1. Probing the Dynamics of Clot-Bound Thrombin at Venous Shear Rates
Laura M. Haynes, Thomas Orfeo, Kenneth G. Mann, Stephen J. Everse, Kathleen E. Brummel-Ziedins 
Biophysical Journal 
Volume 112, Issue 8, Pages (April 2017)
DOI: /j.bpj
Copyright © 2017 Biophysical Society Terms and Conditions

2. Figure 1
Fibrin structure as a function of initial fibrinogen concentration. SEM: Fibrin was formed by incubating (A) 2.5 mg/mL fibrinogen (physiologic concentration) or (B) 0.2 mg/mL fibrinogen (subphysiologic concentration) with thrombin (10 nM) for 2 min at 25°C. SEM was performed as described in Materials and Methods and obtained at an accelerating voltage of 20 kV and a magnification of 23,000×. Scale bar in the upper-right-hand corner of each panel represents 1 μm. (C) Confocal microscopy: Fibrin was formed by incubating 0.2 mg/mL Alexa Fluor 647-conjugated fibrinogen with thrombin (10 nM) for 1 min at 25°C. Scale bar in the upper-right-hand corner represents 10 μm.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

3. Figure 2
Stable incorporation of exogenous thrombin into a preformed fibrin matrix. (A) Flow profiles of SN-59 hydrolysis: Fibrin matrices were formed by incubating fibrinogen (0.2 mg/mL, 588 nM) with thrombin (10 nM) at 25°C in the bottom of the flow chamber. After washing the fibrin matrix with HBS containing 0.1% PEG and 5 mM Ca2+ pH 7.4 (900 μL), it was incubated with additional exogenous thrombin (200 nM, solid, n = 3 or 1400 nM, dark shaded, n = 3 for 15 min; or with a buffer alone, light shaded, n = 3). After further washing, the thrombin-specific fluorescent substrate SN-59 (100 μM) was flowed over the chamber at 92 s−1. Fluorescence intensity values resulting from SN-59 hydrolysis are reported as a function of time as the mean ± SE. (B) Thrombin binding isotherms: The concentration of fibrin bound thrombin is plotted versus the initial or loading concentration of thrombin (total thrombin). For the experimentally determined isotherm (solid circles), rates of SN-59 hydrolysis required to maintain each steady-state level of fluorescence were calculated and converted to concentrations of bound thrombin for a series of thrombin loading concentrations (0–1400 nM). Each data point is the average of two to three independent measurements. For the computationally derived isotherm (open circles), given a preformed fibrin matrix constructed from 588 nM fibrinogen, concentrations of bound thrombin were calculated for a series of thrombin loading concentrations (0–1400 nM) (see Materials and Methods). (Inset) Theoretical binding isotherm reflecting an identical titration in a closed system. Note the difference in scale.
Biophysical Journal  , DOI: ( /j.bpj )
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4. Figure 3
Steady-state fluorescence intensity does not significantly increase as a function of number of thrombin binding sites. (A) Fibrin was formed by incubating fibrinogen (0.1 mg/mL, n = 3, light shaded; 0.2 mg/mL, n = 3, solid; 0.4 mg/mL, n = 3, dark shaded) with thrombin (10 nM) for 1 min at 25°C and an additional 80 pmol of exogenous thrombin were incorporated into the fibrin matrix by incubating for 15 min at 25°C. SN-59 (100 μM) was flowed over the chamber at 92 s−1, and fluorescence intensity resulting from SN-59 hydrolysis (mean ± SE) is reported as a function of time. (B) The maximum fluorescence intensity from each curve was plotted as a function of initial fibrinogen concentration (mean ± SD). The difference in maximum fluorescence intensity between 0.1 and 0.4 mg/mL fibrinogen was statistically significant (Student’s t-test; p = 0.05).
Biophysical Journal  , DOI: ( /j.bpj )
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5. Figure 4
Absolute fluorescence intensity decreases as a function of increasing shear rate. Fibrin was formed by incubating fibrinogen (0.2 mg/mL) with thrombin (10 nM) for 1 min at 25°C and an additional 80 pmol of exogenous thrombin were adsorbed to the fibrin matrix. SN-59 (100 μM) was flowed over the chamber at 46 s−1 (open circles, n = 3), 92 s−1 (solid, n = 3), and 184 s−1 (shaded, n = 3). Fluorescence intensity resulting from SN-59 hydrolysis is reported as a function of time and as the mean ± SE. (Inset) A double reciprocal log plot of flow rate versus steady-state fluorescence intensity (mean ± SD) was fitted to a linear regression that has a slope of −1.0 ± 0.1.
Biophysical Journal  , DOI: ( /j.bpj )
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6. Figure 5
Steady-state fluorescence intensity correlates with recovered active thrombin. Thrombin was recovered from the flow chambers after selected experiments by incubating the fibrin with 1% Triton X-100 for 15 min at 25°C. The recovered thrombin showed a strong correlation with steady-state fluorescence intensity (R2 = , p < 0.001) as shown with the solid line (dotted lines indicate 95% confidence intervals).
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2017 Biophysical Society Terms and Conditions

