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Widespread monitoring of platelet function and the effect of antiplatelet drugs will improve outcomes in cardiovascular patients, but platelet function testing is not routine in clinical practice. We report a rapid, accurate methodology to quantify platelet-protein interactions: a microarray of contact-printed 6-μm fibrinogen dots on a transparent substrate binds platelets from whole blood, one platelet per dot. The fractional occupancy of an array of fibrinogen dots after a predefined incubation time quantitatively assays platelet adhesion to the protein matrix. We demonstrate this technique by measurement of platelet adhesion to fibrinogen as a means to quantify the effect of the P2Y12 and αIIbβ3 receptor inhibitors cangrelor and abciximab, respectively, both in vitro--by incubating the drug with a freshly drawn blood sample--and in blood from patients treated with antiplatelet agents. The effects of single- and dual-antiplatelet therapy are also assessed. Results from this platelet-binding assay are well correlated with standard techniques including flow cytometry and light transmission aggregometry. This assay technology, readily integrated with microfluidic platforms, is generally applicable to the assay of cell-protein interactions and promises more effective, rapid assay of drug effects in cardiovascular disease patients.

We report the development and demonstration of an assay that distinguishes the pharmacological effects of two widely used antiplatelet therapies, aspirin (COX-1 inhibitor) and clopidogrel (P2Y12 inhibitor). Whole blood is perfused through a low-volume microfluidic device in contact with a well-characterized (ellipsometry, atomic force microscopy) acid-soluble type I collagen surface. Whole human blood treated in vitro with a P2Y12 inhibitor 2-methylthioadenosine 5'-monophosphate triethylammonium salt (2-MeSAMP) extended the time to the start of platelet recruitment, i.e., platelet binding to the collagen surface. Treatment with 2-MeSAMP also slowed the rate of aggregate buildup, with an overall reduced average platelet aggregate area after 8 min of constant blood flow. A far smaller effect was observed for in vitro treatment with aspirin, for which the rate of change of surface coverage is indistinguishable from controls. In whole blood obtained from patients under treatment with dual-antiplatelet therapy (aspirin and clopidogrel), a significant extension of time to platelet recruitment was observed along with a slowed rate of aggregate buildup and an average aggregate size approximately half that of control measurements. Differentiation of the pharmacological effects of these two well-targeted antiplatelet pathways suggests a role for this assay in determining the antiplatelet effects of these and related new therapeutics in clinical settings.

We report a microfluidic chip-based hydrodynamic focusing approach that minimizes sample volume for the analysis of cell-surface interactions under controlled fluid-shear conditions. Assays of statistically meaningful numbers of translocating platelets interacting with immobilized von Willebrand factor at arterial shear rates (∼1500 s(-1)) are demonstrated. By controlling spatial disposition and relative flow rates of two contacting fluid streams, e.g., sample (blood) and aqueous buffer, on-chip hydrodynamic focusing guides the cell-containing stream across the protein surface as a thin fluid layer, consuming ∼50 μL of undiluted whole blood for a 2-min platelet assay. Control of wall shear stress is independent of sample consumption for a given flow time. The device design implements a mass-manufacturable fabrication approach. Fluorescent labeling of cells enables readout using standard microscopy tools. Customized image-analysis software rapidly quantifies cellular surface coverage and aggregate size distributions as a function of time during blood-flow analyses, facilitating assessment of drug treatment efficacy or diagnosis of disease state.