STUDIES OF PLATELET FACTOR 4 AND B-THROMBOGLOBULIN

这笔赠款的长期目标是促进 了解基因组组织，生物学作用和 血小板因子4（PF 4）的结构/功能关系， 使用重组人β-血小板球蛋白（β TG） DNA技术克隆、测序和表达这些基因， 细菌 PF 4和β TG是一个家族的成员， 蛋白质（也包括γ IP-10，一种合成的蛋白质） 通过γ-干扰素刺激后的许多细胞系）， 似乎在相互关联的过程中发挥着重要作用， 凝血、趋化性、免疫调节和组织修复。 一 更好地了解这两种基因的生物学功能 蛋白质及其结构/功能关系可以提供 对血栓形成等病理状态的新认识， 心肌梗死期间的动脉粥样硬化血管， 脑血管血栓栓塞 有两个具体目标， 建议的研究：1）进一步表征PF 4和 人和其他哺乳动物中的β TG cDNA和基因组DNA 和鸟类物种使用PF 4 cDNA探针已经分离和 β TG cDNA探针，我建议以类似的方式分离。 这些研究应该会带来关于蛋白质的新信息。 前体，它们的细胞内加工，以及进化 这些蛋白质之间的关系，以及提供氨基酸 序列数据来决定这些蛋白质的哪些修饰 可能会产生新的生物信息。 2)的研究 蛋白质结构与功能的关系 定向氨基酸置换。 天然蛋白质和 前体和诱导突变将在大肠杆菌中合成， 纯化并用于生物测定。 PF 4的修改将 包括a）改变功能上重要的COOH- 人PF 4末端类似于β TG，以研究 这些差异与不同生物学特性之间的关系 B）破坏所述两种蛋白质的次级功能， PF 4的组织通过修改一个或多个四个 占据PF 4、β中同源位置的半胱氨酸残基 TG和伽马IP-10。 待检测的生物学功能包括 肝素亲和力、血小板结合和活化， 免疫调节活性和单核细胞的趋化性， 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.