The impact of LL37 on platelet activation, spreading, and calcium mobilization. (A) The level of fibrinogen binding was analyzed using fluorescein isothiocyanate–conjugated fibrinogen antibodies by flow cytometry in human isolated platelets (i) or PRP (ii). (B) Similarly, the level of P-selectin exposure was measured in human isolated platelets (i) or PRP (ii) using PECy5-labeled P-selectin antibodies. Data represent mean ± SEM (n = 3). (C) Platelet adhesion and spreading on immobilized fibrinogen was analyzed using platelets treated with LL37 (5, 10, and 20 µM) by confocal microscopy (magnification ×60; bar represents 10 µm). The number of adhered (i) and spread (ii) platelets, and the relative surface area of spread platelets (iii) was determined via analyzing the images using ImageJ. Ten random fields of view were recorded and analyzed for each sample. Data represent mean ± SEM (n = 3). (D) Ca²⁺ mobilization was measured using Fluo-4 am dye-loaded human isolated platelets upon stimulation with LL37 by spectrofluorimetry. Data represent mean of maximum level of Ca²⁺ ± SEM (n = 4). (E) The cytotoxic effects of LL37 were measured in human isolated platelets using a lactate dehydrogenase cytotoxicity assay kit. Data represent mean ± SEM (n = 4). The statistical significance was established by 1-way ANOVA followed by Bonferroni’s correction (*P < .05; **P < .01; ***P < .001).