The thrombin inhibitors hirudin and Refludan® activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets
The thrombin inhibitors hirudin and Refludan® activate the soluble guanylyl cyclase and the cGMP pathway in washed human platelets
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DOI：
10.1160/th11-07-0461
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发表时间：
2012-03-01
影响因子：
6.7
通讯作者：
Walter, Ulrich
中科院分区：
文献类型：
--
作者：
Kobsar, Anna;Koessler, Juergen;Walter, Ulrich
许多直接凝血酶抑制剂作为新型抗血栓药和特异性抗凝剂在临床和实验中得到成功应用。它们也用作某些血液采集管中的抗凝剂，用于血小板功能分析。已经出现了一系列血小板功能测试来衡量抗血小板治疗的充分反应。出于比较和实际的原因，对于不同的方法，在采血管中使用相同的抗凝剂将是有利的，例如凝血酶抑制剂。然而，关于凝血酶抑制剂对血小板信号通路的影响的数据很少，这可能会影响结果。我们研究了含凝血酶抑制剂的血液用于VASP测定的血小板反应性指数（PRI）测量的适用性，并研究了两种凝血酶抑制剂（水蛭素和来匹卢定）对洗涤的人血小板中cAMP和cGMP介导的信号传导途径的影响。我们发现，在含有来匹卢定的血液样品中，PGE 1对VASP磷酸化的诱导显著降低。因此，与常规使用的柠檬酸盐血液相比，PRI水平变化很大。令人惊讶的是，血小板与凝血酶抑制剂的体外孵育增加了血小板cGMP水平并诱导了NOS非依赖性sGC/PKG介导的VASP磷酸化。我们得出结论，凝血酶抑制剂激活sGC/PKG依赖性途径，导致VASP磷酸化增加，这有助于PRI测量的偏差。凝血酶抑制剂对sGC和cGMP介导的途径的这些影响（包括VASP磷酸化增加）可能表明存在一种重要的额外血小板机制，可通过凝血酶抑制剂减少血栓形成和血栓栓塞。
A number of direct thrombin inhibitors are successfully used clinically and experimentally as novel antithrombotics and specific anticoagulants. They are also used as anticoagulants in certain blood collection tubes for the analysis of platelet function. A series of platelet function tests have emerged to measure adequate responses to antiplatelet therapy. For comparative and practical reasons, it would be of advantage to use the same anticoagulant in blood collection tubes for different methods, e.g. thrombin inhibitors. However, there are little data on the effects of thrombin inhibitors on platelet signalling pathways that could influence results. We examined the applicability of thrombin inhibitor containing blood for platelet reactivity index (PRI) measurements of the VASP assay and investigated the effects of two thrombin inhibitors (hirudin and lepirudin) on cAMP- and cGMP-mediated signalling pathways in washed human platelets. We show that induction of VASP phosphorylation by PGE1 is markedly reduced in lepirudin containing blood samples. In consequence, PRI levels were highly variable compared to routinely used citrated blood. Surprisingly, in vitro incubation of platelets with thrombin inhibitors increases platelet cGMP levels and induces NOS independent sGC/PKG-mediated VASP phosphorylation. We conclude that thrombin inhibitors activate sGC/PKG-dependent pathways resulting in an increase of VASP phosphorylation which contributes to deviations in PRI measurements. These effects of thrombin inhibitors on sGC- and cGMP-mediated pathways including increased VASP phosphorylation may indicate the presence of an important additional platelet-based mechanism for the reduction of thrombus formation and thromboembolism by thrombin inhibitors.