Microheology of fibrin clots

An active particle tracking microrheology technique has been developed to study the viscoelastic properties of human fibrin and plasma clots. In order to perform microrheology measurements, a magnetic microrheometer device has been adapted and a technique developed, following the procedure of Evans et al., to measure the frequency dependent viscoelastic moduli (G() and G()). This technique has been supported by complementary investigation methodologies, such as protein analysis, turbidity, and multiple microscopy techniques. As a result of this study new insights into the viscoelastic dynamics of fibrin have been revealed. Three stress relaxation mechanisms, as predicted by Morse et al. for networks of semi-flexible fibres, were observed and occur on distinctly different timescales. The scaling of the tension dominated contribution was measured to scale as G ~ c2.7 0.2 in agreement with the prediction of Mackintosh. The presence of FXIII resulted in stiffer less deformable clots but was found to have no effect on the viscoelastic dynamics of clots. Frequency measurements of the loss tangent revealed that on timescales intermediate between stress relaxation modes clots were much more susceptible to permanent deformation. The effect of fibrinogen, thrombin and calcium on the viscoelastic behaviour of clots was also investigated. Increased fibrinogen levels produced clots which displayed predominantly elastic behaviour on shorter time-scales. The molecular mechanism underpinning the role of fibrinogen γ in fibrin clot polymerisation, structure and viscoelasticity was also investigated. We report new data which show that fibrinogen γ is associated with the formation of mechanically weaker, non-uniform clots composed of thinner fibres. This is caused by direct disruption of protofibril formation by and not through thrombin inhibition or binding to FXIII. In addition, the effects of the plasma proteins FVIIa, FIXa, FXIIa and FXIII on clot properties are also reported.