Tissue type plasminogen activator

Thrombus formation under flow was measured as described previously [29 (link)] in whole blood from normal healthy individuals and FXIII-deficient patients. Cellix Vena8 Fluoro+ biochips were precoated with collagen (100 μg/mL) or fibrinogen (100 μg/mL). Whole citrated blood was labeled with 3,3’-dihexyloxacarbocyanine iodide (DiOC6; 4 μg/mL) and treated for 30 minutes prior to perfusion with either TGI (1 mM) or vehicle control (0.01% dimethylsulphoxide). Blood was perfused for 10 minutes to form platelet thrombi at shear rates of 500/s or 1000/s. In some cases, CaCl₂ (5.5 mM) and tissue-type plasminogen activator (20 nM; Sigma-Aldrich) were included with AF647-labeled fibrinogen (75 μg/mL; Life technologies) to form platelet-fibrin thrombi. Thrombi were allowed to form fully before switching flow to Tyrode’s containing tissue-type plasminogen activator (125 nM), and they were lysed to completion. Single z-slice images were taken every 2 to 4 seconds using a Nikon A1R fluorescence confocal microscope at 20× magnification. Data were analyzed using Image J by measuring fluorescence intensity of DiOC6 and AF647-fibrin(ogen) over time, which corresponded to thrombus size.

Clot growth and fibrinolysis in samples of PFP were assessed using a thrombodynamics analyzer and thrombodynamics kit (HemaCore, Moscow, Russia). PFP (120 μl) was thawed in a 37 °C water bath for 5 min. Plasma was placed into a plastic vial containing a lyophilized solution of corn trypsin inhibitor to prevent contact activation, followed by incubation with 4 nM of tissue-type plasminogen activator (Sigma-Aldrich, Dorset, United Kingdom) for 15 min at 37 °C within the thermostat of the analyzer and then the addition of a lyophilized solution of calcium salt. The sample was immediately transferred into an optically transparent cuvette with two thin channels, which were placed into the 37 °C temperature-controlled chambers of the instrument. Finally, an activating insert, the end edges of which were covered with immobilized TF to activate clotting, was gently placed fully into the cuvette. Growth and lysis of the fibrin clot were quantified using video microscopy software over a 60 min period (see Supplementary Figures 2A–C).