To cite this article: Onasoga-Jarvis AA, Puls TJ, O'Brien SK, Kuang L, Liang HJ, Neeves KB. Thrombin generation and fibrin formation under flow on biomimetic tissue factor-rich surfaces. J Thromb Haemost 2014; 12: 373-82.Summary. Background: Blood flow regulates coagulation and fibrin assembly by controlling the rate of transport of zymogens, enzymes and plasma proteins to and from the site of an injury. Objective: The objective of this work was to define the hemodynamic conditions under which fibrin can form under flow on tissue factor (TF)-rich substrates. Methods: TF-coated silica beads (~800 nm) were patterned into 18-85-lm spots. Normal pooled plasma and factors VIII, IX and XI deficient plasmas were perfused over the beads coated with 0.08, 0.8 and 8 molecules-TF lm À2 at shear rates of 50-1000 s À1 . Fibrin deposition and thrombin generation were measured by fluorescence microscopy in a hydrodynamic focusing microfluidic device. Results and Conclusions: Fibrin deposition was supported on patterned bead spots, but not planar TF substrates at the same surface TF concentration. There was a threshold spot size and a shear rate dependent TF concentration that was necessary to support fibrin polymerization. FVIII and FIX had minor effects on fibrin dynamics at 8 molecules-TF lm À2, but were essential at 0.8 molecules-TF lm À2 . The absence of FXI influenced thrombin generation and fibrin deposition at both 0.8 and 8 molecules-TF lm À2 . These results show that fibrin deposition requires perturbations in the flow field that protect reactions from dilution by flow under venous and arterial conditions. FVIII and FIX have a modest effect on fibrin deposition at high TF concentrations, but are necessary for fibrin deposition at low TF concentrations. FXI amplifies thrombin generation under flow at both low and high TF concentrations.

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s−1. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5–30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1–5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.