Effect of exogenous α-Def-1 on the kinetics of fibrin clot formation. (A) Effect of α-Def-1 on dynamic clot turbidity. Clotting of purified fibrinogen was initiated by adding an “activation mix” containing 0.07 U/mL human α-thrombin (see supplemental Methods for details), 0 to 10 µM α-Def-1, and 10 mM calcium chloride. Fibrin formation was evaluated by monitoring the change in turbidity (A₄₀₅) in the presence of 0 µM (solid purple line), 1 µM (green dashed line), 2.5 µM (blue dotted line), and 5 µM (red dashed line) synthetic α-Def-1. One experiment representative of 3 is shown. (B) Effect of α-Def-1 on the lag time and rate of fibrin polymerization. Fibrin formation was initiated as in panel A. Lag time, rate of polymerization, and maximum absorbance (Amax) were determined in reactions containing 0 to 5 µM synthetic α-Def-1 as in panel A. The mean ± SD of 4 experiments is shown. *P < .01, #P < .05. (C) Effect of α-Def-1 on thrombin amidolytic activity. Thrombin was added to PBS containing a chromogenic substrate in the absence (red dashed line and squares) or in the presence of 2 µM (blue line and circles) or 10 µM (green line and circles) α-Def-1. One experiment representative of 3 is shown. (D) Binding of α-defs to fibrinogen and fibrin. ¹²⁵I-α-Def-1 (5 µg/mL; 28 µM), twice the concentration found in fibrin clots (Figure 1G), was incubated with fibrinogen (100 µg/mL) (▲) or soluble fibrin⁸⁷  (100 µg/mL) (△) in 200 μL PBS or PBS alone (○) for 60 minutes at 24°C. The mixture was loaded onto a Sephacryl S-100 gel filtration column, and radioactivity and optical density (OD) at 280 nm in each 0.5-mL fraction eluted from the column were measured. One experiment representative of 3 in shown.