Effects of heme treatment of endothelial cells. (A) HUVECs treated with increasing doses of heme for 20 minutes and detached were labeled with anti-CD55, phycoerythrin-labeled anti-MCP, or anti-CD59 and analyzed by flow cytometry. Results are expressed as the percentage of the initial mean fluorescence intensity measured on untreated cells (mean ± SD; n = 3). (B) HUVECs treated with 100 µM heme for different times were detached at 4°C and labeled with anti–P-selectin (R&D Systems) (mean ± SD; n = 3). (C) Supernatants of cells were tested in a vWF-specific ELISA. Results are expressed as mean ± SD of triplicate wells from one of 3 similar experiments. (D) Schematic view of different nonexclusive mechanisms leading to complement activation on heme-treated cells. (1) Heme intercalates into the C3 molecule (C3*), activates complement in the fluid phase, and deposits C3b on nearby endothelial cells. (2) This C3b initiates the formation of a cell-bound C3 convertase, favored by the decrease of DAF and MCP membrane expressions on heme-treated cells, FH remaining as the major AP control protein. (3) Heme induces the mobilization of Weibel-Palade bodies with the secretion of vWF and the appearance of P-selectin able to bind C3b and to focus the AP amplification cycle. (4) Heme can also bind directly to the cell surface, where it activates complement as in the fluid phase. (5) Heme induces cell retraction, exposing the subendothelial matrix known to efficiently activate complement.