STUDIES OF PLATELET FACTOR 4 AND B-THROMBOGLOBULIN

这笔赠款的长期目标是推动 对基因组组织、生物学作用和 血小板第4因子(PF4)及其受体的结构/功能关系 重组人β-血栓球蛋白(β-TG)的研究 克隆、测序和表达这些基因的DNA技术 细菌。PF4和Beta TG是亲缘关系家族的成员 蛋白质(还包括伽马IP-10，一种合成的蛋白质 通过伽马干扰素刺激后的许多细胞系) 似乎在相互关联的进程中发挥着重要作用 凝血、趋化、免疫调节和组织修复。一个 更好地了解这两个基因的生物学功能 蛋白质及其结构/功能关系可能提供 对血栓形成等病理状态的新见解 心肌梗死时动脉粥样硬化血管和 脑血管血栓栓塞症。有两个具体目标 建议的研究：1)进一步表征PF4和 人和其他哺乳动物的β-甘油三酯基因和基因组DNA 和禽类物种使用已经分离的PF4 cDNA探针和 我建议以类似的方式分离β-TG cdna探针。 这些研究应该会产生关于蛋白质的新信息 前体、它们的细胞内处理和进化 这些蛋白质之间的关系，以及提供氨基酸 用于确定这些蛋白质的哪些修饰的序列数据 可能会产生新的生物信息。2)关于网络技术的研究 用位点法研究蛋白质结构与功能的关系 定向氨基酸替代。天然蛋白质和 前体和诱导突变将在大肠杆菌中合成， 提纯后用于生物检测。对PF4的修改将 包括a)改变功能上重要的COOH- 人PF4末端类似于β-TG的研究 这些差异与不同生物的关系 这两种蛋白质的功能，以及b)二级结构的破坏 通过修改四个中的一个或多个来组织PF4 在PF4，Beta中占据同源位置的半胱氨酸残基 Tg和Gamma IP-10。将被测试的生物功能包括 肝素亲和力，血小板结合和激活， 免疫调节活性和单核细胞趋化活性 成纤维细胞。

The long-term objective of this grant is to further the understanding of the genomic organization, biological roles and structural/functional relationships of platelet factor 4 (PF4) and Beta-thromboglobulin (Beta TG) in humans using recombinant DNA technologies to clone, sequence and express these genes in bacteria. PF4 and Beta TG are members of a family of related proteins (which also includes gamma IP-10, a protein synthesized by many cell lines following gamma-interferon stimulation) that appear to have significant roles in the inter-related processes of coagulation, chemotaxis, immunoregulation and tissue repair. A better understanding of the biological function of these two proteins and their structural/functional relationships may provide new insights into such pathologic states as thrombosis in atherosclerotic vessels during myocardial infarcts and cerebrovascular thromboembolisms. There are 2 specific aims to the proposed research: 1) Further characterization of PF4 and Beta TG cDNA and genomic DNA in humans and other mammalian and avian species using a PF4 cDNA probe already isolated and a Beta TG cDNA probe that I propose to isolate in a similar fashion. These studies should result in new information on protein precursors, their intracellular processing, and the evolutionary relationship between these proteins, as well as provide amino acid sequence data for deciding what modifications of these proteins may yield new biological information. 2) Studies on the relationship between protein structure and function using site- directed amino acid replacement. The natural proteins and precursor and induced mutations will be synthesized in E.coli, purified and used in biologic assays. Modification of PF4 will include a) alteration of the functionally important COOH- terminus of human PF4 to resemble Beta TG in order to study the relationships between these differences and the different biologic functions of the two proteins, and b) disruption of the secondary organization of PF4 by modification of one or more of the four cysteine residues that occupy homologous positions in PF4, Beta TG and gamma IP-10. Biologic functions to be tested include heparin affinity, platelet binding and activation, immunoregulatory activity, and chemotaxis for monocytes and fibroblasts.