Novel Chaperone Mechanism for Platelet Disorder

描述（由申请人提供）：本申请主要涉及广泛的挑战领域（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”。它还解决了广泛的挑战领域（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，为了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复合物与内皮细胞衍生的血管性血友病因子（VWF）之间的相互作用，其在生理上受到严格调节。关于GPIb-IX- V复合物的下游信号传导已经了解了很多。然而，关于GPIb-IX-V大分子复合物是如何组装的，以及分子伴侣在这一过程中的作用还知之甚少。鉴于血小板GPIb-IX-V复合物的缺陷导致临床巨血小板减少症如Bernard-Soulier综合征（BSS），并且GPIb-IX-V复合物一直是血小板疾病的有吸引力的药理学靶点，该领域的缺陷是令人惊讶的。gp 96（grp 94，HSP 90 b）是HSP 90在内质网（endoplasmic reticulum，ER）中的一个特异性配体，在内质网中GPIb-IX-V复合物的组装发生。通过产生条件性gp 96缺失小鼠，我们发现gp 96是多种整合素和Toll样受体（TLR）的必需伴侣。出乎意料的是，我们已经发现，在鼠造血系统中的gp 96消融不会对巨核细胞生成产生负面影响，但会导致出血时间延长、血小板减少症和巨大血小板病症，这些病症在临床上与人BSS难以区分。此外，gp 96的缺失导致细胞表面GPIb的同时减少，但不导致所有IIb 3整合素（GPIIb/IIIa）的同时减少。已经有多种小分子抑制剂可用于靶向HSP 90，包括gp 96。总的来说，我们相信这项研究的成功执行将导致对BSS发病机制的新理解，以及“控制血小板活化的关键点的识别”，这“可能导致新的药物干预（即，抗血小板剂）用于血栓形成和炎症”，如04-HL-103中所规定。根据06-HL-105的要求，该项目还将提供“短时间内有针对性的研究”，“导致开发与人类病理学相关的慢性炎症新动物模型”。该项目致力于研究Bernard-Soulier综合征（BSS）的基本机制，BSS是一种由于血小板及其祖细胞上的几种分子出现问题而导致的出血性疾病。蛋白质必须采用适当的三维形状才能发挥功能。这种过程通常由称为热休克蛋白（HSP）的分子催化。我们首次有数据表明，一种名为gp 96的HSP的丢失可能导致BSS。我们的目标是发现gp 96如何促进BSS蛋白成熟的分子细节。这项研究的成功执行可能会导致对BSS原因的新理解以及BSS和其他血小板疾病的潜在新治疗方法。 公共卫生相关性：该项目致力于研究Bernard-Soulier综合征（BSS）的基本机制，BSS是一种由于血小板及其祖细胞上的几种分子出现问题而导致的出血性疾病。 蛋白质必须采用适当的三维形状才能发挥功能。这种过程通常由称为热休克蛋白（HSP）的分子催化。我们首次有数据表明，一种名为gp 96的HSP的丢失可能导致BSS。我们的目标是发现gp 96如何促进BSS蛋白成熟的分子细节。这项研究的成功执行可能会导致对BSS原因的新理解以及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.