Compositions and methods for evaluation of antiplatelet drug sensitivity
In some aspects, the disclosure relates to compositions and methods for detecting phosphorylation of dynamin-related protein 1 (Dip1) at position Ser-616. In some embodiments, methods described by the disclosure are useful for detecting antiplatelet agent sensitivity in a subject. In some embodiments, methods described by the disclosure are useful for detecting if a subject has been previously exposed to an antiplatelet agent.
1. A method comprising:
(i) stimulating with a platelet agonist, a blood sample, platelet rich plasma sample, or purified platelet sample that has been obtained from a subject, wherein the platelet agonist is selected from arachidonic acid, protease-activated receptor 1 (PAR1) peptide agonists, protease-activated receptor 4 (PAR4) peptide agonists, adenosine diphosphate (ADP), and thromboxane (TP) receptor agonists;
(ii) after stimulation, contacting the sample with an antibody that binds specifically to Dynamin-related protein 1 (Drp1) that is phosphorylated at position Ser616 (phospho-Drp1-Ser616), and a secondary reagent that binds to the antibody; and
(iii) detecting the presence or absence of phospho-Drp1-Ser616, or a ratio of phospho-Drp1-Ser616: total Drp1, in the sample based upon the antibody binding to the phospho-Drp1-Ser616 and to the secondary reagent, wherein absence of phospho-Drp1-Ser616, or a phospho-Drp1-Ser616: total Drp1 ratio below 1.25, in the sample is indicative of the subject having been previously exposed to aspirin, vorapaxar, clopidogrel, cangrelor, prasugrel, ticagrelor, ticlopidine, cilostazol, a COX inhibitor, a non-steroidal anti-inflammatory (NSAID), terutroban, or dipyridamole.
2. The method of claim 1 , wherein the antibody that binds specifically to phospho-Drp1-Ser616 does not bind to phospho-Drp1-Ser637.
3. The method of claim 1 , wherein the secondary reagent is an antibody, single chain antibody (scFv), peptide or peptide fragment, or aptamer.
4. The method of claim 1 , wherein the secondary reagent agent is conjugated to a detectable label.
5. The method of claim 4 , wherein the detectable label is a polymeric particle, a metal particle, or a detectable moiety.
6. The method of claim 5 , wherein the polymeric particle is a latex particle.
7. The method of claim 5 , wherein the metal particle is a gold nanoparticle or magnetic nanoparticle.
8. The method of claim 5 , wherein the detectable moiety is a fluorescent moiety, luminescent moiety, phosphorescent moiety, radiolabeled moiety, or a detectable enzyme.
9. The method of claim 1 , wherein the PAR1 peptide agonist is a SFLLRN (SEQ ID NO: 1) peptide.
10. The method of claim 1 , wherein the PAR4 agonist is a AYPGKF (SEQ ID NO: 2) peptide.
11. The method of claim 1 , wherein the TP receptor agonist is U46619.
12. The method of claim 1 , wherein the subject has or is suspected of having a cardiovascular disease or disorder or a cerebrovascular disease or disorder.
13. The method of claim 12 , wherein the disease or disorder is selected from atherosclerotic disease, coronary artery disease, stable angina, coronary bypass surgery, stroke, thrombosis or thromboembolism, peripheral arterial disease, myocardial ischemia, myocardial infarction, atrial fibrillation (AF), aneurysm, pain, fever, inflammation, heparin-induced thrombocytopenia, Hermansky-Pudlak syndrome, Gray Platelet Syndrome, and impaired platelet function due to myelodysplastic syndrome.
14. The method of claim 1 , wherein the subject is a human subject.
15. The method of claim 1 , wherein the sample is a blood sample.
16. The method of claim 1 , wherein the sample is a platelet rich plasma sample.
17. The method of claim 1 , wherein the sample is a purified platelet sample.