Measuring Single-Cell Platelet Forces via Microcontact-Printed, Reference-Free Traction Force Microscopy Reveals Relationships between Cell Shape, F-Actin Localization, and Force

Abstract

Platelets bind, spread, and contract to stop bleeding and reinforce hemostatic plugs. Platelet contraction occurs when interactions between actin and myosin create cytoskeletal tension and force generation. In this study, single-cell platelet forces were measured using a novel microcontact-printed, reference-free traction force microscopy approach. This approach facilitates single-cell force measurement on a contiguous topology that enables unrestricted platelet spreading without necessitating platelet removal to calculate force. Microcontact printing is used to deposit a grid of fluorescent bovine serum albumin onto a flexible polydimethylsiloxane substrate. Platelets from healthy donors were washed and seeded onto von Willebrand Factor-coated substrates. Platelets were fixed, stained, and imaged via confocal microscopy to simultaneously visualize fluorescently labeled platelets and grid displacement (used to calculate single-cell force.) Because microcontact-printing facilitates printing the grid on large, scalable surfaces, platelet forces were able to be measured on >100 single-cells per donor (N = 6 donors). Donor-to-donor platelet force variability was examined, and some donors produce significantly more force per platelet than other donors (p<0.01). Even within each donor, ∼15-fold variability in single platelet forces exists. A well-established area-force relationship (more spread cells produce more force) accounts for some variability in force generation, but for cells spread equally, there is still ∼5-fold differences in force generation. After examining the relationship between cell shape and force, a relationship was observed wherein platelets that are more circular produce more force. Additionally, amongst spread platelets, platelets with more spread F-actin produce more force. This versatile method that facilitates measurement of single-cell forces over large areas without restricting cell spreading or requiring cell removal reveals donor variability, cell shape, and F-actin localization as contributors to the large variability of single-platelet forces.