Ca1500 system

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.

The Sysmex CA-1500 system (Japan) and fresh citrated plasma samples were used for the following tests: activated partial thromboplastin time (APTT), international normalized ratio (INR), thrombin time (TT) and concentration of fibrinogen. Cell count was performed in EDTA whole blood samples with a hematology analyzer ABX Pentra 60 (Horiba, Japan). The following parameters were analyzed: hemoglobin, erythrocyte, platelet, total leukocyte, neutrophil, eosinophil, basophile, lymphocyte and monocyte counts. Blood chemistry analyses were performed using an RX Imola (Randox, UK) and an Advia 1200 (Siemens, Germany) chemistry analyzers (Siemens, Germany). Glucose concentration, alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine and urea were measured with the RX Imola analyzer. Cholesterol concentration analysis was performed on the Advia 1200 analyzer.