Fibronectin reduces anti-PF4/heparin antibody binding to PF4/heparin complexes by disrupting the complexes. (A) Sera from patients with anti-PF4/heparin IgG were tested in an in-house PF4/heparin ELISA in the presence of increasing concentrations of fibronectin, vitronectin, or heparin, which served as positive control. The signal of anti-PF4/heparin antibody binding without inhibitor was set as 100%. Data are mean ± SD of 5 (vitronectin = 3) independent experiments. (B) The amount of PF4 released from PF4/heparin complexes coated to the microtiter plate by fibronectin was determined by a commercially available ELISA kit. Data are mean ± SD of 3 independent experiments. *P < .05, **P < .01 vs released PF4 without fibronectin. (C) Size of 20 µg/mL PF4 alone and with 0.5 IU/mL UFH plus increasing concentrations of fibronectin was determined by PCS. Data (mean ± SD) represent the results of a minimum of 10 measurements per concentration of fibronectin. One representative experiment is shown. (D) Binding of PF4/heparin complexes (40 µg/mL PF4 + 1 IU/mL UFH) and serial 1:2 dilutions to immobilized fibronectin was monitored by SPR over 7000 seconds. Inset: Half-maximal binding of serial dilutions determines the KD value (dashed line). One representative experiment is shown. *P < .05, **P < .01, ***P < .001 vs antibody binding without inhibitor. d Hyd, hydrodynamic diameter; RU, resonance units.