Novel Chaperone Mechanism for Platelet Disorder

描述（由申请人提供）：本申请主要针对广泛的挑战领域（04）临床研究和特定的挑战主题，04- hl -103，“评估白细胞与血小板、红细胞和内皮细胞相互作用在心脏、肺和血液疾病发病机制中的作用”。它还涉及广泛的挑战领域（06）使能技术和特定的挑战主题06- HL-105，“开发转基因动物模型，为理解人类慢性炎症提供信息”。血小板紊乱直接或间接影响所有人类炎症性疾病。血小板活化的主要途径之一是通过血小板糖蛋白Ib- IX-V复合物与内皮细胞源性血管性血友病因子（VWF）的相互作用，而VWF在生理上受到严格调控。关于GPIb-IX- V复合物的下游信号传导，我们已经了解了很多。然而，对于GPIb-IX-V大分子复合物如何组装以及分子伴侣在此过程中的作用知之甚少。鉴于血小板GPIb-IX-V复合物的缺陷导致临床大量血小板减少症，如Bernard-Soulier综合征（BSS），而GPIb-IX-V复合物一直是血小板疾病的有吸引力的药理靶点，这一领域的不足令人惊讶。gp96 （grp94, HSP90b）是内质网（ER）中发生GPIb-IX-V复合物组装的HSP90的同源物。通过产生条件性gp96缺失小鼠，我们发现gp96是多种整合素和toll样受体（tlr）的重要伴侣。出乎意料的是，我们发现在小鼠造血系统中，gp96消融并不会对巨核生成产生负面影响，但会导致出血时间延长、血小板减少和巨血小板紊乱，这些在临床上与人类BSS没有什么区别。此外，gp96的缺失会导致细胞表面GPIb的同时减少，但不会导致aIIb¿3整合素（GPIIb/IIIa）的同时减少。目前已有多种小分子抑制剂可用于靶向HSP90，包括gp96。总的来说，我们认为本研究的成功实施将导致对BSS发病机制的新认识，并“确定控制血小板活化的关键点”，如04-HL-103所述，这“可能导致新的药物干预（即抗血小板药物）用于血栓和炎症”。根据06-HL-105的要求，该项目还将提供“短期内的针对性研究”，“导致与人类病理相关的慢性炎症的新动物模型的开发”。本项目研究Bernard-Soulier综合征（BSS）的基本机制，BSS是一种由血小板及其祖细胞上的几个分子问题引起的出血性疾病。蛋白质必须采用适当的三维形状才能发挥作用。这种过程通常由称为热休克蛋白（HSPs）的分子催化。我们的数据首次表明，一种名为gp96的HSP的缺失会导致BSS。我们的目标是发现gp96如何促进BSS蛋白成熟的分子细节。这项研究的成功实施可能会导致对BSS病因的新认识，以及BSS和其他血小板疾病的潜在新治疗方法。

DESCRIPTION (provided by applicant): This application primarily addresses broad Challenge Area (04) Clinical Research and specific Challenge Topic, 04-HL-103, "Assess the role of leukocyte interaction with platelets, erythrocytes, and endothelium in the pathogenesis of heart, lung, and blood diseases". It also addresses broad Challenge Area (06) Enabling Techniques and specific Challenge Topic 06- HL-105, "Develop transgenic animal models that are informative for understanding chronic inflammation in humans". Platelet disorder, directly or indirectly, affects all human inflammatory diseases. One of the major pathways for platelet activation is through the interaction between platelet glycoprotein Ib- IX-V complex and the endothelial cell-derived von Willebrand factor (VWF), which is tightly regulated physiologically. A great deal has been learnt on the downstream signaling of GPIb-IX- V complex. However, little is known on how GPIb-IX-V macromolecular complex is assembled and what are the roles of molecular chaperones in this process. The deficiency in this area is surprising in light of the fact that the defect of platelet GPIb-IX-V complex causes clinical macrothrombocytopenia such as Bernard-Soulier syndrome (BSS), and the GPIb-IX-V complex has always been the attractive pharmacological target for platelet disorders. gp96 (grp94, HSP90b) is a paralogue of HSP90 in the endoplasmic reticulum (ER) where the assembly of GPIb-IX-V complex occurs. By generating conditional gp96 null mice, we have found that gp96 is an essential chaperone for multiple integrins and Toll-like receptors (TLRs). Unexpectedly, we have discovered that gp96 ablation in murine hematopoietic system did not negatively affect megakaryopoiesis but resulted in prolonged bleeding time, thrombocytopenia and giant platelet disorder that are clinically indistinguishable from human BSS. Moreover, loss of gp96 causes concurrent reduction of cell surface GPIb but not aIIb¿3 integrin (GPIIb/IIIa). A variety of small molecule inhibitors are already available for targeting HSP90, including gp96. Overall, we believe that the successful execution of this study shall lead to a new understanding of the pathogenesis of BSS, and the "identification of the key points controlling" platelet activation, which "may lead to new pharmaceutical interventions (i.e., anti-platelet agents) for both thrombosis and inflammation", as stipulated in 04-HL-103. This project also will provide "targeted research over short period of time", "lead(ing) to development of new animal models for chronic inflammation that are relevant to human pathology", as called upon by 06-HL-105. This project addresses the fundamental mechanism of Bernard-Soulier syndrome (BSS), a bleeding disorder due to problems in several molecules on platelets and their progenitors. Proteins must adopt a proper 3-dimensional shape in order to function. Such a process is often catalyzed by molecules referred to as heat-shock proteins (HSPs). We have data to implicate for the first time that loss of one HSP called gp96 can cause BSS. We aim to discover the molecular details of how gp96 facilitates the maturation of BSS proteins. Successful execution of this study may lead to new understanding of the causes of BSS and potential novel treatments of BSS and other platelet disorders. PUBLIC HEALTH RELEVANCE: This project addresses the fundamental mechanism of Bernard-Soulier syndrome (BSS), a bleeding disorder due to problems in several molecules on platelets and their progenitors. Proteins must adopt a proper 3-dimensional shape in order to function. Such a process is often catalyzed by molecules referred to as heat-shock proteins (HSPs). We have data to implicate for the first time that loss of one HSP called gp96 can cause BSS. We aim to discover the molecular details of how gp96 facilitates the maturation of BSS proteins. Successful execution of this study may lead to new understanding of the causes of BSS and potential novel treatments of BSS and other platelet disorders.