(A–D) Cellular composition of whole blood and thrombi. (A) Cell types were determined by morphology and cell-specific antibodies in flow cytometry. The diagram presents the percentage of RBCs (white), platelets (black), WBCs (light gray), and cells that could not be precisely identified (other, dark gray) (n = 3). (B) Semithin section through a middle layer of a thrombus. RBCs, some indicated by black arrowheads, are spread throughout the thrombus. Scale bar: 10 μm. (C) Scanning electron microscopy of thrombi. Collagen fibers indicated by white arrowheads, platelets indicated by black arrows, and RBCs indicated by black arrowheads (n = 3). Scale bars as indicated. (D) Thrombi from mice after injury of the carotid artery were isolated and stained with hematoxylin and eosin to visualize RBCs in arterial thrombi. Representative images are shown. Overview in upper panel, details in lower panel. Scale bars: 100 μm (upper panel) and 20 μm (lower panel). (E and F) Effects of hemoglobin on thrombus formation ex vivo. (E) Representative images. Scale bar: 50 μm. (F) Mean surface coverage for PRP (2 × 10⁵ platelets/μl), PRP + RBCs (4 × 10⁶ RBCs/μl), and PRP + hemoglobin (13 mg/ml) at a shear rate of 1,000 s^–1 (n = 3). (G) ATP release from RBCs, platelets, and ghosts was induced by L-arginine and detected by HPLC. A total of 4 × 10⁹ RBCs, an equivalent amount of ghosts, and 2 × 10⁸ platelets were used (n = 3). (H) Ghost preparation was visualized by microscopy for quality inspection. Scale bar: 50 μm. (I and J) Thrombus formation in the presence of RBCs or ghosts and PRP at a shear rate of 1,000 s^–1. (I) Representative images. Scale bar: 50 μm. (J) Mean surface coverage per visual field (n = 3). Bar graphs depict mean values ± SEM. Plts, platelets; WBCs, white blood cells. Student’s t test (J).