Fig. 5. Fibrin polymerization and fibrinolysis in the presence of InsPs.

(A) For turbidity analysis of fibrin polymerization, fibrinogen purified from human plasma was incubated with 16 μM InsP₃ (grey line), InsP₅ (black line) or InsP₆ (red line) in the presence of 5 mM CaCl₂ and 0.1 U/ml human α-thrombin. The control without InsPs is shown by the green line. (B) Fibrinolysis was measured employing an adapted turbidity assay by addition of t-PA (100 pM) and plasminogen (0.24 μM) to the reaction described in (A). Changes in absorbency were monitored at 340 nm, every 12 s for 2 h at room temperature, using a microtiter plate reader. (C) Human plasma from three different donors (P1-P3), CaCl₂ (5 mM), Alexa Fluor 488 labeled fibrinogen (10%) and 16 μM InsP₃ (D), InsP₅ (E) or InsP₆ (F) were diluted in TBS. After addition of human α-thrombin (0.175 U/ml final concentration), the reaction mixture was immediately transferred into the channel of an Ibidi μ-slide VI^0.4. After fibrin network formation was completed, Z-stacks with 20 slices of 1 μm were recorded at room temperature using a confocal microscope (TCS SP5, Leica, Wetzlar, Germany) equipped with an HC PL APO CS2 63.0x1.40 OIL UV objective (Leica) and the following settings: zoom 3x, image size of 512 × 512, laser power of the 488 lasers was set 5 %. 3D reconstruction was performed using the ImageJ software [27]. The scale bars represent 25 μm.