Centre for Blood Research and Department of Pathology and Laboratory Medicine, University of British Columbia , Vancouver, BC, Canada.
Centre for Drug Discovery, Inter University Centre for Biomedical Research & Super Speciality Hospital , Kottayam, Kerala, India.
Biomacromolecules. 2017 Oct 9;18(10):3343-3358. doi: 10.1021/acs.biomac.7b00994. Epub 2017 Sep 19.
Heparins are widely used to prevent blood clotting during surgeries and for the treatment of thrombosis. However, bleeding associated with heparin therapy is a concern. Protamine, the only approved antidote for unfractionated heparin (UFH) could cause adverse cardiovascular events. Here, we describe a unique molecular design used in the development of a synthetic dendritic polycation named as universal heparin reversal agent (UHRA), an antidote for all clinically used heparin anticoagulants. We elucidate the mechanistic basis for the selectivity of UHRA to heparins and its nontoxic nature. Isothermal titration calorimetry based binding studies of UHRAs having different methoxypolyethylene glycol (mPEG) brush structures with UFH as a function of solution conditions, including ionic strength, revealed that mPEG chains impose entropic penalty to the electrostatic binding. Binding studies confirm that, unlike protamine or N-UHRA (a truncated analogue of UHRA with no mPEG chains), the mPEG chains in UHRA avert nonspecific interactions with blood proteins and provide selectivity toward heparins through a combined steric repulsion and Donnan shielding effect (a balance of F and F). Clotting assays reveal that UHRA with mPEG chains did not adversely affect clotting, and neutralized UFH over a wide range of concentrations. Conversely, N-UHRA and protamine display intrinsic anticoagulant activity and showed a narrow concentration window for UFH neutralization. In addition, we found that mPEG chains regulate the size of antidote-UFH complexes, as revealed by atomic force microscopy and dynamic light scattering studies. UHRA molecules with mPEG chains formed smaller complexes with UFH, compared to N-UHRA and protamine. Finally, fluorescence and ELISA experiments show that UHRA disrupts antithrombin-UFH complexes to neutralize heparin's activity.
肝素被广泛用于手术中防止血液凝结和治疗血栓。然而，肝素治疗相关的出血是一个令人关注的问题。鱼精蛋白是唯一被批准用于非分级肝素（UFH）的解毒剂，但可能引起不良心血管事件。在这里，我们描述了一种独特的分子设计，用于开发一种名为通用肝素逆转剂（UHRA）的合成树枝状聚阳离子，它是所有临床使用的肝素抗凝剂的解毒剂。我们阐明了 UHRA 对肝素的选择性和非毒性的机制基础。基于等温滴定量热法的结合研究表明，具有不同甲氧基聚乙二醇（mPEG）刷状结构的 UHRA 与 UFH 的结合，随着溶液条件（包括离子强度）的变化而变化，这表明 mPEG 链对静电结合施加了熵罚。结合研究证实，与鱼精蛋白或 N-UHRA（没有 mPEG 链的 UHRA 的截断类似物）不同，UHRA 中的 mPEG 链避免了与血液蛋白的非特异性相互作用，并通过空间排斥和 Donnan 屏蔽效应（F 和 F 的平衡）提供了对肝素的选择性。凝血试验表明，带有 mPEG 链的 UHRA 不会对凝血产生不利影响，并且可以在广泛的 UFH 浓度范围内中和 UFH。相反，N-UHRA 和鱼精蛋白显示出内在的抗凝活性，并且在 UFH 中和的狭窄浓度窗口内显示。此外，我们发现 mPEG 链调节解毒剂-UFH 复合物的大小，这是通过原子力显微镜和动态光散射研究揭示的。与 N-UHRA 和鱼精蛋白相比，带有 mPEG 链的 UHRA 分子与 UFH 形成较小的复合物。最后，荧光和 ELISA 实验表明，UHRA 破坏抗凝血酶-UFH 复合物以中和肝素的活性。
