The phenomenon of vascular occlusion damages the endothelium of blood vessels, leading to fibrinogen aggregation and microthrombi formation, causing many of the symptoms associated with COVID-19. Previous research has shown that IgG alpha C binding prevents fiber development, and the fibrin molecule has a binding site for fibronectin on the αC domain at Aa 221-391. Since fibrin polymerizes by binding to its αC domain, we propose that fibronectin-derived oligopeptides such as P12 can prevent non-thrombogenic fibrinogen fiber formation by binding to the αC domain and blocking its interactions. In order to translate the use of P12 in vivo, it is important to study the effectiveness in vitro by (a) confirming that the peptide is also effective in preventing fiber formation associated with viral infection, and (b) determining the minimum dosage required for observing the effect. <br/>In order to test the effectiveness of P12, H1N1 (PR8) was used to infect gingival epithelial cells for 24 hours, and their infected conditioned medium was incubated with HUVEC cells for 6 hours, since endothelial cells lack the receptor for H1N1 infection. The viral conditioned medium was replaced with a 4 mg/mL fibrinogen and 50 μM P12 solution and incubated for 6 additional hours. The control samples without any P12 had fibers covering almost 80% of the surface while 50 μM of P12 dropped the fiber area coverage to less than 10%, giving evidence supporting further studies of P12 for use clinically. <br/>To test the minimum P12 concentration needed for these results, solutions were made of fibrinogen with 50 μM, 10 μM, 2 μM, 0.5 μM, and 0 μM (control) P12. 1 ml of each solution was added onto polystyrene spun cast on 1 cm x 1 cm silicon wafers.  After fibrinogen adsorption overnight, the samples were dried and viewed with an optical microscope, and fiber formation was analyzed with AFM imaging. Fibers were observed on the control sample as well as samples containing the 0.5 μM, but a reduction of 18.1% and 66.0% in fiber width was observed with 2 μM and 10 μM P12, resepctively. There were hardly any fibers on the 50 μM sample. Thus, it can be concluded that for in vitro testing, 2~10 μM P12 concentration solution is the threshold for preventing fiber formation. This data can provide a guide for further in vivo experiments, which could prove critical in the prevention of thrombosis complications associated with viral infection.

Effectiveness of P12: potential anti-thrombogenic drug for COVID-19

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.