Structure and Function of the Platelet Integrin

本项目的目的是研究血小板膜整合素αIIb/β3的结构和功能，αIb/β3是一种钙依赖的异源二聚体，其与纤维蛋白原和yon Willebrand因子等配体的结合部位通过血小板刺激而暴露。由于与AlphaIIb/Beta3的配体结合负责血小板聚集，调节AlphaIIb/Beta3上Tigand结合部位的暴露是血小板功能的关键步骤。尽管经过了几十年的深入研究，但使alphaIIb/beta3能够与配体结合的机制尚不清楚，alphaIIb/beta3中的TIGG和结合位点也没有令人信服地确定。这个项目的具体目标就是解决这些问题。在具体目标1中，将确定调节AlphaIIb/Beta3激活状态的Alphallb和Beta3跨膜和细胞质结构域的结构特征。这些研究基于我们的观察，首先，与αIIb和β3的跨膜区和细胞质结构域相对应的蛋白质分散在脂质胶束中，分别以单体-二聚体和单体-三聚体的平衡存在，其次，在Beta3跨膜螺旋的特定位置引入极性天冬酰胺导致了结构性的AlphaIIb/Beta3配体结合活性。拟议的实验将使用转基因小鼠的转基因CHO细胞和淋巴细胞和血小板来测量特定跨膜和胞浆结构域突变体的配体结合活性。在特定的目标2中，将再次使用转基因CHO细胞和转基因小鼠来研究alphaIIb和beta3胞外域中负责配体结合和异源二聚体形成的序列基序。拟议的实验将利用最近报道的Alphaupsilon beta3胞外域的晶体结构来确定特定的AlphaIIb/BATA3区域和感兴趣的残基。在具体目标3中，我们将使用激光镊子来测量AlphaIIb/Beta3、其他血小板受体和其他整合素的功能。激光镊子是一种光学系统，它使用激光来捕获和操纵介电粒子，如小珠子或细胞。使用该系统，我们已经在激动剂刺激的活体人和小鼠血小板上以及在转基因的组织培养细胞上，分别重复性和准确地测量了纤维蛋白原和骨桥蛋白分别与αIIb/β3和αuPβ3结合的强度。为此，我们将把这些测量扩展到alphaIIb/beta3突变体的功能，von Willebrand因子与alphaIIb/beta3和GPIB-IX-V的相互作用，以及Beta2整合素与ICAM-1等配体的相互作用。

The objective of this project is to correlate the structure and function of the platelet membrane integrin alphaIIb/beta3, alphalIb/beta3 is a calcium-dependent heterodimer whose binding site for ligands such as fibrinogen and yon Willebrand factor is exposed by platelet stimulation. Because ligand binding to alphaIIb/Beta3 is responsible for platelet aggregation, regulation of the exposure of the tigand binding site on alphaIIb/Beta3 is a critical step in platelet function. Despite several decades of intensive study, the mechanism that enables alphaIIb/Beta3 to bind ligands is not known, nor have the tigand binding sites in alphaIIb/Beta3 been convincingly identified. The Specific Aims of this project addresses these questions. In Specific Aim 1, structural features of the alphallb and Beta3 transmembrane and cytoplasmic domains that regulate the alphaIIb/Beta3 activation state will be identified. The studies are based on our observations first, that proteins corresponding to the transmembrane and cytoplasmic domains of alphaIIb and Beta3, dispersed in lipid micelles, are present in monomer-dimer and monomer-trimer equilibria, respectively, and second, that the introduction of a polar asparagine at specific sites in the Beta3 transmembrane helix results in constitutive alphaIIb/Beta3 ligand binding activity. Proposed experiments will measure the igand binding activity of specific transmembrane and cytoplasmic domain mutants using transfected CHO cells and lymphocytes and ptatelets from transgenic mice. In Specific Aim 2, sequence motifs in the extracellular domains of alphaIIb and Beta3 that are responsible for ligand binding and for heterodimer formation will be studied, again using transfected CHO cells and transgenic mice. The proposed experiments will take advantage of the recently reported crystal structure of the extracellular domain of alpha upsilon Beta3 to identify specific alphaIIb/Bata3 regions and residues of interest. In Specific Aim 3, we will use laser tweezers to measure the function of alphaIIb/Beta3, other platelet receptors, and other integrins. Laser tweezers are an optical system that use laser light to trap and manipulate dielectric particles such as small beads or cells. Using this system, we have reproducibly and accurately measured the strength of fibrinogen and osteopontin binding to alphaIIb/Beta3 and alpha upsilon Beta3, respectively, both on agonist-stimulated living human and murine platelets, as well as on transfected tissue culture cells. In this Aim, we will extend these measurements to the function of alphaIIb/Beta3 mutants, to the interaction of von Willebrand factor with both alphaIIb/Beta3 and GPIb-IX-V, and to the interaction of Beta2 integrins with ligands such as ICAM- 1.