The in vitro effect of antirheumatic drugs on platelet function.

Several antirheumatic drugs lower the cardiovascular risk among rheumatoid arthritis patients. It is, however, unknown whether inhibition of platelet function contributes to this risk reduction. Only few studies have investigated the potential role of platelets as a target of antirheumatic drugs. In this study, platelet function was tested in vitro in samples from 24 healthy individuals spiked with antirheumatic drugs in clinically relevant concentrations or vehicle. Platelet aggregation was tested with 96-well light transmission aggregometry (LTA), and when an effect ≥20% compared to vehicle was observed, flow cytometric platelet aggregation and activation were evaluated and closure time was measured by Platelet Function Analyzer (PFA-200). When evaluated by LTA, teriflunomide (the active metabolite of leflunomide), tocilizumab, and prednisolone reduced ADP- and collagen-induced platelet aggregation ≥20%, while adalimumab increased TRAP-induced platelet aggregation ≥20%. Using flow cytometry, agonist-induced platelet aggregation with teriflunomide or vehicle was mean ± standard deviation (SD); 30.7% ± 5.8 vs. 41.7% ± 6.5, p = 0.02 using ADP, and 34.7% ± 13.9 vs. 55.8% ± 3.9, p = 0.01 using collagen. Results indicate that teriflunomide, prednisolone, and tocilizumab inhibit, and adalimumab increases platelet aggregation. The study suggests that the majority of antirheumatic drugs mainly reduced cardiovascular risk through indirect effects (e.g., reducing inflammation).

Light transmission aggregometry using pre-coated microtiter plates and a Victor X5 plate reader.

BACKGROUND: Light transmission aggregometry (LTA) can be performed with microtiter plates (96-well LTA). When conducting LTA, an agonist is added to platelet-rich plasma and the sample is shaken for minutes after which absorbance readings are done. Platelet aggregation is detected as decrease in absorbance. However, the classical method is cumbersome and therefore microtiter plates can be used for concomitant testing of multiple samples. Furthermore, it would be convenient to prepare the plate in advance of platelet aggregation testing. Aim: The aim of the present study was to establish a simplified 96-well LTA protocol, where plates were pre-coated with agonists and stored at -80 C until use. RESULTS: We developed and validated a protocol for 96-well LTA using a Victor X5 plate reader and pre-coated microtiter plates. The minimum requirement of platelet-rich plasma was 45 µL per sample and the sample platelet count should not be below 100 x109/L. Optimal absorbance reading was 595 nm wavelengths. Platelet aggregation results were higher at 37°C than at room temperature. Platelet adherence to wells after stimulation was observed; it was not avoided by pre-coating of the wells with gelatin. A range of up to 7 concentrations for each agonist (collagen, arachidonic acid, adenosine diphosphate, thrombin receptor-activating peptide and protease-activated receptor-4) was tested concomitantly. A transient rise in platelet aggregation was observed after 2 minutes of shaking in some samples with low agonist concentration, and platelet aggregation was optimal after 10 minutes of shaking for samples with high agonist concentration. Plates could be stored at -80°C for 15 days without significant change in the platelet aggregation results. CONCLUSION: The 96-well LTA is suitable for platelet aggregation testing and a range of agonist concentrations can be concomitantly tested.