The role of protease activated receptors on platelets

 描述（由申请方提供）：血小板定义为在止血和病理性血栓形成中的重要作用。因此，血小板研究的主要焦点集中在鉴定和开发更安全和更有效的抗血栓形成疗法上。第二个目标是能够预测特定疗法在人群中的有效性。药物基因组学可以让我们预测谁会对治疗产生反应或耐药。为了使其成功，需要在特定的多态性和生理输出之间有明确的关系。最后，等位基因需要以足够高的频率存在，以保证在治疗开始前进行检测。在目前的项目中，我们将检查血小板受体（PAR 4）的常见多态性，以解释在分子水平上对拮抗剂的反应性和反应性的差异。我们将使用结构质谱法（酰胺氢氘交换（HDX）、组氨酸HDX和羟基自由基足迹法）用纯化的PAR 4和细胞和血小板上的PAR 4确定在凝血酶活化后PAR 4的结构重排。这些研究将确定拴系配体如何影响受体的整体构象。在人血小板中的药理学研究表明，PAR 4序列变体具有显著不同的反应。例如，PAR 4 - 120 T是高反应性的，PAR 4 - 296 V对信号传导具有抗性。我们将确定PAR 4变体如何影响配体结合位点以及在分子水平上向活性构象的转变。此外，PAR 4 - 296 V抑制其他等位基因的活性，表明其形成显性负性同源二聚体。我们将研究PAR 4变体，PAR 1和P2 Y12之间的物理相互作用，以确定这些受体如何影响信号通路。最后，我们将确定PAR 4序列变异如何影响急性冠状动脉综合征患者的血小板反应性，以及PAR 1拮抗剂vorapaxar是否会加剧PAR 4变异引起的血小板反应性改变。通过分析凝血酶激活后PAR 4的结构重排，这些研究将首次详细描述 PAR家族的拴系配体激活机制，并有可能揭示可用于治疗的变构位点。更具体地说，了解自然发生的序列变异如何影响PAR 4的反应，可能使我们能够根据患者的基因型预测最合适的抗血小板治疗。

 DESCRIPTION (provided by applicant): Platelets are defined by their essential roles in hemostasis and the formation of pathological thrombi. Therefore, the major focus of platelet research is focused on identifying and developing safer and more effective anti- thrombotic therapies. A second goal is to have the ability to predict the effectiveness of specific therapies across populations. Pharmacogenomics may allow us to predict who will respond to or be resistant to therapies. In order for this to be successful, there needs to be a clear relationship between specific polymorphisms and physiological output. Finally, the alleles need to be present at high enough frequency to warrant testing prior to the start of therapy. In the current project w will examine common polymorphisms of a platelet receptor (PAR4) to explain the differences in reactivities and response to an antagonist at the molecular level. We will determine the structural rearrangement of PAR4 upon activation by thrombin using structural mass spectrometry (amide hydrogen deuterium exchange (HDX), histidine HDX, and hydroxyl radical foot-printing) with purified PAR4 and PAR4 on cells and platelets. These studies will determine how the tethered ligand influences the overall conformation of the receptor. Pharmacological studies in human platelet show that the PAR4 sequence variants have dramatically different responses. For example, PAR4-120T is hyper-reactive and PAR4-296V is resistant to signaling. We will determine how the PAR4 variants affect the ligand binding site and the transition to the active conformation at the molecular level. Further, PAR4-296V suppresses the activity of other alleles suggesting that it forms dominant negative homodimers. We will examine the physical interaction between PAR4 variants, PAR1 and P2Y12 to determine how these receptors influence signaling pathways. Finally, we will determine how the PAR4 sequence variants influence platelet reactivity in acute coronary syndrome patients on the current standard of care (aspirin and a P2Y12 antagonist) and if the altered platelet reactivity due to PAR4 variants is exacerbated with the PAR1 antagonist vorapaxar. By analyzing the structural rearrangements of PAR4 following activation by thrombin, these studies will provide the first detailed description of the tethered ligand activation mechanism for the PAR family and have the potential to uncover allosteric sites that can be exploited therapeutically. More specifically, understanding how naturally occurring sequence variants influence PAR4's response may allow us to predict the most appropriate antiplatelet therapy for patients depending on their genotype.