PF4/heparin complexes bind preferentially to B cells in the peripheral blood. Whole blood from healthy donors was incubated with buffer or antigen (PF4 with or without heparin) followed by staining for cell-specific markers and KKO. Mean ± standard deviation (SD) of 3 independent experiments is shown. (A) Flow cytometric analyses of peripheral blood leukocytes. Leukocyte subpopulations were defined by forward scatter and side scatter characteristics (lymphocytes, neutrophils, and monocytes). Gated subpopulations were identified by cell-surface markers for T lymphocytes (CD3), B lymphocytes (CD19), neutrophils (CD66b), and monocytes (CD14) on the x-axis and for KKO staining on the y-axis for each antigen. The percentage of KKO-positive cells for each cell lineage appears in the upper right corner of each graph. (B) Graph of cell lineage–specific staining of flow data from (A). Binding of KKO to cell lineages incubated with antigen is shown. (C) ImageStream analysis of PF4/heparin binding to B cells. Cell-surface binding of PF4/heparin complexes is shown by imaging flow cytometry using fluorescently labeled cell-surface markers (CD3-APC, CD19-PE, CD14-APC, and CD66b-APC) and KKO-AF488 (green). For clarity, colors were reassigned to show T cells in red, B cells in yellow, monocytes in purple, and neutrophils in blue. (D) Graph of cell lineage–specific staining of ImageStream data from (C). (E) PF4/heparin complexes bind preferentially to B cells from multiple healthy donors. CD19⁺ cells were gated for KKO binding for various antigens. The graphs show percentage of KKO-positive B cells (mean ± SD) in various conditions from 6 healthy donors. Each colored symbol represents % KKO + B cells in an individual donor. (F) Kinetics of PF4/heparin binding to B cells. Whole blood was incubated with antigen for defined time points and stained with KKO/CD19. For each condition, time is shown on the x-axis and binding of KKO shown on the y-axis. **P < .005 compared with other data in graph.