Development of Novel Ex Vivo Assays to Characterize the Mechanical Properties of Blood Plasma Clots

Blood coagulation is a life preserving process that operates in a narrow regime between thrombosis and hemorrhage. Inappropriate and inadequate blood clotting can lead to heart attack, stroke, and surgical bleeding, all potentially fatal conditions. Due to the large negative impact of inappropriate blood clotting, it is necessary to characterize normal plasma clot structure and properties to better understand abnormal conditions as well as develop strategies to control bleeding and prevent thrombosis.
Activation of the coagulation cascade transforms blood from a viscous liquid to a porous viscoelastic gel of crosslinked fibrin fibers and platelets. The ability, or inability, of a blood clot to perform its necessary function is linked to the mechanical properties of the fibrin/platelet network. This work describes two novel ex vivo assays to characterize the mechanical properties of blood plasma clots; a passive microrheology assay and an optical microscopy – ultrasound acoustic radiation force (ARF) coupled assay are detailed here.
The passive microrheology assay utilizes single particle tracking of microspheres dispersed within a small volume plasma sample (approximately 15 μL) to calculate the mean squared displacement (MSD) of the particle. MSD can be used to calculate the diffusivity of the particle and the viscoelastic modulus of the plasma sample. The passive microrheology assay can also be coupled with fluorescence microscopy of the fibrin network to establish structure-function relationships.
The ARF-based assay is a non-contact method of strain application in blood plasma clots. Acoustically reflective microspheres dispersed in the sample act as force transducers and strain gauges. Ultrasound ARF is used to induce microsphere displacement within a plasma sample and the displacement, coupled with a known magnitude of ARF application, is used to calculate clot stiffness. The assays described in this work can be used to characterize blood plasma clots under normal as well as perturbed conditions and demonstrate discernible differences in clot mechanical properties when platelet and fibrin polymerization inhibitors are used.