Measuring Platelet Contributions to Blood Clot Viscoelasticity Using Acoustic Radiation Force

Blood clotting is an enzymatically and cellularly regulated process of stiffening the blood to prevent blood loss. Despite the life-preserving function blood clotting serves in the body, inappropriate blood clot stiffness has been associated with life-threatening diseases such as stroke, hemorrhage, and heart attack. The relationship between clot stiffness and vascular diseases underscores the importance of identifying the mechanisms by which blood clots stiffen. The primary mediators of clot stiffening are fibrin and platelets. Fibrin, when polymerized in a blood clot, provides a protein scaffold to which platelets subsequently bind. Mechanical properties of fibrin have mainly been investigated in the absence of platelets. Research on platelet activation has mainly been examined outside of the context of a fibrin network. It would be desirable to link fibrin clot properties and platelet biology. My dissertation describes the development of an in vitro method to measure blood clot stiffness and its application to quantifying the contributions of both fibrin network formation and platelet activation. What is unique about my approach is that I apply ultrasound acoustic radiation force to push on microscopic beads suspended in blood and use microscopy imaging to analyze the resulting bead motion to assess blood clot mechanical properties. To link macro scale clot mechanics to clot microstructure, I have also established an in vitro model of blood plasma clotting where platelet-fibrin interactions are visible by several microscopy techniques. With the clot stiffness assessment device and clot microstructure assays I have been investigating the role of platelet enzymatic or adhesive function on clot stiffness and clot structural properties. The experiments differentiate the modulation of clot mechanical properties and structures by platelet thrombin generation from the clot changes due to integrin IIb-IIIa mediated platelet adhesion. Linking platelet thrombin generation and adhesion to modulations of clot mechanical properties and fibrin clot architecture will facilitate identification of targets of anti-platelet drugs that could ultimately prevent thrombosis without interrupting hemostasis.