After vascular injury numerous chemical signals are released to induce platelet activation, coagulation, and post-hemostatic events. This thesis aims to investigate the interplay between thrombus structure and the spatiotemporal distribution and transport of biologically relevant solutes, and how this impacts thrombus formation in vivo. Using intravital microscopy we have previously described a characteristic architecture of thrombi formed in vivo. The architecture consists of a core of highly-activated and tightly packed platelets covered by a loose shell of less activated platelets. Initially, we developed a novel platelet-targeted sensor capable of reporting on thrombin activity, a potent platelet agonist, within thrombi formed ex vivo or in vivo. We found that thrombin activity was high in the core region, but restricted from the shell. We then designed another sensor capable of tracking soluble protein transport within thrombi formed in vivo, and found significant retention of soluble proteins within the platelets that would go on to form the core region. Using computational methods we found that the platelet packing density between the platelets restricted the diffusion of proteins within the core region, and allowed for rapid elution of proteins that made it to the shell. To test this in vivo we used mice with a defect in platelet retraction, but not platelet sensitivity to agonists. The mutant mice showed a much faster rate of solute elution using our transport sensor, and we also observed decreased platelet activation and thrombin activity within the thrombus. Next, we extended this model of thrombi as regulators of protein transport by examining how thrombus architecture altered the leakage of plasma proteins into the surrounding tissue. We found that extravascular solute gradients were sensitive to commonly used anti-platelet agents as well as small changes in platelet packing densities. Finally, we developed a new intravital imaging technique to visualize thrombus architecture formation in the mouse femoral artery and vein to extend our observations into the macrocirculation. Together, this thesis proposes a novel mechanism of thrombus regulation, which is dependent upon molecular transport properties shaped by the local hemodynamics and the intrathrombus microenvironment.