Thrombus formation with different platelet fractions. Platelet- and leukocyte-depleted whole blood was reconstituted with unsorted, large, small, or small (mass adjusted) platelets and perfused for 4 minutes at a wall shear rate of 1000 s⁻¹ over a coated microspot containing collagen type I. Thrombus build-up was evaluated by image capturing every 30 seconds during the 4 minutes of blood perfusion and end point analysis after postlabeling and perfusion. (A) Representative brightfield images (upper row) and fluorescent images of DiOC₆ (middle row) or P-selectin expression (lower row) of the different platelet fractions after 4 minutes of perfusion. (B-C) Quantification of thrombus build-up (kinetics, n = 6) with brightfield images of platelet deposition (B) and fluorescence images of DiOC₆ deposition (C). Correction for platelet surface differences (∼30% between large and small platelets¹⁰ ) would increase surface area coverage (SAC) of small platelets to similar values as the determined SAC for mass-adjusted small platelets. (D-E) Quantification of endpoint analysis (n = 6) of brightfield images of multilayer deposition (D) and fluorescence images of P-selectin expression (E). (F) Heatmap representing each parameter for the respective platelet size subpopulation at the end of the measurement. Unsorted platelets were set at 0 for reference. n = 4. *P < .05 and ****P < .0001 vs unsorted.