Novel Chaperone Mechanism for Platelet Disorder

描述(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，“开发对了解人类慢性炎症有信息的转基因动物模型”。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膜糖蛋白Ib-IX-V复合体与内皮细胞衍生的血管性血友病因子(VWF)相互作用，后者在生理上受到严格调控。关于GPIB-IX-V复合体的下游信号转导机制已经有了大量的研究。然而，关于GPIB-IX-V大分子复合体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。这方面的不足令人惊讶，因为血小板GPIb-IX-V复合体的缺陷会导致临床上的大血小板减少症，如Bernard-Soulier综合征(BSS)，而GPIb-IX-V复合体一直是治疗血小板疾病的有吸引力的药理靶点。Gp96(grp94，HSP90b)是HSP90在内质网(ER)中的一种平行分布，GPIB-IX-V复合体在内质网中组装。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(TLRs)的必需伴侣。出乎意料的是，我们发现，在小鼠的造血系统中，gp96的去除并没有对巨核细胞产生负面影响，而是导致出血时间延长，血小板减少和巨大的血小板紊乱，这些在临床上与人类的血瘀证无法区分。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)的减少。已经有多种小分子抑制剂可用于靶向HSP90，包括gp96。总体而言，我们相信这项研究的成功将导致对血瘀证的发病机制有一个新的认识，并按照04-HL-103的规定，“识别控制”血小板激活的关键点，这“可能导致针对血栓形成和炎症的新的药物干预(即，抗血小板药物)”。该项目还将按照06-HL-105的要求，“在短时间内进行有针对性的研究”，“引导开发与人类病理学相关的新的慢性炎症动物模型”。该项目探讨了伯纳德-苏利埃综合征(BSS)的基本机制，这是一种由于血小板及其祖细胞上的几个分子存在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作用。这一过程通常由被称为热休克蛋白(HSPs)的分子催化。我们第一次有数据表明，失去一个名为gp96的热休克蛋白会导致血瘀证。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血瘀证病因的新理解，以及血瘀证和其他血小板疾病的潜在新治疗方法。 与公共卫生相关：该项目解决了伯纳德-苏利埃综合征(BSS)的基本机制，这是一种由于血小板及其祖细胞上的几个分子存在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作用。这一过程通常由被称为热休克蛋白(HSPs)的分子催化。我们第一次有数据表明，失去一个名为gp96的热休克蛋白会导致血瘀证。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血瘀证病因的新理解，以及血瘀证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.