Probing the Dynamics of Clot-Bound Thrombin at Venous Shear Rates
In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes to clot stability and is resistant to inhibition by antithrombin/heparin while still susceptible to small, active-site inhibitors. Each molecule of fibrin can bind ~1.6 thrombin molecules at low-affinity binding sites (K^sub d^ = 2.8 μM) and ~0.3 molecules of thrombin at high-affinity binding sites (K^sub d^ = 0.15 μM). The goal of this study is to assess the stability of fibrin-bound thrombin under venous flow conditions and to determine both its accessibility and susceptibility to inhibition. A parallel-plate flow chamber (7 x 50 x 0.25 mm) for studying the stability of thrombin (0-1400 nM) adhered to a fibrin matrix (0.1-0.4 mg/mL fibrinogen, 10 nM thrombin) under a variety of venous flow conditions was developed using the thrombin-specific, fluorogenic substrate SN-59 (100 μM). The flow within this system is laminar (R^sub e^ < 1) and reaction rates are driven by enzyme kinetics (P^sub e^ = 100, Da = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46-184 s^sup -1^) for upwards of 30 min, and this population is saturable at loads >500 nM and sensitive to the initial fibrinogen concentration. These observations were also supported by a mathematical model of thrombin adhesion to fibrin, which demonstrates that thrombin initially binds to the low-affinity thrombin binding sites before preferentially equilibrating to higher affinity sites. Antithrombin (2.6 μM) plus heparin (4 U/mL) inhibits 72% of the active clot-bound thrombin after ~10 min at 92 s^sup -1^, while no inhibition is observed in the absence of heparin. Dabigatran (20 and 200 nM) inhibits (50 and 93%) clot-bound thrombin reversibly (87 and 66% recovery). This model illustrates that clot-bound thrombin stability is the result of a constant rearrangement of thrombin molecules within a dense matrix of binding sites..
| Medienart: |
Artikel |
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| Erscheinungsjahr: |
2017 |
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| Erschienen: |
2017 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:112 |
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| Enthalten in: |
Biophysical journal - 112(2017), 8, Seite P1634 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Laura M Haynes [VerfasserIn] |
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| Links: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
OLC1994697830 |
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| 520 | |a In closed system models of fibrin formation, exosite-mediated thrombin binding to fibrin contributes to clot stability and is resistant to inhibition by antithrombin/heparin while still susceptible to small, active-site inhibitors. Each molecule of fibrin can bind ~1.6 thrombin molecules at low-affinity binding sites (K^sub d^ = 2.8 μM) and ~0.3 molecules of thrombin at high-affinity binding sites (K^sub d^ = 0.15 μM). The goal of this study is to assess the stability of fibrin-bound thrombin under venous flow conditions and to determine both its accessibility and susceptibility to inhibition. A parallel-plate flow chamber (7 x 50 x 0.25 mm) for studying the stability of thrombin (0-1400 nM) adhered to a fibrin matrix (0.1-0.4 mg/mL fibrinogen, 10 nM thrombin) under a variety of venous flow conditions was developed using the thrombin-specific, fluorogenic substrate SN-59 (100 μM). The flow within this system is laminar (R^sub e^ < 1) and reaction rates are driven by enzyme kinetics (P^sub e^ = 100, Da = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46-184 s^sup -1^) for upwards of 30 min, and this population is saturable at loads >500 nM and sensitive to the initial fibrinogen concentration. These observations were also supported by a mathematical model of thrombin adhesion to fibrin, which demonstrates that thrombin initially binds to the low-affinity thrombin binding sites before preferentially equilibrating to higher affinity sites. Antithrombin (2.6 μM) plus heparin (4 U/mL) inhibits 72% of the active clot-bound thrombin after ~10 min at 92 s^sup -1^, while no inhibition is observed in the absence of heparin. Dabigatran (20 and 200 nM) inhibits (50 and 93%) clot-bound thrombin reversibly (87 and 66% recovery). This model illustrates that clot-bound thrombin stability is the result of a constant rearrangement of thrombin molecules within a dense matrix of binding sites. | ||
| 650 | 4 | |a Thrombin | |
| 650 | 4 | |a Flow | |
| 650 | 4 | |a Matrix methods | |
| 650 | 4 | |a Mathematical models | |
| 650 | 4 | |a Stability analysis | |
| 650 | 4 | |a Fibrinogen | |
| 650 | 4 | |a Flow stability | |
| 650 | 4 | |a Enzyme kinetics | |
| 650 | 4 | |a Molecules | |
| 650 | 4 | |a Inhibition | |
| 650 | 4 | |a Affinity | |
| 650 | 4 | |a Fibrin | |
| 650 | 4 | |a Antithrombin | |
| 650 | 4 | |a Mathematical analysis | |
| 650 | 4 | |a Biophysics | |
| 650 | 4 | |a Reaction kinetics | |
| 650 | 4 | |a Laminar flow | |
| 650 | 4 | |a Kinetics | |
| 650 | 4 | |a Binding sites | |
| 650 | 4 | |a Loads (forces) | |
| 650 | 4 | |a Balancing | |
| 650 | 4 | |a Heparin | |
| 700 | 0 | |a Thomas Orfeo |4 oth | |
| 700 | 0 | |a Kenneth G Mann |4 oth | |
| 700 | 0 | |a Stephen J Everse |4 oth | |
| 700 | 0 | |a Kathleen E Brummel-Ziedins |4 oth | |
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