Identification of disulfide bond-linking sites in biosynthesized platelet factor 4 by establishing a partial reduction method without alkylation

Abstract

Platelet factor 4 (PF4), a specific protein primarily found in megakaryocytes and platelet α-granules, plays an essential role in the coagulation process. It carries a high positive charge and thus has a unique ability to readily form complexes with negatively charged heparin. This interaction between PF4 and heparin plays a crucial role in platelet aggregation and thrombosis, resulting in heparin-induced thrombocytopenia (HIT). HIT is often diagnosed through various diagnostic tests that utilize exogenous PF4 detecting antibodies against PF4/heparin complexes. Besides, PF4 was recently found to have the potential to restore cognitive function. Therefore, a comprehensive characterization of biosynthetic PF4 standards is crucial for the diagnosis and management of HIT. In this study, a bacterial expression system was established to efficiently produce recombinant human PF4 (rhPF4). This PF4 was characterized using liquid chromatography-high resolution mass spectrometry, confirming it is completely identical to native PF4 in terms of disulfide connectivity and sequence. The in vitro chemotaxis assay indicates that it possesses biological activity. Furthermore, PF4 contains two disulfide bonds, which are crucial for its structural integrity and function. A partial reduction method was successfully developed to assign the disulfide bond connectivity as Cys10–Cys36 and Cys12–Cys52.