Self-Assembly of\nHuman Fibrinogen into Microclot-Mimicking\nAntifibrinolytic Amyloid Fibrinogen Particles

Recent clinical studies have highlighted the presence\nof microclots\nin the form of amyloid fibrinogen particles (AFPs) in plasma samples\nfrom Long COVID patients. However, the clinical significance of these\nabnormal, nonfibrillar self-assembly aggregates of human fibrinogen\nremains debated due to the limited understanding of their structural\nand biological characteristics. In this study, we present a method\nfor generating mimetic microclots in vitro. Using this approach, the\nself-assembly process, structural organization of AFPs, and their\ninteractions with human plasma components were elucidated. The amyloid\ntransition of fibrinogen occurs under acidic conditions within a pH\nrange of 2.3–3.2. Well-dispersed amyloid oligomers of fibrinogen,\nranging in size from 1 to 5 μm, can be prepared at pH 2.8 after\n1 h of incubation. We tracked the dynamic self-assembly process at\nthe single-molecule level using high-speed atomic force microscopy\n(HS-AFM). The arrangement of amyloid oligomers manifests as well-ordered,\nstacked nanodomains with striped patterns, growing perpendicular to\nthe primary axis of the fibrinogen monomer. Upon transfer to physiological\nsolution conditions or human plasma, these amyloid oligomers further\naggregate into nonfibrillar structures at the micrometer scale, resembling\nthe microclots observed in the bloodstream of Long COVID patients.\nNotably, these AFPs exhibit characteristics consistent with microclots,\nincluding positive staining in thioflavin T (ThT) assays and resistance\nto fibrinolysis. Proteomic analysis suggests that AFPs interact with\nvarious components of human plasma and have an enhanced binding affinity\nwith complement C3 compared to native fibrinogen. This study enables\nthe in vitro preparation of mimetic microclots exhibiting amyloid\nfeatures. It is anticipated to facilitate further researches on the\nmechanisms, detection, and treatment of diseases associated with fibrinogen\namyloidogenesis.