CAREER: Understanding the Contraction Biomechanics of Platelets at the Single-Cell Level

Emory University, Atlanta GA

Investigators

Abstract

1150235 lam The goal of this project is to understand the contraction mechanics and dynamics of individual platelets, the blood cells responsible for the initiation of clot formation, in order to improve the fields of medicine and bioengineering. During the formation of blood clots, activated platelets interact with growing networks of fibrin polymers and contract against this fibrin scaffold. Although this platelet-driven clot retraction is known to be acto-myosin mediated, extremely little is known about the underlying biomechanical aspects of platelet contraction, due in part to technological limitations. Bulk assays of clot retraction exist but cannot directly measure platelet contractility at the single cell level, which is necessary to obtain a mechanistic understanding of the contraction process. In addition, as fibrin has recently been shown to have extremely complex material and mechanical properties, single platelet studies would enable the decoupling of the fibrin effects from platelets when examining the cellular biomechanics of clot formation. Therefore, a clear need exists for robust, single cell assays of platelet contraction. We recently published the first technique to achieve this goal using a modified atomic force microscopy system to directly and quantitatively measure the contraction mechanics and dynamics of single platelets. Although the atomic force micrscopy technique is highly sensitive, given the known wide physiologic heterogeneity and variability among individual platelets, the throughput must be improved to obtain a comprehensive understanding of the underlying cellular biomechanical mechanisms of platelet contraction. To those ends, Dr. Wilbur Lam and his laboratory will apply microfabrication techniques to develop a high-throughput "biomechanical flow cytometer" that simultaneously measures the contractility of multiple platelets in a single experiment. Then, Dr. Lam and his colleagues will quantitatively investigate the mechanistic relationship between the biomechanical and biological aspects of platelet contraction to obtain a comprehensive, cellular biomechanical understanding of that process. The education objectives of this proposal are to: create a K-12 science outreach program for hospitalized children in which their own specific diseases are used as motivation and springboards for learning about science; use hospital-based supplies and equipment these children are accustomed to for hands-on science enrichment as part of this outreach; enable undergraduate and graduate students to implement this outreach program; and integrate cellular biomechanics concepts into this program, emphasizing that medicine is interdisciplinary and involves biology, physics, chemistry, and math. Intellectual Merit: Clot formation occurs in three phases: 1) platelet aggregation at the site of injury, 2) formation of a fibrin polymer embedding platelets at that site, and 3) platelet-driven clot retraction/contraction. While the first two phases have been well characterized, extremely little is known about the last phase. As clots are exposed to a wide range of external forces in a hemodynamic environment and are spatially non-uniform, leading to a heterogeneity of mechanical microenvironments platelets might encounter, applying the concepts of cellular biomechanics to platelet contraction will vastly improve the overall basic understanding of clot formation. Indeed, our previous data suggest that platelet contraction is dependent on the mechanical properties of the underlying substrate. We will test the hypothesis that the mechanical microenvironment interacts with the known signaling pathways that mediate platelet contraction. To that end, we will build upon Dr. Lam's atomic force microscopy technique to enable the higher throughput experiments needed to quantitatively investigate the mechanics of platelet contraction. Broader Impact: These will be the first reported experiments that quantitatively investigate the cellular biomechanics of platelet contraction and the results will significantly improve the overall understanding of platelet physiology and clot formation. In addition, these studies will have broad reaching implications as platelets are not only involved in clotting but also in numerous other biological processes (e.g., infections, inflammation, cardiovascular disease, stroke, and cancer) and the biocompatibility of implanted biomaterials. Diagnostics assessing platelet function are currently based only on platelet aggregation and as such, the proposed microsystem will form the basis for a new category of platelet function testing. Furthermore, the microsystem will potentially serve as a drug discovery platform for diseases associated with dysfunction in platelet contractility, such as cardiovascular disease and stroke. Combined with the innovative education program geared towards science education of hospitalized children, this interdisciplinary program will have a lasting impact on cellular biomechanics, basic hematology, and biomedical engineering, and biomaterials.