Enhancing effect of serum is not complement, varies between NHS, and requires divalent cations for interaction with platelets. (A) Respiratory burst activity of PMN toward B2G1-opsonized platelets in NHS was not diminished after heat inactivation of complement. NHS, deficient in mannan-binding lectin, was also capable of enhancing phagocyte responses to B2G1-opsonized platelets. NHS served as a negative control. (B) The respiratory burst activity of PMN toward B2G1-opsonized platelets was enhanced by some but not all 14 different NHS compared with unopsonized platelets. (C) Chelation of divalent cations from NHS with EDTA (5 mM) ablates the ability of serum to induce respiratory burst of PMN triggered by B2G1-opsonized platelets. (D) The serum factor, enhancing IgG-specific PMN respiratory burst activity toward platelets binds directly to platelets in a calcium-dependent manner. Platelets were preincubated with NHS or anti-HPA-1a FNAIT sera, washed with HEPES with or without Ca²⁺, and resuspended in HEPES with or without Ca²⁺ as indicated. Data are representative of 3 independent experiments, showing mean ± standard deviation. Statistical comparisons were performed as follows: (A) 1-way ANOVA with Tukey’s posttest; (B) paired t test; and (C-D) 2-way ANOVA with Bonferroni posttest. *P ≤ .05; **P ≤ .01; ***P ≤ .001.