Multiple mechanisms mediate thrombin generation and consequently the thrombin concentration present during fibrin formation. First, the levels of pro-and anticoagulants present during coagulation strongly influence procoagulant activity. For example, elevated levels of prothrombin are associated with increased thrombin generation, 35, 42 formation of dense fibrin networks, 35 and increased venous thrombus weight in mice. 43 These studies, designed to model the clinical situation in humans with the G20210A prothrombin mutation associated with increased circulating prothrombin levels, 44 suggest that increased thrombin generation enhances venous thrombosis risk in part by promoting abnormal fibrin deposition and structure. Second, the location of thrombin generation impacts fibrin network formation. Effective assembly and activity of the prothrombinase complex (factors Xa, Va, and prothrombin) requires a lipid surface. 45 Localization of prothrombinase on a cell surface establishes a thrombin concentration gradient that influences both fibrin formation and network structure. In vitro experiments using in situ thrombin generation on fibroblasts and endothelial cells reveal a significantly denser fiber network proximal versus distal to the cell surface. 29 These structural differences give rise to substantially different fibrinolytic susceptibilities in different regions of the clot; fibrin located near the cell surface is significantly more resistant to lysis than fibrin located distal to the cell surface. 29 Third, blood flow (shear) present during fibrin formation influences local thrombin concentrations by (re) supplying procoagulant proteins and removing activated enzymes. 46 Flow also aligns fibrin fibers, 31, 32 which may have profound effects on fibrin formation and mechanical and fibrinolytic stability. 47 Furthermore, the shear rate affects clot formation triggered on tissue factor-plus collagen-coated plates, resulting in different fibrin deposition in different regions of a thrombus. 48 Nanoindentation analysis to evaluate clot biophysical properties shows that this fibrin distribution pattern determines clot microelasticity, which may impact thrombus stability and risk of embolization. 48 Fourth, thrombin movement through the thrombus is substantially influenced by solute transport mechanisms mediated by cell packing density; this may also influence the amount of fibrin deposition in different regions of the clot. 49