Microfluidic Assay of Platelet Deposition on Collagen by Perfusion of Whole Blood from Healthy Individuals Taking Aspirin

BACKGROUND Microfluidic devices can create hemodynamic conditions for platelet assays. We validated an 8-channel device in a study of interdonor response to acetylsalicylic acid (ASA, aspirin) with whole blood from 28 healthy individuals. METHODS Platelet deposition was assessed before treatment or 24 h after ingestion of 325 mg ASA. Whole blood (plus 100 μmol/L H-d-Phe-Pro-Arg-chloromethylketone to inhibit thrombin) was further treated ex vivo with ASA (0–500 μmol/L) and perfused over fibrillar collagen for 300 s at a venous wall shear rate (200 s−1). RESULTS Ex vivo ASA addition to blood drawn before aspirin ingestion caused a reduction in platelet deposition [half-maximal inhibitory concentration (IC50) approximately 10–20 μmol/L], especially between 150 and 300 s of perfusion, when secondary aggregation mediated by thromboxane was expected. Twenty-seven of 28 individuals displayed smaller deposits (45% mean reduction; range 10%–90%; P < 0.001) from blood obtained 24 h after ASA ingestion (no ASA added ex vivo). In replicate tests, an R value to score secondary aggregation [deposition rate from 150 to 300 s normalized by rate from 60 to 150 s] showed R < 1 in only 2 of 28 individuals without ASA ingestion, with R > 1 in only 3 of 28 individuals after 500 μmol/L ASA addition ex vivo. At 24 h after ASA ingestion, 21 of 28 individuals displayed poor secondary aggregation (R < 1) without ex vivo ASA addition, whereas the 7 individuals with residual secondary aggregation (R > 1) displayed insensitivity to ex vivo ASA addition. Platelet deposition was not correlated with platelet count. Ex vivo ASA addition caused similar inhibition at venous and arterial wall shear rates. CONCLUSIONS Microfluidic devices quantified platelet deposition after ingestion or ex vivo addition of aspirin.

24-hour time-dependent aspirin efficacy in patients with stable coronary artery disease

SummaryAspirin-induced cyclooxygenase (COX)-1 acetylation is irreversible and it is assumed that the platelet thromboxane-A2 aggregation pathway is inhibited for at least 24 hours (h) after aspirin ingestion. However, time course of biological efficacy of daily low-dose aspirin has rarely been assessed in patients with coronary artery disease (CAD). We aimed to assess the 24-h biological efficacy of daily low-dose aspirin in CAD patients. The peak and trough (2 h –24 h) effect of a chronic treatment with once daily dose aspirin were studied in 150 consecutive stable CAD patients. The main outcome measure was light transmission aggregometry (LTA) triggered with 0.5 mg/ml arachidonic acid (AA). In the last 47 consecutive patients, additional tests were conducted at 6, 12, 16, 20 h after last aspirin administration. 4.7% of the patients had significant aggregation (>20% maximal intensity LTA-AA) 2 h after aspirin ingestion and 24.7% at 24 h (p<0.0001). The more precise assessments in the last 47 patients showed that significant platelet aggregation progressively reappeared with time after aspirin intake (2 h – 4% of patients, 6 h – 4%, 12 h – 11%, 16 h – 16%, 20 h – 19% and 24 h – 28%). Concordant results were observed using production of thromboxane-B2 and other techniques evaluating AA-induced platelet aggregation/activation. No significant differences were found between lower (75–100 mg/day) and higher (>100 mg/day) dose aspirin. Such aspirin «resistance» at 24 h after ingestion was related to biological inflammatory markers, current smoking and diabetes. In conclusion, once daily aspirin does not provide stable 24-h antiplatelet protection in a significant proportion of CAD patients. Any biological assessment of aspirin efficacy should take time since last aspirin intake into consideration.