Haynes Laura M, Orfeo Thomas, Mann Kenneth G, Everse Stephen J, Brummel-Ziedins Kathleen E
Department of Biochemistry, Robert Larner M.D. College of Medicine, University of Vermont, Colchester, Vermont.
Haematologic Technologies, Essex Junction, Vermont.
Biophys J. 2017 Apr 25;112(8):1634-1644. doi: 10.1016/j.bpj.2017.03.002.
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 = 2.8 μM) and ∼0.3 molecules of thrombin at high-affinity binding sites (K = 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 × 50 × 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 < 1) and reaction rates are driven by enzyme kinetics (P = 100, D = 7000). A subpopulation of active thrombin remains stably adhered to a fibrin matrix over a range of venous shear rates (46-184 s) 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, 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.
在纤维蛋白形成的封闭系统模型中，外位点介导的凝血酶与纤维蛋白结合有助于凝块稳定，并且对抗凝血酶/肝素的抑制具有抗性，同时仍易受小的活性位点抑制剂的影响。每个纤维蛋白分子可以在低亲和力结合位点（K = 2.8 μM）结合约1.6个凝血酶分子，在高亲和力结合位点（K = 0.15 μM）结合约0.3个凝血酶分子。本研究的目的是评估静脉血流条件下纤维蛋白结合的凝血酶的稳定性，并确定其对抑制的可及性和敏感性。使用凝血酶特异性荧光底物SN-59（100 μM）开发了一种平行板流动腔（7×50×0.25 mm），用于研究在各种静脉血流条件下粘附于纤维蛋白基质（0.1 - 0.4 mg/mL纤维蛋白原，10 nM凝血酶）的凝血酶（0 - 1400 nM）的稳定性。该系统内的流动是层流（R < 1），反应速率由酶动力学驱动（P = 100，D = 7000）。在一系列静脉剪切速率（46 - 184 s）下，有一部分活性凝血酶在纤维蛋白基质上稳定粘附超过30分钟，并且在负载>500 nM时该部分是可饱和的，且对初始纤维蛋白原浓度敏感。这些观察结果也得到了凝血酶与纤维蛋白粘附的数学模型的支持，该模型表明凝血酶最初结合到低亲和力凝血酶结合位点，然后优先平衡到更高亲和力位点。抗凝血酶（2.6 μM）加肝素（4 U/mL）在92 s时约10分钟后抑制72%的与凝块结合的活性凝血酶，而在没有肝素的情况下未观察到抑制作用。达比加群（20和200 nM）可逆地抑制（50%和93%）与凝块结合的凝血酶（恢复率87%和66%）。该模型表明，与凝块结合的凝血酶稳定性是凝血酶分子在密集结合位点基质中不断重排的结果。
