Analysis of Morphology of Platelet Aggregates Formed on Collagen Under Laminar Blood Flow

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Abstract
In a focal injury model, platelets adhere and activate under flow on a collagen-coated surface, creating a field of individual platelet aggregates. These aggregates exhibit distinct structural characteristics that are linked to the local flow conditions. By combining image analysis techniques and epifluorescence microscopy, we developed a robust strategy for quantifying the characteristic instantaneous width and length of a growing platelet deposit. We have confirmed the technique using model images consisting of ellipsoid objects and quantified the shear rate-dependent nature of aggregate morphology. Venous wall shear rate conditions (100 s−1) generated small, circular platelet deposits, whereas elevated arterial shear rates (500 and 1000 s−1) generated platelet masses elongated twofold in the direction of flow. At 2000 s−1, an important regime for von Willebrand Factor (vWF)-mediated recruitment, we observed sporadic platelet capture events on collagen that led to rapidly growing deposits. Furthermore, inter-donor differences were investigated with respect to aggregate growth rate. After perfusion at elevated shear rates (1000 s−1) for 5 min, we identified a twofold increase in aggregate size (81.5 ± 24.6 μm; p < 0.1) and a threefold increase in growth rate parallel to the flow (0.40 ± 0.09 μm/s; p < 0.01) for an individual donor. Suspecting a role for vWF, we found that this donor had a twofold increase in soluble vWF relative to the other donors and pooled plasma. Microfluidic devices in combination with automated morphology analysis offer new tools for characterizing clot development under flow.