Effect of the WBC/platelet ratio on cell-surface–bound PF4 and HIT antigenicity in vitro. (A) PRP was prepared from PF4^null mouse blood. Recombinant hPF4 (100 µg/mL) and varying numbers of isolated total WBCs were added. PF4 bound to the surface of platelets was measured using KKO. Mean ± 1 standard deviation is shown. N = 3 per arm, each in duplicate; P < .05 compared with baseline value (no WBC cells added, dashed line) using Wilcoxon matched-pairs test. (B) Monocytes were depleted in PF4^null mice using clodronate liposomes. The indicated amounts of PF4 were added to monocyte-depleted or control nondepleted PF4^null blood, and platelet-bound PF4 was measured based on binding of KKO to platelets by flow cytometry. Relative binding in the monocyte-depleted blood compared with the nondepleted blood is shown at the indicated PF4 concentrations with 1 = no difference (dashed line). Mean ± 1 standard deviation is shown. N = 3 mice per arm. P < .05 compared with baseline value (nondepleted blood) using Wilcoxon matched-pairs test.