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This paper will be introduced recent advances in the molecular biology of platelet factor 4 (PE4). Since PF4 has been reported to be an immunological regulator by inhibiting suppressor T cell activity and to have .an autocrine effect of megakaryocyte differentiation. I also discuss our research at expressing a recombinant PE4 (rPE4) and our present progress in studying its biological properties, as well as the cloning and organization of the human PF4 and a highly homologous gene Malt. PF4 is a 70 amino acid protein released from ´the alpha graules of platelets following activation. The exact biological function of this protein is unknown. PF4 binds to heparin with high affinity. This property ;allowed initial purification of PF4 from platelets, and may reflect its key biological role. By neutralizing heparin. PF4 may inhibit heparin-induced anti-thrombin III activation, and permit platelets to bind to endothelial cells, thus supporting the early phases of coagula-Jion. PF4 is also a strong chemoattractant for neutrophils, monocytes and fibroblasts. It binds to platelet ;surfaces, and permits platelet activation at lower agonist concentrations. Recently, studies have suggested that PF4 may also effect megakaryocyte maturation in the marrow as a negative autocrine. A repeating structure of two lycinoe followed by two hydrophobic amino acid residues in mature protein is present near the C-terminus. Based on studies using short C-terminal oligopeptides these lysine repeats are thought to be critical for PF4´s heparin binding and chemoattractant properties. We have previously cloned the cDNA forhuman PF4 from a human erythroleukemic (IIEL) cell expression library and showed that the inital translated product is 100 amino acids in length consisting of a 30 amino acid residue signal peptide followed by the 70 amino acid mature protein. "These studies also de-monstrated that in HEI, cells, the PF4 mRNA is approximately 800 nucleotides (n) long. PF4 also appears to have a number of other properties in vitro whose biological significance is unknown. Since some of these properties require fairly high concentrations of PF4, it is also possible that some of these activities may be due to contaminating proteins. One of the values of synthesizing rPF4 is to confirm proposed biological functions of the native protein. We have constructed an expression vector for rPF4 in the T7-based promo-ter vector pT7-7, to better the relationship between PF4 structure and function. The protein was expressed in E. soli and purified to homogeneity. Biological studies have demonstrated that rPF4 has identical neutrophil chemotactic properties as the native protein. These support PF4´s role as an important chemotactic agent in the human body. Site-directed mutations of rPF4 will further demonstrate whether these many biological properties depend on the C-terminal region of PF4 which contains heparin-binding activity. Initial studies with rPF4 as a negative autocrine agent in megakaryocyte development also suggest that rPF4 has identical biological function to native PF4 in regulating megakaryocyte development, supporting the conclusion that the negative effect seen with native PF4 is not due to a contaminating factor. Furthermore, over expression vector should prove useful for the construction of recombinant forms of PF4 to investigate structure function relationships of this biologically important protein.
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