Increased platelet density helps sustain calcium flux in aggregating platelets. Washed platelets loaded with the caged calcium chelator NP-EGTA and calcium indicator dyes (see “Analysis of cytosolic calcium flux”) were reconstituted with RBCs (to a final concentration of 150 × 10⁶/mL or 5 × 10 × ⁶ /mL) and perfused through microcapillary tubes coated with VWF (50 μg/mL) at 1800 s⁻¹. A transient elevation of cytosolic calcium was induced in translocating platelets by uncaging with brief exposure to near UV light. (A) Proportion of high Δ[Ca²⁺]_c platelets (displaying transient or sustained calcium oscillations at a platelet density of 5 × 10⁶/mL or 150 × 10⁶/mL [n = 3]). Notably, all of the cells were observed to undergo transient stationary adhesion events at a platelet density of 5 × 10⁶/mL. (B) Single-channel Oregon green fluorescence images showing translocating platelets prior to uncaging (0 seconds) and the elevation in intracellular calcium levels following UV exposure (1.8 seconds). The left panels show a representative platelet uncaged at low density, where the platelet remains stationary for a period and then continues translocation when calcium levels decrease. The middle panels show a representative platelet at high density, where calcium levels remain elevated after uncaging and the platelet remains stationary and becomes a focal point for recruitment of other platelets into an unstable aggregate (highlighted in the higher-magnification panels on the right-hand side). (C) After uncaging, platelets remained stationary while calcium levels remained elevated. The duration of the stationary period was less in platelets at low density compared with those uncaged at high density (n = 50; bar represents the median; cutoff time was 48 seconds).