BIOLOGY OF HUMAN PLATELET FC(GAMMA) RECEPTORS

我们已经确定人类的血小板和巨核细胞有受体 对于FcGammaRIIA基因编码的Ig G的Pc部分。接洽 免疫复合物介导的血小板FcGamma受体与抗血小板作用 抗体导致加速血小板清除和血小板 活化、生物活性产物的释放和血栓形成。 干预这些病理过程的治疗将需要 了解受体表达水平的调节和受体的表达 受体激活信号的传递机制。这个 本研究项目的目的是为了更好地了解 血小板FcGamma受体在血小板活化中的生物学作用这个 第一个特定目的是检查血小板表达的调节 FcGamma受体。血小板膜FcGamma水平的变化 表达导致受体介导的激活的改变 血小板。我们已经确定了顺式作用序列和反式作用序列 FcGammaRIIA基础转录的重要因子并将识别 那些负责FcGammariA转录调控的人 巨核细胞中细胞因子和糖皮质激素的报告基因分析 细胞。我们的转基因小鼠模型将能够确定这种影响 这些调节分子对体内血小板FcGammaRIIA表达的影响。 第二个具体目标是检查结构/功能关系 血小板/巨核细胞FcGamma受体信号传导。FcGammaRIIA有三个 ITAM免疫受体酪氨酸激活中的细胞质酪氨酸 基序)-类序列。我们已经确定这三种细胞质 酪氨酸和酪氨酸激酶Syk在早期事件中很重要 在受体交联体之后。SYK反义寡核苷酸将 用于鉴定Syk消融对蛋白质酪氨酸的影响 巨核细胞的磷酸化和钙信号转导。这些信号 巨核细胞中的转导事件将被研究 与野生型和突变型FcGammariIA细胞质结构域序列。我们 还将使用我们的FcGammaRIIA转基因小鼠的血小板来研究 FcGammaRIIA介导的分泌反应。最后，第三个具体目标 检测FcGammaRIIA在血小板清除和激活中的作用 活着。我们的假设是人类FcGammaRIIA的表达导致 更广泛的血小板减少是由抗血小板抗体和 通过免疫复合体和增强血管内血小板活化。 在用抗血小板抗体和免疫复合体挑战之后， 我们的血小板计数和血管内血小板活化标志 FcGammaRIIA转基因小鼠将与野生型小鼠和小鼠进行比较 FcGammaRIIA仅在血小板中表达。

We have determined that human platelets and megakaryocytes have receptors for the Pc portion of IgG encoded by the FcgammaRIIA gene. Engagement of the platelet Fcgamma receptor by immune complexes and anti-platelet antibodies leads to accelerated platelet clearance and platelet activation, the release of biologically active products and thrombosis. Therapy to intervene in these pathologic processes will require understanding the regulation of receptor expression level and the mechanism of the transmission of the receptor's activation signals. The goal of this research project is to gain a greater understanding of the biologic role of the platelet Fcgamma receptor in platelet activation. The first specific aim examines the regulation of expression of the platelet Fcgamma receptor. Alterations in the level of platelet surface FcgammaRIIA expression leads to alterations in receptor mediated activation of platelets. We have identified cis-acting sequences and trans-acting factors important for FcgammaRIIA basal transcription and will identify those responsible for the modulation of FcgammaRIIA transcription by cytokines and glucocorticoids using reporter gene assays in megakaryotic cells. Our transgenic mouse model will enable determination of the effect of these regulatory molecule on platelet FcgammaRIIA expression in vivo. The second specific aim examines structure/function relationships in platelet/megakaryocyte Fcgamma receptor signaling. FcgammaRIIA has three cytoplasmic tyrosines in an ITAM-(immunoreceptor tyrosine-based activation motif)-like sequence. We have established that all three cytoplasmic tyrosines and the tyrosine kinase Syk are important in the early events following receptor crosslinking. Syk antisense oligodeoxynucleotides will be used to identify the effect of Syk ablation on protein tyrosine phosphorylation and Ca2+ signaling in megakaryocytic cells. These signal transduction events will be studied in megakaryocytic cells transfected with wild-type and mutant FcgammaRIIA cytoplasmic domain sequences. We will also use platelets from our FcgammaRIIA transgenic mice to study the FcgammaRIIA mediated secretory response. Finally, the third specific aim examines the role of FcgammaRIIA in platelet clearance and activation in vivo. Our hypothesis is that the expression of human FcgammaRIIA leads to more extensive thrombocytopenia triggered by anti-platelet antibodies and by immune complexes and to enhanced intravascular platelet activation. Following challenge with anti-platelet antibody and immune complex, platelet counts and a marker of intravascular platelet activation in our FcgammaRIIA transgenic mice will be compared to wild-type mice and mice expressing FcgammaRIIA only in platelets.