Free Hb increases platelet adhesion to ECM, collagen, and fibrin(ogen) at high shear stress. (A) Citrated whole blood containing purified human HbA (0.0-500 mg/dL) was perfused over a surface coated with ECM at high shear stress (60 dyne/cm²). Each anti-GPIbα antibody (SZ2, AN51, or 6D1, 20 μg/mL) effectively blocked (85%-95%) platelet adhesion in the presence of acellular Hb. Under similar flow conditions, blood mixed with 500 mg/dL (∼300 μM heme) free Hb was perfused over (B) human collagen type III, or (C) human fibrinogen. Buildup of the fluorescently labeled platelets was recorded during the 3-minute perfusion. The graphs depict adhered/accumulated fluorescently labeled platelets (AF) as a function of time, and are representative of 4 to 10 separated experiments. (D) The enhancement of platelet adhesion to collagen by free Hb (500 mg/mL) was significantly reduced when VWF was diminished in the perfused whole blood. The bar graph shows the adhesion of platelets after 3-minute perfusion (mean ± SD) (*P < .0001).