Fibrin Clot Formation and Characterization

Fibrin clots of 50 μl volume were prepared in duplicate: fibrinogen (at concentration in the range 1.5 – 6 μM) in 10 mM HEPES buffer pH 7.4 containing 150 mM NaCl and the additives (arginine or CPB) was clotted with 20 nM thrombin at 37 °C for 30 min. Thereafter clots were placed into 10 mL 100 mM Na-cacodylate pH 7.2 buffer for 24 h at 4 °C. Following repeated washes with the same buffer, samples were fixed in 1%(v/v) glutaraldehyde for 16 h. The fixed samples were dehydrated in a series of ethanol dilutions (20 – 96%(v/v)), 1:1 mixture of 96%(v/v) ethanol/acetone and pure acetone followed by critical point drying with CO₂ in E3000 Critical Point Drying Apparatus (Quorum Technologies, Newhaven, UK). The specimens were mounted on adhesive carbon discs, sputter coated with gold in SC7620 Sputter Coater (Quorum Technologies, Newhaven, UK) and images were taken with scanning electron microscope EVO40 (Carl Zeiss GmbH, Oberkochen, Germany). The SEM images were analyzed to determine the diameter of the fibrin fibers using self-designed program functions running under the Image Processing Toolbox v. 8.2 of Matlab 8.1.0.604 (R2013a) (The Mathworks, Natick, MA) as previously described [12] (link), [31] (link).