Characterization of thrombus consolidation in vitro. Lepirudin-anticoagulated human whole-blood was perfused through collagen-coated microslides at 1800 s−1. (A) To quantify thrombus volume, whole blood was preincubated with DiIC12 prior to perfusion, and three-dimensional images captured in real-time using an inverted Leica DMIRB confocal microscope, followed by off-line analysis to quantify thrombus volume, as described in “Three-dimensional volumetric thrombus analysis.” This graph depicts thrombus volume over time from 4 individual thrombi taken from 4 independent experiments. (B) Quantification of thrombus consolidation was performed by “spiking” whole blood with DiIC12-labeled platelets prior to perfusion, followed by capture of consecutive DIC and fluorescence images in real-time. (i) Images are taken from 1 flow representative of 12 independent flows. (ii) The decrease in distance between firmly adherent platelets was quantified as described in “Two-dimensional quantification of thrombus consolidation” and used as an indirect marker of thrombus contraction. Results are expressed as the mean plus or minus SD of 36 individual thrombi, from 12 independent experiments (n = 12; —).
