In vitro distribution of endogenous hPF4 released from activated platelets in whole human blood in the absence and presence of heparin. TFLLR-NH2 was added to whole human blood. Total cell-surface–bound PF4 on RBCs, platelets (plts), monocytes (mono), neutrophils (neu), and lymphocytes (lymph) was detected using RTO and HIT antigenicity using KKO. (B) Effect of increasing amounts of heparin (0, 0.1, 0.4, and 100 U/mL) on RTO and KKO binding. (A,B) Data are expressed as increase of MFI after stimulation compared with unstimulated samples using distribution resolution metric (R_D), where value 0 means complete overlap and value ≥1 means complete separation of the distribution curves; values >0 mean increase in binding. Mean ± 1 standard deviation of the change in surface-bound PF4 against a hypothetical value of 0 for no change is shown. (A) N = 6. (B) N = 3. Distinct experiments performed in duplicate. *P < .05, ***P < .0005, ****P < .0001 using 2-way analysis of variance with Dunnett’s multiple comparisons test.