Histone H3 and H4 binding to F1 and F2 of prothrombin competes with FVa. Computer prediction using the ZDOCK server shows that H3 (PDB: 4HGA) (A), H4 (PDB: 4HGA) (B), and H2B (PDB: 5FUG) (C) bind to prothrombin (PDB: 4HZH). Specifically, H3 and H4 recognize prothrombin F1 and F2, while H2B recognizes the protease domain (thrombin). (D) Schematic representation of F1, F2, and protease domains of prothrombin. (E) Coomassie blue–stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel showing purified prothrombin F1 and F2 produced in BL21 bacteria. (F) Binding affinities (K_d) of H2B, H3, and H4 to prothrombin F1 and F2 by SPR kinetic assay. Means ± SD are presented from 3 independent experiments. N/C, none calculable. Prothrombinase activity from FXa activation of prothrombin in the presence of phospholipids (40% phosphatidylcholine, 20% phosphatidylserine, and 40% phosphatidylethanolamine) and FVa, with or without either H4 (G) or H3 (H). All reactions were performed for 90 seconds in the presence of calcium (5 mmol/L) and terminated by the addition of EDTA (10 mmol/L). Data are represented as a percentage of classical prothrombinase activity (mean ± SD from 3 independent experiments). Student t test shows a significant decrease in prothrombinase activity compared with the absence of histones (*P < .05) and a significant increase in prothrombinase activity compared with H3 or H4 alone (^#P < .05). Arrow indicates the order in which the competing reagents were added. (I) Thrombin generation in recalcified FV-deficient PPP ± H4 (50 µg/mL).