7. Figure 6
Inhibition of clot-bound thrombin by AT-UFH and dabigatran. Fibrin was formed by incubating fibrinogen (0.2 mg/mL) with thrombin (10 nM) for 1 min at 25°C and an additional 80 pmol of exogenous thrombin was incorporated into the fibrin matrix by incubating for 15 min at 25°C. (A) SN-59 (100 μM) was flowed (92 s−1) through the chamber and allowed to establish equilibrium for ∼10 min. AT (2.6 μM; dark shaded) or AT with heparin (4 U/mL, light shaded) was added to the flowing SN-59 solution (first arrow). After an additional 10 min, the AT or AT with heparin was removed from the flowing solution (second arrow) and allowed to reequilibrate for 10 min. (B) In analogous experiments to those in (A), dabigatran (20 nM, dark shaded; 200 nM, light shaded) was introduced to the system. All data are presented as the mean ± SE (n = 3). The solid trace is reproduced from Fig. 2 and corresponds to the reaction in the absence of either AT (±UFH) or dabigatran.
Biophysical Journal  , DOI: ( /j.bpj )
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8. Figure 7
Models of both thrombin association and dissociation from the fibrin matrix reveal the dynamics of thrombin binding within a clot. (A) Thrombin binding to fibrin. Exogenous thrombin (200 nM) binding to the fibrin matrix was modeled as described in Materials and Methods assuming a total volume of 400 μL and the complete conversion of fibrinogen (0.2 mg/mL) to fibrin. Free thrombin (●) binds primarily to low-affinity thrombin binding sites (▼) but redistributes to high-affinity thrombin binding sites (▲) until equilibrium is reached. (B) Dissociation of thrombin from fibrin. The dissociation of thrombin was modeled using the equilibrium binding site occupations calculated in (A), in the absence of free thrombin except in the volume of the flow chamber (87.5 μL). The initial burst in free thrombin (●) is a result of thrombin dissociation from low-affinity thrombin binding sites (▼). This is followed by a slower partial repopulation of the low-affinity sites from the thrombin occupied high-affinity binding sites (▲). (C–E) Stepwise dissociation of thrombin from fibrin. The calculation shown in (B) was repeated after allowing the system to come to equilibrium for 13 s (flow cell volume passage time at 92 s−1) and resetting the free thrombin with each iteration. The majority of fibrin-bound thrombin is associated with high-affinity thrombin binding sites (▲) with the low-affinity thrombin binding sites (▼) providing an additional reserve of thrombin. The total occupied thrombin binding sites are also shown (■). After an initial reequilibration in the absence of thrombin, the free thrombin was reset to (C) 0%, (D) 10%, or (E) 90%. The washing steps present in the experimental procedure were not accounted for in these simulations. To see this figure in color, go online.
Biophysical Journal  , DOI: ( /j.bpj )
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