Force Generation and Cytoskeletal Structure of Single Platelets

Abstract

Cardiovascular disease is the most common cause of death worldwide, and one in four deaths is related to dysfunctional blood clotting. Platelet forces are an emerging metric for the balance of clotting and bleeding due to recent demonstrations of their powerful abilities to predict bleeding risk in trauma patients and detect bleeding dysfunction more sensitively than all existing clinical tests. While existing methods have indicated the clinical and scientific potential of platelet forces, they have been hampered by low-yield, inability to co-measure immunofluorescent cell markers, and/or arbitrary restriction of cell spreading. To address these limitations, we developed a technique (dubbed “black dots”) that enables high-yield co-measurement of cellular forces and immunofluorescent-labeled cell markers in a single image without constraining cell spreading. Applying black dots to measure single-platelet forces, we identify biophysical factors that associate with force generation, determine the effects of platelet storage conditions on function, and identify unique cytoskeletal morphologies induced by different key blood proteins. As a result of the high yield and high resolution of data obtainable with black dots, approaches including multivariate mixed effects modeling, K-means clustering, and machine learning were able to be applied to elucidate complex relationships between platelet activation, structure, and force generation. Identifying factors that associate with platelet forces, characterizing effects of storage temperature on platelet force generation, and elucidating platelets’ complex reaction to activating proteins has implications in bleeding, clotting, and transfusion medicine.