BIOLOGY OF HUMAN PLATELET FC(GAMMA) RECEPTORS

我们已经确定人类血小板和巨核细胞有受体 对于由Fc γ RIIA基因编码的IgG的Pc部分。接合 血小板Fc γ受体通过免疫复合物和抗血小板 抗体导致血小板清除加速， 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需要 了解受体表达水平的调节和 受体激活信号的传递机制。的 该研究项目的目标是更好地了解 血小板Fc γ受体在血小板活化中的生物学作用。的 第一个具体目的是检查血小板表达的调节 Fc γ受体。血小板表面Fc γ RIIA水平的改变 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 对Fc γ RIIA基础转录重要的因子，并将鉴定 那些负责调节Fc γ RIIA转录的蛋白， 巨噬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 这些调节分子对血小板Fc γ RIIA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Fc γ受体信号传导。FcgammaRIIA有三个 ITAM-（基于免疫受体酪氨酸的活化）中的细胞质酪氨酸 基序）样序列。我们已经确定，所有三个细胞质 酪氨酸和酪氨酸激酶Syk在早期事件中是重要的 在受体交联之后。Syk反义寡核苷酸将 用于鉴定Syk消融对蛋白酪氨酸的影响 磷酸化和Ca2+信号转导。这些信号 将在转染的巨核细胞中研究转导事件 与野生型和突变体Fc γ RIIA胞质结构域序列。我们 我们还将使用来自我们的Fc γ RIIA转基因小鼠的血小板来研究 Fc γ RIIA介导的分泌应答。第三，具体目标 研究了Fc γ RIIA在血小板清除和活化中的作用， vivo.我们的假设是，人Fc γ RIIA的表达导致 抗血小板抗体引发的更广泛的血小板减少症， 通过免疫复合物和增强的血管内血小板活化。 用抗血小板抗体和免疫复合物攻击后， 血小板计数和血管内血小板活化的标志物， 将Fc γ RIIA转基因小鼠与野生型小鼠和小鼠对照进行比较。 仅在血小板中表达Fc γ RIIA。

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.