טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
M.Sc Thesis
M.Sc StudentShamma Gharam
SubjectThe Effect after Loading Doses of Irreversible Inhibitor
of P2Y12 Receptor on Platelets Activity in Flow
Cytometry Test Comparing to Aggregation
Test
DepartmentDepartment of Medicine
Supervisors Dr. Meir Preis
Professor Moshe Flugelman
Full Thesis textFull thesis text - English Version


Abstract

Clopidogrel is a pro-drug that belongs to the thienopyridines family. The active metabolite of Clopidogrel irreversibly blocks the P2Y12 platelet ADP receptor, thus preventing the formation of blood clots. As of today, there is no simple and reliable assay to test the drug effectiveness. The research goal is to find a simple and reliable test for platelet activation in patients undergoing percutaneous coronary intervention (PCI) and using Clopidogrel.

The gold standard test for platelet activation is light transmission aggregometry (LTA). This test is long and operator dependent. The test we are going to examine in this research is Flow cytometry. We choose to test platelet activity based on expression of CD41 an identifying indicator for thrombocytes, and CD62, a P-Selectin as an indicator for platelet activity.
 

Thirty subjects who had been schedule for PCI were tested for platelet activity before taking Clopidogrel and 12 hours after loading dose of Clopidogrel.  In parallel, a control group of 21 healthy subjects were also tested. In addition we tested gene expression in platelets of 8 patients using Real Time Polymerase Chain Reaction (RT-PCR) as another indicator for platelet activation.

Comparing the 60 tests done before and after loading as one group, a significant correlation was found in the platelet activity between the flow cytometry and the aggregometry tests.  Our findings imply that the Flow cytometry can be used as a test for quantifying platelet activity and response to therapy.

The RT-PCR method for the gene expression quantification revealed that 3 out of 4 patients that were resistant to Clopidogrel have high levels of DAG, P2Y1-R, P2Y12-R, PLCbeta2, PLCbeta4 and PIP5K1C (genes involved in the ADP platelet activity pathway), as compared to the 4 patients that showed response to Clopidogrel.  This RT-PCR based genetic analysis requires further analysis of polymorphism mutations in Clopidogrel resistant patients.

Based on our findings that we were able to identify reduced platelet activity with FACS, we plan to validate the results on a larger number of patients and test the use of FACS as a potential substitute to aggregometry.