Cellular activation exposes phoshorylcholine after oxidation, enabling CRP binding and phagocytosis. (A) Platelet FcγRIIa was not involved in the opsonization of platelets with FNAIT serum. Platelets were pretreated with blocking anti-CD32-Fab antibody (clone 7.3), opsonized with anti-HPA-1a–containing FNAIT sera (NHS as negative control), washed, and resuspended in PBS (negative control) or NHS. Thereafter, the platelets were added to PMN and the respiratory burst was measured. (B) CRP fluorescein isothiocyanate bound directly to B2G1-opsonized platelets, and this process required both calcium and platelet activity at 37°C, as measured by flow cytometry. (C) Neutralization of CRP with pneumococcal CWPS in NHS disabled CRP induction of the respiratory burst toward B2G1-opsonized platelets. (D) Binding of FNAIT serum-opsonized platelets (HPA-1a⁺/HPA-5b⁻) to CRP was further investigated by cSPR imaging, with CRP spotted to 3 sensor surface spots and 3 bovine serum albumin control spots. A specific response was observed for platelets to the CRP spots, which was enhanced if the platelets were opsonized with anti-HPA-1a antibodies from FNAIT serum (also compared with platelets incubated with anti-HPA-5b antibodies from FNAIT serum), but blocked by CWPS, indicating that platelet phosphorylcholine is the ligand for CRP that is exposed by antibody binding. (E) Binding of platelets to CRP was inhibited by DPI, a broad spectrum oxidation inhibitor including NADPH oxidase, as well as by Rotenone, which more specifically inhibits the mitochondrial electron transport chain (F). Conversely, stimulation of platelet oxidation by glucose and glucose oxidase enhanced platelet binding to CRP (G). Each line in D-G represents the average sensorgrams and standard deviation from 3 spots monitored simultaneously in real time. Statistical comparisons were performed as follows: (H) CRP enhancement of IgG-mediated phagocytosis of platelets by neutrophils was inhibited by DPI and not by Rotenone. Data are representative of 3 independent experiments, showing mean ± standard deviation. (A,C,D,H) 1-way ANOVA with Tukey’s posttest; (B) 2-way ANOVA with Bonferroni posttest; (E-G) 1-tailed paired t test. *P ≤ .05; **P ≤ .01; ***P ≤ .001.