Effect of IAIP, HMW-HA, and bikunin on histone-induced platelet aggregation. (A) Washed platelets were incubated with r-H4 (20 µg/mL) with or without IAIP (50 to 200 µg/mL) or HMW-HA (100 to 500 µg/mL) before aggregometry. Thrombin (0.5 U/mL)–induced aggregation was measured as positive control. H4-induced platelet aggregation was decreased by IAIP in a concentration-dependent manner when platelets were suspended in plasma. (B) Washed platelets were incubated with integral (total), cryo-poor (CP) or IAIP-depleted (depl.) CP plasma containing or not 20 µg/mL r-H4. In some experiments, IAIP-depleted CP plasma was supplemented with IAIP (100 µg/mL), CRP (250 µg/mL), or albumin (Alb; 5 mg/mL). H4-induced aggregation was dependent on the amount of IAIP but not on CRP or albumin in plasma. (C-D) Washed platelets were incubated with r-H4 with or without increasing concentrations of (C) HMW-HA or (D) bikunin. (C) HMW-HA had a dose-dependent inhibitory effect on histone-induced platelet aggregation, whereas (D) preincubation of H4 with bikunin did not affect platelet aggregation. Data are presented as mean ± SEM of 3 independent experiments using one-way ANOVA with Bonferroni’s multi-comparison test. ****P < .0001, ***P < .001, and **P < .01 compared with the H4 challenge group.