Thromboxane B2 and Platelet Function Testing as Measures of Aspirin Resistance in Hong Kong Chinese Patients

Fasting blood samples will be obtained from patients in the study centre after fasting overnight for at least 10 hours immediately before and 1 hour after the 80 mg aspirin dose. A 5-ml blood sample will be obtained in a serum separator tube and allowed to clot at room temperature followed by centrifugation at 4 degrees C. Separated serum will be stored in aliquots at -80°C until analysis of TXB2. A 3-ml blood sample will be obtained in a hirudin blood tube for the platelet aggregation test with the Multiplate® analyzer which will be performed within 3 hours after blood collection. A 10 ml blood sample will be obtained in an ethylene diamine tetra acetic acid (EDTA) tube to be used for DNA extraction. During the study, a total of 26 ml blood will be taken from each participant. Morning urine samples before and 1 hour after aspirin ingestion will also be collected and stored at -80 °C until measurement.

The platelet activity of the samples will be measured with the hirudin blood using the Multiplate analyzer from Roche (Roche Diagnostics International Ltd, CH-6343 Rotkreuz, Switzerland) according to the manufacturer's instructions for the arachidonic acid induced platelet aggregation (ASPI) test. It will be analyzed within 0.5-3 hours after blood collection.

The serum samples will be assayed for TXB2 with EIA kits from Cayman (Item no. 501020, Cayman Chemical, MI, USA) according to the manufacturer's instructions.

The urine will be assayed for 11-dehydro TXB2 using the enzyme-linked immunosorbent assay (EIA) kit from Cayman (Item no. 519510, Cayman Chemical, MI, USA) according to the manufacturer's instructions. The data will then be standardized with urinary creatinine measured with Cayman creatinine (urinary) colorimetric assay kit (item no 500701).

The 11-dehydro TXB2 data will be standardized with the urinary creatinine levels measured by Creatinine (urinary) Colorimetric Assay kit from Cayman (Item no. 500701, Cayman Chemical, MI, USA) according to the manufacturer's instructions.

Genomic DNA of the patients will be extracted from EDTA blood by using phenol chloroform. The following six single nucleotide polymorphisms (SNPs) will be investigated initially: ITGA2 rs1126643, ITGA2B rs5911, PTGS1 rs1330344, ADK rs16931294, PEAR1 rs12041331 and COX2 rs20417. The SNPs will be assayed with TaqMan SNP genotyping assays from Applied Biosystems (Applied Biosystems, Foster City, CA, USA) by Thermo Fisher Scientific according to the product inserts.

Inclusion criteria

• Patients aged ≥ 18 years who have a history of stable CHD receiving long-term mono-antiplatelet therapy with aspirin (80 mg once daily) for reducing cardiovascular risk will be recruited from the outpatient clinic.

Exclusion criteria

• Patients who are currently taking ticlopidine, clopidogrel, dipyridamole or other antiplatelet / antithrombotic agents will not be recruited.
• Patients who are on regular therapy with anti-inflammatory drugs, or others drugs containing aspirin or non-steroid anti-inflammatory drugs (NSAIDS), or traditional Chinese medicine that have direct effects on the haemostatic system, such as angelica, danshen, garlic, ginger, ginkgo and motherwort will not be included unless they are willing to stop these treatments for at least 2 weeks
• Patients will not be recruited if they had MI, stroke, coronary artery bypass surgery or other revascularization procedure, unstable angina or angioplasty within 3 months of screening.