Space and Time Resolved Detection of Platelet Activation and von Willebrand Factor Conformational Changes in Deep Suspensions

Abstract

Tracking cells and proteins’ phenotypic changes in deep suspensions is critical for the direct imaging of blood-related phenomena in in vitro replica of cardiovascular systems and blood-handling devices. This paper introduces fluorescence imaging techniques for space and time resolved detection of platelet activation, von Willebrand factor (VWF) conformational changes, and VWF-platelet interaction in deep suspensions. Labeled VWF, platelets, and VWF-platelet strands are suspended in deep cuvettes, illuminated, and imaged with a high-sensitivity EM-CCD camera, allowing detection using an exposure time of 1 ms. In-house postprocessing algorithms identify and track the moving signals. Recombinant VWF-eGFP (rVWF-eGFP) and VWF labeled with an FITC-conjugated polyclonal antibody are employed. Anti-P-Selectin FITC-conjugated antibodies and the calcium-sensitive probe Indo-1 are used to detect activated platelets. A positive correlation between the mean number of platelets detected per image and the percentage of activated platelets determined through flow cytometry is obtained, validating the technique. An increase in the number of rVWF-eGFP signals upon exposure to shear stress demonstrates the technique’s ability to detect breakup of self-aggregates. VWF globular and unfolded conformations and self-aggregation are also observed. The ability to track the size and shape of VWF-platelet strands in space and time provides means to detect pro- and antithrombotic processes.