Department of Pathology, University of Wisconsin-Madison, Madison, WI, USA.
J Thromb Haemost. 2012 Oct;10(10):2086-98. doi: 10.1111/j.1538-7836.2012.04892.x.
Although heparin possesses multiple mechanisms of action, enhanced factor Xa inhibition by antithrombin is accepted as the predominant therapeutic mechanism. The contribution of FIXa inhibition to heparin activity in human plasma remains incompletely defined.
To determine the relevance of FIXa as a therapeutic target for heparins, particularly serpin-independent inhibition of intrinsic tenase (FIXa-FVIIIa) activity.
PATIENTS/METHODS: Thrombin generation was detected by fluorogenic substrate cleavage. The inhibitory potencies (EC(50) s) of low molecular weight heparin (LMWH), super-sulfated LMWH (ssLMWH), fondaparinux and unfractionated heparin (UFH) were determined by plotting concentration vs. relative velocity index (ratio ± heparin). Inhibition was compared under FIX-dependent and FIX-independent conditions (0.2 or 4 pm tissue factor [TF], respectively) in normal plasma, and in mock-depleted or antithrombin/FIX-depleted plasma supplemented with recombinant FIX.
UFH and fondaparinux demonstrated similar potency under FIX-dependent and FIX-independent conditions, whereas LMWH (2.9-fold) and ssLMWH (5.1-fold) demonstrated increased potency with limiting TF. UFH (62-fold) and fondaparinux (42-fold) demonstrated markedly increased EC(50) values in antithrombin-depleted plasma, whereas LMWH (9.4-fold) and ssLMWH (two-fold) were less affected, with an EC(50) within the therapeutic range for LMWH. The molecular target for LMWH/ssLMWH was confirmed by supplementing FIX/antithrombin-depleted plasma with 90 nm recombinant FIX possessing mutations in the heparin-binding exosite. Mutated FIX demonstrated resistance to inhibition of thrombin generation by LMWH and ssLMWH that paralleled the effect of these mutations on intrinsic tenase inhibition.
Therapeutic LMWH concentrations inhibit plasma thrombin generation via antithrombin-independent interaction with the FIXa heparin-binding exosite.
肝素具有多种作用机制，抗凝血酶增强因子 Xa 抑制作用被认为是主要的治疗机制。FIXa 抑制作用对人血浆中肝素活性的贡献仍不完全明确。
确定 FIXa 作为肝素治疗靶点的相关性，特别是对内在凝血酶原酶（FIXa-FVIIIa）活性的无丝氨酸蛋白酶抑制剂抑制作用。
患者/方法：通过荧光底物裂解检测凝血酶生成。通过绘制浓度与相对速度指数（比率±肝素）的关系图，确定低分子量肝素（LMWH）、超硫酸盐化 LMWH（ssLMWH）、磺达肝癸钠和未分级肝素（UFH）的抑制效价（EC50s）。在正常血浆中，分别在 FIX 依赖性和 FIX 非依赖性条件下（分别为 0.2 或 4 pm 组织因子 [TF]）比较抑制作用，并在补充重组 FIX 的模拟耗尽或抗凝血酶/FIX 耗尽血浆中进行比较。
UFH 和磺达肝癸钠在 FIX 依赖性和 FIX 非依赖性条件下显示出相似的效价，而 LMWH（2.9 倍）和 ssLMWH（5.1 倍）在 TF 限制时显示出更高的效价。在抗凝血酶耗尽的血浆中，UFH（62 倍）和磺达肝癸钠（42 倍）的 EC50 值显著增加，而 LMWH（9.4 倍）和 ssLMWH（两倍）的影响较小，其 EC50 值在 LMWH 的治疗范围内。通过向 FIX/抗凝血酶耗尽的血浆中补充带有肝素结合外位点突变的 90nm 重组 FIX，证实了 LMWH/ssLMWH 的分子靶标。突变 FIX 对 LMWH 和 ssLMWH 抑制凝血酶生成的作用表现出抗性，这与这些突变对内在凝血酶原酶抑制的作用一致。
治疗性 LMWH 浓度通过与 FIXa 肝素结合外位点的抗凝血酶非依赖性相互作用抑制血浆凝血酶生成。
