Hepatic Lectin Receptors in Glycoprotein Homeostasis

描述（申请人提供）：Gilbert Ashwell和Anatol Morell在40多年前的经典研究中报道了第一个细胞受体和第一个哺乳动物凝集素的鉴定和分离。他们的发现表明，肝细胞的唾液酸糖蛋白受体，现在被称为Ashwell-Morell受体（AMR），结合并内化外源性给药的循环糖蛋白，这些糖蛋白已经被神经氨酸酶（唾液酸酶）活性重塑。神经氨酸酶对糖蛋白的重塑将唾液酸从附着的聚糖链的末端移除，揭开了底层半乳糖的面纱。由此产生的暴露的半乳糖键有助于AMR配体的形成。amr介导的高容量和快速清除这些asialal糖蛋白的动力学表明，这种功能已经进化到消除循环中潜在的有害血液成分。然而，在发现AMR后的几十年里，内源性配体仍然不确定，也没有确定AMR的生理目的。最近，首席研究员的实验室发现血小板表达AMR的内源性配体。这一发现包括发现肝细胞在肺炎链球菌（SPN）引起的脓毒症过程中通过amr依赖性血小板清除引起血小板减少。此外，amr依赖性血小板减少症与细菌的NanA神经氨酸酶的血小板去唾液化有关。本文提出的研究将确定血小板减少对AMR功能在调节小鼠SPN败血症的凝血功能和致死率方面所提供的保护的贡献。利用amr依赖性和amr非依赖性血小板清除机制，重组NanA神经氨酸酶和抗cd41治疗将同样建立血小板减少症，比较SPN败血症期间凝血功能障碍和死亡率的结果。此外，初步数据表明NanA和抗cd41在SPN败血症过程中具有实质性的治疗活性。因此，这些研究将同时描述一种新的和潜在有效的未来治疗方法来治疗败血症。这些研究将进一步阐明AMR在重组NanA和抗cd41给药治疗SPN败血症中的作用。据推测，amr -血小板受体-配体相互作用需要高度唾液化的血小板GpIba蛋白。因此，本文提出的研究将在GpIb？并将进一步确定GpIb是否导致血小板减少？在SPN脓毒症的过程中缺乏同样具有保护作用。AMR在哺乳动物中高度保守并进化出快速诱导血小板减少的能力的原因表明，血小板在某些情况下可能具有致病性，例如在败血症期间产生的血小板中诱导颗粒酶- b表达。颗粒酶- b引起细胞死亡和组织损伤，导致败血症致死。因此，本研究将检验amr依赖性血小板减少症的保护作用是否可归因于对表达颗粒酶- b的血小板的消除。

DESCRIPTION (provided by applicant): The classical studies of Gilbert Ashwell and Anatol Morell over 40 years ago reported the identification and isolation of the first cellular receptor ad the first mammalian lectin. Their findings revealed that the asialoglycoprotein receptor of hepatocytes, now termed the Ashwell-Morell receptor (AMR), binds and internalizes exogenously administered circulating glycoproteins that have been remodeled by neuraminidase (sialidase) activity. This remodeling of glycoproteins by neuraminidase removes sialic acid from the termini of the attached glycan chains, unmasking the underlying galactose. The resulting exposed galactose linkages contribute to the formation of AMR ligands. The high capacity and rapid kinetics of AMR-mediated clearance of such asialoglycoproteins suggests that this function has evolved to eliminate potentially deleterious blood components from circulation. However, in the decades since the discovery of the AMR, endogenous ligands remained undefined and no physiological purpose for the AMR was identified. Recently, the Principal Investigator's laboratory identified platelets as expressing endogenous ligands of the AMR. This discovery included the finding that hepatocytes cause thrombocytopenia by AMR-dependent platelet clearance in the course of sepsis induced by Streptococcal pneumoniae (SPN). In addition, AMR-dependent thrombocytopenia was linked to platelet de-sialylation by the bacterium's NanA neuraminidase. Research proposed herein will determine the contribution of thrombocytopenia to the protection afforded by AMR function in moderating the coagulopathy and lethality of SPN sepsis in the mouse. Using AMR-dependent and AMR-independent platelet clearance mechanisms, recombinant NanA neuraminidase and anti-CD41 treatment will similarly establish thrombocytopenia in comparing the outcomes on coagulopathy and mortality during SPN sepsis. Moreover, preliminary data indicates substantial therapeutic activity of NanA and anti-CD41 upon their administration during the course of SPN sepsis. Therefore these studies will be simultaneously characterizing a novel and potentially effective future therapeutic approach to treat sepsis. These studies will further resolve the role of the AMR in the therapeutic activities of recombinant NanA and anti-CD41 administration during SPN sepsis. The AMR-platelet receptor-ligand interaction has been hypothesized to require the highly sialylated platelet GpIba protein. Therefore, research proposed herein will test this hypothesis among GpIb?-deficient platelets, and will further determine whether thrombocytopenia due to GpIb? deficiency in also protective in the course of SPN sepsis. The reason why the AMR is highly conserved among mammals and evolved an ability to rapidly induce thrombocytopenia suggests that platelets may become pathogenic in some circumstances, such as the induction of Granzyme-B expression among platelets produced during sepsis. Granzyme-B causes cell death and tissue damage leading to lethality in sepsis. Therefore this proposal will test whether the protective effect of AMR-dependent thrombocytopenia can be ascribed to the elimination of platelets expressing Granzyme-B.