Complement mediates binding of PF4/heparin complexes to B cells. (A) Effect of complement inhibition on PF4/heparin binding to B cells. Plasma and blood cells were separated from blood by centrifugation, subjected to conditions associated with complement inactivation (heat inactivation and treatment with 10 mM EDTA or ice), and exposed to antigen or buffer. Conditions A to E correspond to various incubation conditions. Mean ± SD from 3 independent experiments. **P < .005 compared with condition B. N, normal; HI, heat inactivated. (B) and (C) PF4/heparin binding to B cells correlates with C3c/C4c deposition. Percentage of double-positive cells, which represent KKO and C3c or C4c binding, appears in the right upper quadrant of dot plots (B) and as quantified in (C) from 3 independent experiments. *P < .05, and **P < .005 compared with buffer condition. (D) Complement fixation occurs primarily in the solution phase and not on the B-cell surface. Plasma and blood cells were separated, and sequence of B-cell binding was determined as described in “Methods.” Overlay histogram for KKO staining on the CD19-gated B cells is shown by the sequence of incubations as indicated in the key. (E) Binding of PF4/heparin complexes and complement to B cells from heparinized patients. Blood from 2 patients (P-1 and P-2) was stained concurrently for CD19, KKO, C3c, or C4c. Dot plots show binding of KKO and C3c/C4c binding on CD19⁺ gated cells. (F) Binding of PF4/heparin, C3c, and C4c to B cells over time in a patient treated with heparin. Shown is the percentage of KKO-positive/C3c-positive/C4c-positive B cells in the circulation of a heparinized patient during the course of heparin therapy. The x-axis shows time from start of heparin therapy, the y-axis shows % KKO + B cells, and the 2-headed arrow in the figure indicates duration of UFH therapy.