Maurizio Tomaiuolo - US grants

Project summary Thrombin is a critical element of the hemostatic/thrombotic response, as evidenced by the large number of clinically relevant pro- and anti-coagulant therapies designed to regulate its generation or activity. Thrombin regulation is not a purely biochemical matter, but rather it emerges from the interaction of the biochemical cascade with the evolving physical microenvironment (i.e., platelet deposition). As such, in order to determine how reaction rates of the coagulation cascade may be impacted inside of a hemostatic (or thrombotic) mass we need to study the tightly-woven interaction between the biochemical reactions responsible for thrombin generation and the physical environment in which they occur. Our primary objective is to answer a fundamental question: can the narrow pores of a hemostatic mass operate as a ?molecular barrier? and terminate thrombin generation? If so, this would represent an understudied mechanism mediated by platelets and/or fibrin, and the structure they form following accumulation, at a site of injury. The hypothesized molecular barrier results from the hindered movement of soluble species through the evolving hemostatic mass microenvironment. Hemostatic masses are defined by a complex network of mesoscopic scale pores with dimensions of a few to tens of nanometers, and as a result, biochemical reactions relevant to clotting occur in extremely confined spaces. Previous studies explored the idea that the physical environment of a hemostatic plug may contribute to regulating the hemostatic response, but an accurate knowledge of the microstructure of a hemostatic mass remains elusive. Our proposed studies will address this bottleneck by combining novel volume imaging electron microscopy methods of hemostatic masses with artificial intelligence methods to create anatomically realistic domains for simulations of coagulation biochemistry. In Aim #1, in collaboration with Dr. Weisel (letter attached), we will acquire sequential image stacks of hemostatic masses formed in vivo using correlative multi-photon fluorescence and Focused Ion Beam Scanning Electron microscopy. In Aim #2, we employ artificial intelligence methods to perform accurate image-driven 3D reconstruction of hemostatic mass microarchitectures, using the image stacks generated in Aim #1. As part of a related research project, we have already acquired an initial set of transmission electron microscopy images of hemostatic thrombi at single-platelet resolution to guide our initial computational efforts. In Aim #3, we will use the reconstructions obtained to examine how the hemostatic mass microarchitecture impedes molecular transport. We will evolve simulations to systematically evaluate how pore size and molecule size interact to regulate molecular diffusion. Finally, we will ask whether limitations in molecular transport through the hemostatic mass are responsible for the termination of thrombin production at a local level. If confirmed, this mechanism will represent a fundamental shift in the way we understand the role of platelet activation and accumulation, the hallmarks of hemostasis and thrombosis.