The PF4 binding inhibitor is heat sensitive and binds to Sepharose. (A) GFPs were incubated with 25 µg/mL PF4 in the presence of 20% untreated or heat-treated (56°C or 100°C) serum. Data are mean ± SD of 3 independent experiments. (B) GFPs were incubated with 25 µg/mL PF4 in the presence of 20% untreated serum, IgG-depleted serum (using protein G Sepharose), or IgG-depleted serum substituted with purified IgG according to the original IgG serum concentration. Data are mean ± SD of 10 independent experiments. (C) GFPs were incubated with 25 µg/mL PF4 in the presence of 20% untreated serum, depleted serum (using heparin Sepharose), or depleted serum substituted with 100 µg/mL fibronectin (FN) or 50 µg/mL vitronectin (VN). Data are mean ± SD of 3 (depleted serum + FN or VN, n = 2) independent experiments. (A-C) PF4 binding was detected with an FITC-labeled anti-human PF4 antibody using flow cytometry, and antibody binding was quantified by GMFI. Surface-bound PF4 in the presence of buffer was defined as 100%. *P <.05, **P < .01 vs buffer.