Effect of FXII-concentration on fibrin structure in plasma. (A) FXII-deficient plasma was reconstituted with purified FXII, and clotting was initiated via contact activation with sulfatides, in the presence of phospholipid vesicles and CaCl2. Turbidity was monitored every 15 seconds at 405 nm at 37°C. Final concentrations were 76% plasma, variable FXII concentrations (0%-100% of normal plasma concentration), 4μM sulfatides, 4μM phospholipid vesicles, and 16mM CaCl2. Data are mean ± range of 3 measurements. (B) Representative figures of immunofluorescent staining of fibrin clots. FXII-deficient plasma was reconstituted with purified FXII. Alexa Fluor–488 fibrinogen was added, and clotting was initiated with sulfatide, phospholipid vesicles, and CaCl2. Final concentrations were 25% plasma, 5% Alexa Fluor–488 fibrinogen of the total fibrinogen concentration, a range of FXII (0%-100% of normal plasma concentration), 0.4μM sulfatides, 4μM phospholipid vesicles, 5mM CaCl2, 25mM HEPES (pH 7.4), and 150mM NaCl. (C) Fiber density was calculated from the data shown in panel B. Per condition, 2 separate clots were made, images were taken in different areas of the clot, and fiber density was determined in 5 images by counting the number of fibers that cross a line of 100 μm. Bars represent mean ± SEM. *P < .05. Scale bars represent 25 μm.
