Downstream Molecular Signals of P2Y12 Receptors in Hyporeactive Patients Under Clopidogrel Treatment (A Possible Mechanism of HOTPR:High On- Treatment Platelet Reactivity)
Conditions: Stable Angina
Sponsor: Taipei City Hospital

TITLE OF STUDY 
Downstream molecular signals of P2Y12 receptors in hyporeactive patients 
under clopidogrel treatment 
 
NCT03190005   Unique Protocol ID: TCHIRB-10603117-E 
 
PRINCIPAL INVESTIGATOR  
[Name] Chen Yueh Chung 
[Department] Taipei City hospital Division of Cardiovascular section 
[Address] Ren-Ai Rd, No 10, sec 4, Taipei, Taiwan, ROC  
[Phone] 0933060177 
[Email] chenyuehchung.tw@yahoo.com.tw 
 
Date: 2017/6/6 
 
Methods 
 
This study enrolled 35 patients(subgroup 1 and subgroup 2) who had undergone 
elective PCI and stent implantation and were receiving routine dual antiplatelet 
treatment (aspirin + clopidogrel). The study was registered under ClinicalTrials.gov 
(ID: NCT03190005) and approved by the Institutional Review Board of the Taipei 
City hospital Reserch Ethics Committee(ID: TCHIRB-10603117-E) 
In subgroup 1 
Blood samples were obtained from all study patients, and platelets were separated as 
previously described. The experimental procedure is described in Supplementary 
Figure A. PRU values were determined for control patients and for patients treated 
with DAPT after 7 days of treatment. Based on PRU values, patients were classified 

as 1) normal reactive (defined as PRU=85-207), 2) hyporeactive (defined as 
PRU>208), or 3) hyper-reactive groups (defined as PRU < 85). The Western blot 
responses were determined for each group after treatment with the different drugs, and 
these values were represented as a bar graph for statistical and comparative purposes. 
in-vitro experiments were performed using platelet-rich plasma (PRP) samples which 
were treated with different combinations of drugs. These included 1) ADP (10µM), 2) 
PGE1 (0.1 µM), 3) aggrastat (glycoprotein IIb/IIIa receptor inhibitor; 0.25µg/ml), 4) 
AR-C 66096 (P2Y12 receptor antagonist; 1 µM), 5) OPC-13013 (PDE3A tyrosine 
kinase inhibitor; 10 µM). The control group received no drugs. Reactions were 
terminated by addition of Indomethacin (50 μM) and EDTA (2 mM) 4 minutes after 
the drugs were added. Total protein was prepared from untreated and treated samples, 
and subjected to Western blotting according to standard protocols.   
In order to eliminate the effect of liver enzymes, we design subgroup 2: 
In this subgroup 2, we developed an ex-vivo system to investigate the role of 
P2Y12 signaling on HOTPR in patients with different reactivities to DAPT.  
PRU measurements 
PRU values were determined in 2 ml blood samples using the VerifyNow test 
(Accumetrics, San Diego, CA, USA) according to the manufacturer’s instructions. In 
this study HOTPR was defined as PRUs ≥208 as recently described 21. 

Western blotting 
Protein expression was analyzed in PRP samples obtained from patients in the 
different groups. Briefly, PRP was obtained from 15cc blood after addition of 1.5cc of 
3.8% sodium citrate. The samples were centrifuged at 1000 rpm , and complete blood 
counts were performed on the PRP samples in order to determine the levels of WBC 
contamination. If the WBC to platelet ratio was > 0.4%, the sample was 
re-centrifuged at 1000 rpm for 5 minutes repeatedly until the WBC to platelet ratio 
was < 0.4%. Total protein was prepared from the PRP samples as described. Total 
proteins were separated by SDS-PAGE, and immunoblotted to evaluate the expression 
of p-p38, total P38, p-PKCζ, total PKCζ, p-Akt/PKB, total AKT, p-GSK3β, total 
GSK3β, p-VASP, total VASP, p-Syk, total syk, P2Y12 receptor, PLCβ-1, and Rap1b  
(Supplementary Table 1). The protein quantifications from western blot images were 
done using the Image J program (NIH, MD).