CDL Research, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
Van Creveldkliniek, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands.
J Thromb Haemost. 2022 Feb;20(2):353-365. doi: 10.1111/jth.15554. Epub 2021 Oct 29.
Occlusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN-TaSER), consisting of a function-blocking V H against glycoprotein Ibα (GPIbα) and a thrombin-inhibiting serine protease inhibitor (SERPIN; α1-antitrypsin AIAR ) to interfere with platelet-driven thrombin formation.
To evaluate the antithrombotic properties of TaSER.
Besides TaSER, we generated three analogous control variants with either a wild-type antitrypsin subunit, a non-targeting control V H, or their combination. We investigated TaSER and controls in protease activity assays, (platelet-dependent) thrombin generation assays, and by western blotting. The effects of TaSER on platelet activation and von Willebrand factor (VWF) binding were studied by fluorescence-activated cell sorting, in agglutination studies, and in ATP secretion experiments. We studied the influence of TaSER in whole blood (1) on platelet adhesion on VWF, (2) aggregate formation on collagen, and (3) thrombus formation (after recalcification) on collagen and tissue factor.
TaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor-triggered thrombin generation and ATP secretion in platelet-rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet-dependent thrombin activity.
The synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications.
闭塞性血栓在组成上并不均匀。血栓的核心富含活化的血小板和纤维蛋白，而外壳则含有静止的血小板。这个核心无法与血浆蛋白接触。我们制备了一种融合蛋白（靶向 SERPIN-TaSER），由针对糖蛋白 Ibα（GPIbα）的功能阻断 V H 和抑制凝血酶的丝氨酸蛋白酶抑制剂（SERPIN；α1-抗胰蛋白酶 AIAR）组成，以干扰血小板驱动的凝血酶形成。
评估 TaSER 的抗血栓特性。
除了 TaSER，我们还生成了三种类似的对照变体，它们分别具有野生型抗胰蛋白酶亚基、非靶向对照 V H 或它们的组合。我们在蛋白酶活性测定、（血小板依赖性）凝血酶生成测定中以及通过 Western blot 研究了 TaSER 和对照物。通过荧光激活细胞分选、凝集研究和 ATP 分泌实验研究了 TaSER 对血小板活化和血管性血友病因子（VWF）结合的影响。我们研究了 TaSER 在全血中的影响：（1）对 VWF 上血小板黏附的影响，（2）对胶原蛋白上聚集物形成的影响，以及（3）对胶原蛋白和组织因子上血栓形成（再钙化后）的影响。
TaSER 结合血小板并抑制血小板表面的凝血酶活性。它阻断 VWF 结合并解聚血小板聚集物。TaSER 以靶向方式延迟血小板富含血浆中组织因子触发的凝血酶生成和 ATP 分泌。在流动研究中，由于 GPIbα 阻断，TaSER 干扰血小板黏附和聚集物形成，并由于靶向抑制血小板依赖性凝血酶活性而限制血栓形成。
TaSER 的个体特性之间的协同作用使其成为一种高效的抗血栓药物，可能具有临床意义。
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