Signaling pathways activating adhesion in sickle cells

描述（由申请人提供）：激动剂诱导的信号传导是调节血小板、白细胞和其他细胞粘附性的主要机制。然而，红细胞通常被认为对激动剂刺激的粘附增强是惰性的；因此，镰状红细胞（SS）中的信号通过任何激动剂直接激活SS红细胞粘附性的研究相对较少。血小板反应蛋白（TSP）是一种黏附蛋白，在镰状细胞患者血浆中异常升高。虽然已知TSP对其他细胞具有激动剂特性，但在镰状细胞疾病中，它被认为是一种纯粹的粘附分子，通过将粘附的SS红细胞桥接到内皮细胞和亚内皮细胞基质上。在这里，我们证实，与正常（AA）红细胞相比，SS红细胞对TSP的激动剂刺激作出反应，变得明显更具黏附性。我们还在TSP中发现了一个位点，当固定时可以支持SS红细胞粘附，或者当溶解时，可以激活SS红细胞粘附。我们进一步确定了整合素相关蛋白（TAP或CD47）在SS红细胞上作为固定TSP的基础粘附受体和响应可溶性TSP的信号转导受体的作用，并检测了SS红细胞上TAP的潜在物理差异，这可能导致SS红细胞比AA红细胞具有更高的粘附性。还有证据表明tap介导的信号传导和剪切应力诱导的信号传导之间存在独特的协同作用，包括大g蛋白和酪氨酸激酶的激活，最终激活a4B1整合素依赖性SS红细胞粘附的增加。因此，我们建议：1)确定a4b1介导的SS红细胞粘附激活的近端事件，2)定义镰状细胞TAP明显物理差异的基础，并探讨这种差异是否有助于增强基础和刺激的镰状细胞粘附，3)描述由lAP刺激诱导的SS红细胞粘附增强的特定信号通路，4)确定TAP介导的SS红细胞活化在SS红细胞粘附内皮细胞中的作用和机制。5)通过大鼠肌模型系统研究人镰状细胞流动，验证TAP参与SS红细胞粘附和体内病理的假设。因此，这些数据为镰状细胞粘附提供了新的基础模型，并确定了下调镰状细胞粘附和潜在预防血管闭塞危像的多种潜在治疗靶点。

DESCRIPTION (provided by applicant): Agonist-induced signaling is a primary mechanism regulating adhesiveness of platelets, leukocytes and other cells. However, RBCs are generally considered to be inert to agonist-stimulated enhancement of adhesion; thus signaling in sickle (SS) RBCs by any agonist to directly activate SS RBC adhesiveness is relatively unexplored. Thrombospondin (TSP) is an adhesive protein that is abnormally elevated in the plasma of sickle cell patients. While TSP has known agonist properties towards other cells, it has been proposed in sickle cell disease to function purely as an adhesive molecule, by bridging adherent SS RBCs to the endothelium and subendothelial matrix. Here we establish that SS RBCs, in contrast to normal (AA) RBCs, respond to agonist stimulation by TSP to become significantly more adhesive. We also identify a site within TSP, which when immobilized can support SS RBC adhesion, or when soluble, can activate SS RBC adhesion. We further establish a previously unappreciated role for integrin-associated protein (TAP or CD47) on SS RBCs as both a basal adhesion receptor for immobilized TSP and a signal-transducing receptor in response to soluble TSP, and detect a potential physical difference in TAP on SS RBC that may contribute to the higher adhesion of SS versus AA RBCs. Evidence is also provided for a unique synergy between TAP-mediated signaling and shear stress-induced signaling, involving activation of large G-proteins and tyrosine kinases, to ultimately activate an a4B1 integrin-dependent increase in SS RBC adhesion. We therefore propose to: 1) identify proximal events in the activation of a4B1-mediated SS RBC adhesion, 2) define the basis for the apparent physical difference in sickle cell TAP and ask whether that difference contributes to the enhanced basal and stimulated sickle cell adhesion, 3) delineate the specific signaling pathway(s) induced by stimulation of lAP that results in increased SS RBC adhesiveness, 4) determine the role and mechanism of TAP-mediated SS RBC activation in SS RBC adhesion to endothelial cells, and 5) test the hypothesis that TAP contributes to SS RBC adhesion and pathology in vivo in studies of human sickle cell flow through a rat cremaster muscle model system. These data therefore provide new fundamental models of sickle cell adhesion and identify multiple potential therapeutic targets for down-regulating sickle cell adhesiveness and potentially preventing vaso-occlusive crises.