Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis.
Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis.
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DOI：
10.1161/atvbaha.117.310588
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发表时间：
2018-04
期刊：
影响因子：
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通讯作者：
Majumder R
中科院分区：
文献类型：
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作者：
Plautz WE;Sekhar Pilli VS;Cooley BC;Chattopadhyay R;Westmark PR;Getz T;Paul D;Bergmeier W;Sheehan JP;Majumder R
S蛋白(PS)是一种在体外直接抑制凝血因子IXA(FixA)的血浆蛋白。由于FixA升高与静脉血栓栓塞症的风险增加有关，因此确定PS如何在体内抑制FixA功能是很重要的。本工作的目的是在体内证实PS与FixA的直接结合，鉴定在体内与PS结合所需的FixA氨基酸残基，并使用一个不能与PS结合的具有酶活性的FixA突变体来衡量PS-FixA相互作用在止血中的意义。我们证明PS在体内通过与FixA肝素结合的外切酶结合来抑制FixA。我们使用荧光标记、免疫组织化学和蛋白质-蛋白质交联来显示FixA和PS之间的体内相互作用。重要的是，血小板共定位需要两种蛋白质之间的直接相互作用。FixA和PS也从血浆中免疫共沉淀，证实了它们在生理环境中的相互作用。PS与FixA的结合和PS对固有Xase复合体的抑制需要FixA与肝素结合的外切体中的K132、K126和R170残基。双突变体K132A/R170A保持了全部活性，但不能与PS结合。至关重要的是，与野生型FixA相比，注射FixA K132A/R170A的血友病B鼠显示出更快的纤维蛋白凝块形成速度。我们的发现证实PS是一种重要的体内FixA抑制剂。破坏PS和FixA之间的相互作用会导致小鼠血栓形成的速度增加。这一新发现的PS功能暗示了抗血栓治疗的一个未被开发的靶点。
Protein S (PS) is a plasma protein that directly inhibits the coagulation factor IXa (FIXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this work is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS-FIXa interaction in hemostasis. We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein-protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet co-localization required a direct interaction between the two proteins. FIXa and PS also co-immunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126 and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild type FIXa. Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.