The Molecular Basis for Non-Thrombogenic Clots Following Viral Infection

Fibrinogen is a plasma-soluble glycoprotein integral to the coagulation process. Typically, it is cleaved into fibrin monomers, which polymerize into fibers to promote platelet aggregation and, by extension, clot formation. Nonetheless, fibrinogen may also convert into fibrin via additional mechanisms, including interaction with hydrophobic materials. Such conversion pathways involve the deposition and adsorption of fibrinogen onto hydrophobic surfaces, leading to exposure of the molecule’s αC domain and thus, recruitment of other fibrin molecules to promote polymerization of fibers considerable in size. As the synthesis of fibrin networks progresses, thrombus formation occurs, potentially obstructing vasculature and critical blood flow.

It has been shown that the development of viral infections, such as COVID-19 and swine flu (H1N1), is correlated with severe, extensive blood clotting, a quality attributed to intensification of lipid droplet synthesis associated with acute infection. Lipid droplets (LDs), dynamic storage organelles involved in protein sequestration and transfer mechanisms as well as signaling cascades, begin accumulating on tissue surfaces within two hours of infection onset, increasing hydrophobicity of the bodily environment¹. This disturbance associated with LD synthesis has been hypothesized to promote fiber formation and thus, hypercoagulability during and after patient infection. As such, viral-induced thrombosis has emerged at the forefront of medical research, with investigation into potential solutions to mitigate these risks ongoing. This study aims to characterize the role of viral infection in thrombosis.

To begin, we cultured human gingival keratinocytes in well plates to obtain a confluent layer of cells for analysis. Cells were seeded at a density of 1 x 10⁷/well, and once confluency was reached, keratinocyte growth media was added to all cultures. Then, select cultures were infected with H1N1. These selected cultures were incubated for two hours, allowing the viral agent to attach to and thus, successfully infect the requisite wells. Following infection, cells were incubated for an hour in the presence of a 4 mg/mL fibrinogen solution. Uninfected cells were also incubated with a fibrinogen solution to establish a basis for comparison. To observe fiber formation, cells were fixed and stained for immunofluorescence imaging. This procedure was subsequently repeated with human umbilical vein endothelial cells (HUVEC) to further simulate conditions for clot formation and validate the keratinocyte findings.

Fluorescent microscopy images were taken for both keratinocyte and HUVEC cells. Fibers of the usual size and length formed in uninfected wells of each cell type. With the addition of a 488 stain, increased lipid deposition was observed in wells infected with H1N1, substantiating the effect of viral infections on modulating bodily environments. Moreover, fibers synthesized within the infected medium were enlarged and elongated when compared with control samples. By demonstrating that viral infection is correlated with increased fiber polymerization–which in turn promotes the platelet adhesion, thrombin release, and compounded platelet aggregation necessary for coagulation–these findings provide a strong basis by which to understand and combat viral-induced thrombosis in vivo.

This study illustrates how the upregulation of LDs upon viral infection generates an environment favoring abnormal thrombus development by altering the surface properties of cells and vascular walls. Further experimentation is required to elucidate underlying mechanisms and confirm experimental findings.

We would like to acknowledge the Louis Morin Charitable Trust for their support in our research.

[1] Monson, E.A., et al. Nat Commun 12, 4303 (2021).