During coagulation, thrombin rapidly generates fibrin while fibrin potently sequesters thrombin. It has been shown previously that thrombin becomes trapped within fibrin fibers during coagulation but not much is known about the mechanisms of thrombin retention, its impact on the stability of fibrin, or thrombin’s activity while bound. This co-regulation was studied in fibrin gels formed under isotropic conditions and whole blood and plasma clots formed under flow. A 384-well plate thermal shift assay (TSA) with SYPRO-orange provided melting temperatures (Tm) of thrombin, PPACK-thrombin, fibrinogen, fibrin monomer, and fibrin. Treatment of fibrinogen with fibrin polymerization inhibitor GPRP increased fibrinogen stability, similar to the ΔTm when fibrinogen was converted to fibrin monomer or to fibrin. Addition of PPACK-thrombin at high 5:1 molar ratio to fibrin (or fibrinogen) had little effect on fibrinogen or fibrin Tm values, indicating that thrombin binding does not detectably stabilize fibrin via a putative bivalent E-domain to γ’-domain interaction. In a subsequent study, the capabilities of clot-bound thrombin were probed with a microfluidic whole blood clotting on collagen/tissue factor, followed by buffer wash, and a start/stop cycling flow assay using the thrombin fluorogenic substrate, Boc-Val-Pro-Arg-AMC. After 3-min of clotting (100 s-1) and 5-min buffer wash, non-elutable thrombin activity was easily detected during cycles of flow cessation. Non-elutable thrombin was similarly detected in plasma clots or arterial whole blood clots (1000 s-1). Reaction-diffusion simulations predicted 108 nM thrombin within the clot. Addition of heparin-antithrombin (AT) required over 6 min to inhibit the thrombin for whole blood clots, indicating a substantial diffusion limitation. In contrast, heparin-AT rapidly inhibited thrombin within microfluidic plasma clots, indicating marked differences in fibrin structure and functionality between plasma clots and whole blood clots. Perfusion of AF647-fibrinogen over washed FITC-fibrin clots resulted in an intense red layer around, but not within, the original FITC-fibrin. Similarly, introduction of plasma/AF647-fibrinogen generated substantial red fibrin masses that did not penetrate the original green clots, demonstrating that fibrin cannot be re-clotted with fibrinogen. Overall, thrombin within fibrin is non-elutable, easily accessed by peptides, slowly accessed by average-sized proteins (heparin/AT), and not accessible to fresh fibrinogen.