RESUMO
BACKGROUND: Arterial thrombosis triggered by vascular injury is a balance between thrombus growth and thrombus fragmentation (dethrombosis). Unbalance towards thrombus growth can lead to vascular occlusion, downstream ischemia and tissue damage.Here we describe the development of a simple methodology that allows for continuous real time monitoring and quantification of both processes during perfusion of human blood under arterial shear rate conditions. Using this methodology, we have studied the effects of antiplatelet agents targeting COX-1 (aspirin), P2Y12 (2-MeSAMP, clopidogrel), GP IIb-IIIa (eptifibatide) and their combinations on the kinetics of thrombosis over time. RESULTS: Untreated samples of blood perfused over type III collagen at arterial rates of shear promoted the growth of stable thrombi. Modulation by eptifibatide affected thrombus growth, while that mediated by 2-MeSAMP and aspirin affected thrombus stability. Using this technique, we confirmed the primacy of continuous signaling by the ADP autocrine loop acting on P2Y12 in the maintenance of thrombus stability. Analysis of the kinetics of thrombosis revealed that continuous and prolonged analysis of thrombosis is required to capture the role of platelet signaling pathways in their entirety. Furthermore, studies evaluating the thrombotic profiles of 20 healthy volunteers treated with aspirin, clopidogrel or their combination indicated that while three individuals did not benefits from either aspirin or clopidogrel treatments, all individuals displayed marked destabilization profiles when treated with the combination regimen. CONCLUSIONS: These results show the utility of a simple perfusion chamber technology to assess in real time the activity of antiplatelet drugs and their combinations. It offers the opportunity to perform pharmacodynamic monitoring of arterial thrombosis in clinical trials and to investigate novel strategies directed at inhibiting thrombus stability in the management of cardiovascular disease.
RESUMO
Low-coherence interferometry is presented as a method for nondestructive, noncontact, and high-resolution measurement of liquid volumes and fill heights in microplates. A Meniscense (Bolton, MA) prototype system captures both fill height and meniscus shape and combines them into a volume measurement, allowing for the measurement of a wide range of liquids with different meniscus shapes. The system has a liquid fill height resolution of 0.7 µm, corresponding to a volume resolution of 0.02 µL in a typical 96-well plate, demonstrated in an ideal model system. Initial data on the gravimetric verification of volume measurements on aliquots of distilled water between 50 and 350 µL in a 96-well plate suggest an inaccuracy of volume measurement of <2%. The Meniscense system offers accuracy comparable to ratiometric photometry, the only commercially available high-resolution volume measurement system. Its fill height resolution is substantially better than that of ultrasonic ranging, the only other noncontact, nondestructive method for fill height measurement.