Platelet traction forces are highly sensitive to partial myosin IIA inhibition by blebbistatin (BBT). (A) Assay format for traction force microscopy. Elastomeric polydimethylsiloxane (PDMS) micropost arrays were microcontact-printed with fibrinogen at their tops and passivated with bovine serum albumin (BSA) elsewhere. A single platelet specifically adheres to, spreads over, and pulls at several microposts, as mediated by αIIbβ3 integrins and the actomyosin cytoskeleton. Scale bars: 2 μm. (B) Fluorescence images of f-actin (green) of fixed washed human platelets on fibrinogen-functionalized micropost arrays (red) after spreading for 60 minutes in the absence/presence of subsaturating concentrations of BBT. Indicated are total single-cell traction forces. Scale bars: 5 μm. (C) Total traction force per cell (left), mean traction force per post (middle), and spreading area (right) of washed platelets on micropost arrays at subsaturating concentrations of BBT. The dashed line indicates the measurement force resolution of individual posts. Mean values and SD from 4 independent biological replicates with 24 to 94 cells per condition are shown. Statistical tests were performed using one-way analysis of variance with Bonferroni correction for multiple comparisons; only P values of <.05 are indicated. (D) Frequency histograms of total traction forces per platelet at subsaturating concentrations of BBT. Histograms were subdivided into noncontractile cells (<7 nN; light gray) and cells with medium (7-40 nN; dark gray) or high (>40 nN; pink) contractility. The frequency of subpopulations with different levels of contractility is indicated; note that the peak of noncontractile cells was cropped to maintain a consistent y-axis scaling. Data were pooled from 4 independent biological replicates; the total number of cells is indicated as n.