Abstract
Platelets play a crucial role in the immunological response and are involved in the pathological settings of vascular diseases, and their adhesion to the extracellular matrix is important to bring leukocytes close to the endothelial cells and to form and stabilize the thrombus. Currently there are several methods to study platelet adhesion; however, the optimal parameters to perform the assay vary among studies, which hinders their comparison and reproducibility. Here, a standardization and validation of a fluorescence-based quantitative adhesion assay to study platelet-ECM interaction in a high-throughput screening format is proposed. Our study confirms that fluorescence-based quantitative assays can be effectively used to detect platelet adhesion, in which BCECF-AM presents the highest sensitivity in comparison to other dyes.
Highlights
Platelet adhesion is a crucial step in bleeding control and in the thrombosis process
The balance between hemostasis and thrombosis depends on platelet-extracellular matrix (ECM) interaction: inefficient adhesion leads to bleeding, whereas excessive adhesion followed by platelet activation can prompt thrombus formation [2]
At high concentrations of BCECF-acetoxymethyl ester (AM) (16 μg/mL), there was no statistical difference between platelet adhesion to collagen-I compared to non-coated wells. These results demonstrate the feasibility of platelet adhesion measurement using BCECF-AM and confirm that platelet and BCECF-AM concentrations are important parameters to detect statistical differences between platelet adhesion to ECM-coated and non-coated wells
Summary
Platelet adhesion is a crucial step in bleeding control and in the thrombosis process. Circulating platelets show no interaction between them, neither with the internal surface of normal vessels, platelets promptly adhere to exposed extracellular matrix (ECM) proteins upon vascular injury or in pathological settings [1]. The balance between hemostasis and thrombosis depends on platelet-ECM interaction: inefficient adhesion leads to bleeding, whereas excessive adhesion followed by platelet activation can prompt thrombus formation [2]. Upon endothelial cell damage, depending on the depth of the injury, platelets come into contact with different ECM constituents [2]. Platelets can adhere to damaged endothelial cells [3], ECM is their main adhesive substrate. Platelet adhesion might influence atherosclerotic plaque progression and stability during the development of atherosclerosis [6]
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