VITT Ab–mediated PLT-Leuk interactions are predominantly driven by the procoagulant platelet subpopulation. (A) Representative confocal images of WB from healthy individuals costained with anti-CD41 (PLTs, cyan), anti-CD15 (Neuts, yellow), and GSAO-AF647 (procoagulant PLTs, red) after incubation with SFLLRN (5 μM) and plasma from patients with VITT or HCs. As indicated, WB samples were pretreated with SYK inhibitor lanraplenib (5 μM) or vehicle control before incubation with SFLLRN and VITT plasma. Images were acquired at magnification ×63 with additional ×1 or ×8 digital zoom (n = 4). Scale bars, 20 and 2 μM, respectively. Representative images of independent experiments are shown. White arrows highlight GSAO-AF647–positive (red) ballooned procoagulant PLTs interacting with Neuts (yellow). (B-C) Dot plots and quantification of FC analysis from corresponding WB samples from panel [A]. CD62p (P-selectin)–positive PLTs were compared with (CD62p/GSAO double positive) procoagulant PLT events in the presence of lanraplenib (5 μM) or vehicle in (B) PLT-Leuk aggregates (CD41/CD45 double positive, [PLTs in aggregates]) and (C) single PLTs (CD41⁺/CD45⁻ events, [PLTs not in aggregates]). (D) Effect of lanraplenib pretreatment (expressed as ratio of PLT phenotype lanraplenib to control) on P-selectin expression and procoagulant PLT subpopulation within the PLT-Leuk aggregates (CD41/CD45 double positive) and PLTs not in aggregates (CD41⁺/CD45⁻ events) was compared. Note that CD62p–positive events are the combination of CD62p single-positive and CD62p/GSAO double-positive events. Combined data reported in bar graphs showing the distribution of the values from n = 4 individual plasma samples. Unpaired or paired t test is shown ∗P < .05. ns, nonsignificant.