Effect of FXIIa on fibrin fiber density, independent from additional thrombin formation. (A-B) Plasma deficient in FXII and prothrombin was reconstituted with FXII. Sulfatides and Alexa Fluor–488 fibrinogen were added, and clotting was initiated with 0.625nM thrombin, in the presence of phospholipid vesicles and CaCl₂. Final concentrations were 25% plasma, 5% Alexa Fluor–488 fibrinogen of the total fibrinogen concentration, FXII (0% or 100% of normal plasma concentration), 0.4μM sulfatides, 4μM phospholipid vesicles, 5mM CaCl₂, 25mM HEPES (pH 7.4), and 150mM NaCl. Per condition, 2 separate clots were made, and pictures were taken in different areas of the clot. (C-D) Prothrombin-deficient plasma, in the presence of hirudin, was incubated with the FXIIa inhibitor CTI or with α-FXIIa. Alexa Fluor–488 fibrinogen was added to the plasma, and clotting was initiated by the addition of ancrod and CaCl₂. Final concentrations were 25% plasma, 30nM hirudin, 75 μg/mL CTI, 30nM α-FXIIa, 5% Alexa Fluor–488 fibrinogen of the total fibrinogen concentration, 0.1 U/mL ancrod, 5mM CaCl₂, 25mM HEPES (pH 7.4), and 150mM NaCl. (B,D) Fiber density was calculated from the data shown in the corresponding panel. Per condition, 2 separate clots were made, pictures were taken in different areas of the clot, and fiber density was determined in 5 pictures by counting the number of fibers that crosses a line of 100 μm. Bars represent mean ± SEM. *P < .05. Scale bars represent 25 μm.