Fibrinogenesis and Fibrinolysis Followed with Nano-Thrombelastography

Abstract

Hemostasis is a complex process that relies on a sensitive balance between the formation and breakdown of the thrombus, a three-dimensional polymer network of the fibrous protein fibrin. Neither the details of the fibrinogen-fibrin transition, nor the exact mechanisms of thrombus degradation are fully understood at the molecular or supramolecular level. We investigated nanoscale changes in the viscoelasticity of the 3D-fibrin network during fibrinogenesis and streptokinase (STK)-induced fibrinolysis by using a novel, atomic-force-microscope (AFM)-based application of force spectroscopy, named nano-thrombelastography.Clot formation was initiated by adding Ca2+ to fresh, anti-coagulated mixed human plasma droplet on a glass surface. In order to induce fibrinolysis, STK, at a final enzyme activity of up to 10,000 IU was applied in situ. For measuring the nanoscale elastic and viscous properties of the fibrin network, the tip of an AFM cantilever was immersed in the plasma droplet and oscillated vertically with a constant rate. The cantilever bending was correlated with fibrin-clot elasticity and viscosity in time. Morphological changes were followed by scanning AFM on polymerized fibrin deposited on mica surface.Whereas the global features of the time-dependent change in cantilever deflection corresponded well to a macroscopic thrombelastogram, the underlying force spectra revealed large, sample-dependent oscillations in the range of 3-50 nN and allowed the separation of elastic and viscous components of fibrin behavior. Upon STK treatment the nano-thrombelastogram signal decayed gradually. The decay was driven by a decrease in thrombus elasticity, whereas thrombus viscosity decayed with a time delay. In scanning AFM images mature fibrin appeared as 17-nm-high and 12-196-nm-wide filaments. STK-treatment resulted in the decrease of filament height and the appearance of a surface roughness with 23.7 nm discrete steps that corresponds well to the length of a fibrinogen monomer.