PB1172 - Using both passive and active microrheology to investigate local network mechanics of the fibrin clot

Background: The viscoelastic behaviour of the blood clot and its fibrin network involve a complex interplay of the mechanical behaviour at multiple levels of molecular and structural organisation. Previously we have investigated the mechanics of sub-micron single fibres by atomic force microscopy, and millimetre-centimetre sized clots on a bulk rheometer.

Aims: To investigate the viscoelastic behaviour at an important intermediate step, the microscale.

Methods: Clots were prepared from either fibrinogen or plasma samples. Final concentrations were 0.5 mg/ml fibrinogen or 1:10 diluted plasma, 0.1 IU/ml thrombin, 5 mM calcium. For active microrheology, 4.5 µm superparamagnetic beads were embedded in the clots at 1:250 (v/v), while for passive microrheology, 1 µm dark red polystyrene microparticles were embedded at 1:1000 dilution.

Results: In active rheology using magnetic tweezers, frequency-dependent viscoelastic properties (loss and storage moduli) of the clot were calculated from the resistance of the bead against the applied force, and its time-dependent recovery. This allowed application of higher forces advantageous for stiff clots later in development. However, the frequency range is limited by the speed the magnetic field can be modulated. For passive microrheology high frame-rates (6000 Hz) were used to track the thermal Brownian motion of multiple microbeads to a spatial precision of 10 nm. This extended the range of viscoelastic properties to frequencies >1kHz, into a more physiologically relevant regime not investigated before by rheological techniques. Additionally, passive microrheology can clearly quantify the increasing viscosity and early transition to increasing elastic modulus upon gelation, overlapping with the active micro-rheology and bulk rheology.

Conclusion(s): Traditional active microrheology measures the stress-response of fibrin clots at a relatively low range of frequencies from 0.01 Hz-25 Hz. High speed particle tracking substantially extends the range of our measurement to physiologically relevant frequencies while also providing new insights into the gelation of fibrin clots.