B4218 1

We used a one-stage aPTT-based clotting assay to measure the F8 coagulation activity with a Sysmex CA1500 system (Sysmex, Kobe, Japan). Siemens reagents (Siemens; Marburg, Germany) including Dade Actin activated cephaloplast in reagent (Siemens; B4218-1) and coagulation F8-deficient plasma (Siemens; OTXW17) were used. The mouse plasma samples were diluted with Dade Owren’s Veronal Buffer (Siemens; B4234-25) by a factor of 4. The F8 activity was performed by mixing 5 μl of diluted mouse sample plasma with 45 μl of Dade Owren’s Veronal Buffer and 50 μl of F8-deficient plasma and 50 μl of aPTT reagent (Dade Actin activated cephaloplast in reagent), followed by a 120-s incubation at 37 °C. Coagulation was initiated after the addition of 50 μl of 25 mM calcium chloride. Time to clot formation was measured by the Sysmex CA1500 system. A standard curve was prepared by diluting the human calibration plasma (Siemens), and the plasma obtained from wild-type mice served as a positive control.

Factor VIII coagulation activity (FVIII:C) was assessed using a one-stage clotting assay on a Sysmex CA1500 system analyzer (Sysmex, Kobe, Japan). First, plasma samples were diluted fourfold and tested with the aPTT reagent (Dade Actin activated cephaloplastin reagent; Siemens; B4218-1) and factor VIII deficient plasma (Siemens; OTXW17) from Siemens (Siemens; Marburg, Germany), with a normal range of 74–112%. During the measurement, 5ul of diluted mouse plasma sample was mixed with 45 µl of Dade Owren’s Veronal Buffer (Siemens; B4234-25), 50 µl of factor VIII deficient plasma, and 50 µl of aPTT reagent. The mixture was incubated at 37 °C for 120 s to generate factor XIa. Subsequently, 50 µl of 25 mM calcium chloride was added for factor IXa and thrombin generation, leading to clot formation. The Sysmex CA1500 system recorded the clot formation time, which was compared to a standard curve obtained by diluting human calibration plasma (Siemens).