Previous studies have shown effects of thrombin and fibrinogen ?' on clot structure. However, structural information was obtained using electron microscopy, which requires sample dehydration. Our aim was to investigate the role of thrombin and fibrinogen ?' in modulating fibrin structure under fully hydrated conditions. Fibrin fibers were studied using turbidimetry, atomic force microscopy, electron microscopy, and magnetic tweezers in purified and plasma solutions. Increased thrombin induced a pronounced decrease in average protofibril content per fiber, with a relatively minor decrease in fiber size, leading to the formation of less compact fiber structures. Atomic force microscopy under fully hydrated conditions confirmed that fiber diameter was only marginally decreased. Decreased protofibril content of the fibers produced by high thrombin resulted in weakened clot architecture as analyzed by magnetic tweezers in purified systems and by thromboelastometry in plasma and whole blood. Fibers produced with fibrinogen ?' showed reduced protofibril packing over a range of thrombin concentrations. High-magnification electron microscopy demonstrated reduced protofibril packing in ?' fibers and unraveling of fibers into separate protofibrils. Decreased protofibril packing was confirmed in plasma for high thrombin concentrations and fibrinogen-deficient plasma reconstituted with ?' fibrinogen. These findings demonstrate that, in fully hydrated conditions, thrombin and fibrinogen ?' have dramatic effects on protofibril content and that protein density within fibers correlates with strength of the fibrin network. We conclude that regulation of protofibril content of fibers is an important mechanism by which thrombin and fibrinogen ?' modulate fibrin clot structure and strength. ? 2016 by The American Society of Hematology.