Signaling pathways activating adhesion in sickle cells

描述(申请人提供)：激动剂诱导的信号转导是调节血小板、白细胞和其他细胞粘附性的主要机制。然而，红细胞对激动剂刺激的粘附性增强通常被认为是惰性的；因此，任何激动剂通过信号转导直接激活SS红细胞粘附性的研究相对较少。凝血酶原蛋白(TSP)是一种黏附蛋白，在镰状细胞患者的血浆中异常升高。虽然TSP对其他细胞具有已知的激动剂特性，但在镰状细胞病中，它被认为纯粹作为一种黏附分子，通过将黏附的SS红细胞连接到内皮和内皮下基质上来发挥作用。在这里，我们建立了SS红细胞，与正常(AA)红细胞相比，对TSP的激动剂刺激做出反应，使其显著变得更粘连。我们还确定了TSP中的一个部位，当固定时可以支持SS红细胞的黏附，或者当溶解时可以激活SS RBC的黏附。我们进一步建立了SS红细胞上整合素相关蛋白(TAP或CD47)作为固定化TSP的基础黏附受体和响应可溶性TSP的信号转导受体的先前未被认识的作用，并检测到SS RBC上TAP的潜在物理差异，这可能有助于SS与AA红细胞更高的黏附。也有证据表明，TAP介导的信号和剪切力诱导的信号之间存在独特的协同作用，涉及激活大G蛋白和酪氨酸激酶，最终激活依赖a4B1整合素的SS红细胞黏附增加。因此，我们建议：1)确定激活4B1介导的SS红细胞黏附的近端事件，2)确定镰状细胞TAP明显物理差异的基础，并询问这种差异是否有助于增强基础和刺激的镰状细胞黏附，3)描绘由LAP刺激诱导的导致SS RBC黏附增加的特定信号通路(S)，4)确定TAP介导的SS RBC激活在SS RBC与内皮细胞黏附中的作用和机制，5)在大鼠提睾肌模型系统中检验TAP促进SS RBC黏附和体内病理变化的假说。因此，这些数据提供了新的镰状细胞黏附的基本模型，并确定了下调镰状细胞黏附和潜在预防血管闭塞危机的多个潜在治疗靶点。

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.