Self-Assembly of Human Fibrinogen into Microclot-Mimicking Antifibrinolytic Amyloid Fibrinogen Particles.

Abstract

Recent clinical studies have highlighted the presence of microclots in the form of amyloid fibrinogen particles (AFPs) in plasma samples from Long COVID patients. However, the clinical significance of these abnormal, nonfibrillar self-assembly aggregates of human fibrinogen remains debated due to the limited understanding of their structural and biological characteristics. In this study, we present a method for generating mimetic microclots in vitro. Using this approach, the self-assembly process, structural organization of AFPs, and their interactions with human plasma components were elucidated. The amyloid transition of fibrinogen occurs under acidic conditions within a pH range of 2.3-3.2. Well-dispersed amyloid oligomers of fibrinogen, ranging in size from 1 to 5 μm, can be prepared at pH 2.8 after 1 h of incubation. We tracked the dynamic self-assembly process at the single-molecule level using high-speed atomic force microscopy (HS-AFM). The arrangement of amyloid oligomers manifests as well-ordered, stacked nanodomains with striped patterns, growing perpendicular to the primary axis of the fibrinogen monomer. Upon transfer to physiological solution conditions or human plasma, these amyloid oligomers further aggregate into nonfibrillar structures at the micrometer scale, resembling the microclots observed in the bloodstream of Long COVID patients. Notably, these AFPs exhibit characteristics consistent with microclots, including positive staining in thioflavin T (ThT) assays and resistance to fibrinolysis. Proteomic analysis suggests that AFPs interact with various components of human plasma and have an enhanced binding affinity with complement C3 compared to native fibrinogen. This study enables the in vitro preparation of mimetic microclots exhibiting amyloid features. It is anticipated to facilitate further researches on the mechanisms, detection, and treatment of diseases associated with fibrinogen amyloidogenesis.