Histone-induced platelet aggregation requires β3-integrins. (A) Histones induce platelet aggregation. PRP was mixed at t = 0 minutes with histones (200 μg/mL), ADP (50μM), or collagen (25 μg/mL) or left unstimulated. Histones induced aggregation as efficiently as ADP or collagen. (B) Histones bind to platelets. Fluorescence microscopy of platelets labeled in red incubated with BSA or histones labeled fluorescently in green. Histones localize to the platelet surface. Scale bar = 2 μm. (C) Quantification of histone or BSA binding to washed platelets by flow cytometry (***compared with BSA; n = 3). (D) Histones induce calcium influx into platelets. Platelets loaded with the calcium-sensitive dye Fluo-4 and stimulated with indicated concentrations of histones for 5 minutes. Platelets were resuspended in medium with or without CaCl₂, and fluorescence was analyzed by flow cytometry (**compared with or without Ca²⁺; n = 6). (E) Comparison of histone-induced aggregation of control mice (C57, red), CalDAG-GEF1 (black), or β3-integrin (green) deficient platelets, 5 minutes after stimulation with indicated concentrations of histones or ADP (50μM). CalDAG-GEF1 deficiency leads to an impaired platelet aggregation in response to histones, but it is less severe than β3-integrin deficiency. (F) Histone-induced platelet aggregation requires plasma proteins. Dose-dependent response of platelets to histones in the presence of plasma (black circles), 200 μg/mL fibrinogen (gray circles), or the response of washed platelets with buffer (white circle); n = 3; ***plasma compared with fibrinogen. (G) Exogenous CaCl₂ (2mM) enhanced HiPA. Data show extent of platelet aggregation 3 minutes after stimulation (n = 4). (H) Aggregation of washed platelets stimulated with 1μM recombinant histone H1, H2A, H2B, H3, or H4 in the presence of 200μg/mL fibrinogen. Histone H4 induced platelet aggregation potently. Data presented are representative of ≥ 3 independent experiments. *P < .05, **P < .01, and ***P < .001.