood in a condition suitable for platelet aggregation.
1. A method for measuring inhibition of platelet reactivity by a P2Y12 antagonist in an individual treated with a drug-eluting stent (DES), comprising the steps of:
a) providing a platelet containing blood sample from an individual treated with a DES and a P2Y12 antagonist;
b) contacting said platelet containing blood sample with particles comprising a GPIIb/IIIa receptor ligand immobilized thereon, adenosine diphosphate (ADP) and prostaglandin E1 (PGE1) under conditions suitable for agglutination of said particles mediated by said platelet in said blood sample; and
c) assessing said agglutination to determine the presence, absence and/or degree of inhibition of platelet aggregation by said P2Y12 antagonist in said individual, wherein absence or a reduction of said agglutination indicates that said individual has reduced platelet reactivity in response to said P2Y12 antagonist treatment.
2. The method of claim 1 , wherein the platelet containing blood sample is a whole blood sample.
3. The method of claim 1 , wherein the platelet containing blood sample is a plasma sample.
4. The method of claim 3 , wherein the plasma sample is a platelet rich plasma (PRP) sample.
5. The method of claim 1 , wherein the P2Y12 antagonist is a thienopyridine.
6. The method of claim 5 , wherein the thienopyridine is clopidogrel.
7. The method of claim 5 , wherein the thienopyridine is ticlopidine.
8. The method of claim 1 , wherein the individual is further treated with aspirin.
9. The method of claim 1 , wherein the particles comprise polystyrene or latex.
10. The method of claim 1 , wherein the particles comprise an infrared dye, the contacting step forms an assay mixture between the platelet containing blood sample and the particles, assessing the agglutination of the particles comprises irradiating the assay mixture with a light in the infrared spectrum and assessing the transmission of infrared light from the assay mixture.
11. The method of claim 1 , wherein the GPIIb/IIIa receptor ligand comprises a substance selected from 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, or a peptidomimetic that mimics the RGD sequence.
12. The method of claim 1 , wherein the GPIIb/IIIa receptor ligand is fibrinogen.
13. The method of claim 1 , wherein the ADP has a final concentration of 2 to 35 μM and the PGE1 has a final concentration of 2 to 30 nM.
14. The method of claim 1 , wherein the ADP has a final concentration of 15 to 20 μM.
15. The method of claim 1 , wherein the PGE1 has a final concentration of 20 to 25 nM.
16. The method of claim 1 , wherein the particles, ADP and PGE1 are contained in an assay medium.
17. The method of claim 16 , wherein the assay medium has high absorption at about 800 nm.
18. 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.
19. The method of claim 5 , wherein the thienopyridine is prasugrel.
20. The method of claim 1 , further comprising assessing the risk level of the individual for stent thrombosis.