Lejla Hagimola

The physiology of SARS-CoV-2 virus/host interactions is not well understood. To better understand host/virus interactions, we performed a CRISPR activation screen to identify host genes that confer resistance to authentic SARS-CoV-2. This highlighted 34 new candidate genes that may alter the course of infection. We validated that 7 of these genes can suppress authentic SARS-CoV-2 infection, including the innate immune receptor P selectin, which increases SARS-CoV-2 spike-dependent binding to cells, while protecting from infection. P selectin also promotes binding to SARS-CoV-2 variants, SARS-CoV-1, and Middle East respiratory syndrome spike proteins, suggesting a general role for P selectin in highly pathogenic coronavirus infections. Importantly, P selectin protein expression driven by synthetic mRNA can block SARS-CoV-2 infection. Naturally, P selectin is expressed on platelets, and we show that it promotes spike-mediated platelet aggregation. P selectin is also expressed on the endothelium, where SARS-CoV-2 spike interactions are also P selectin dependent. In vivo, SARS-CoV-2 uses P selectin to home to capillary beds where the virus interacts with platelets and endothelium, and blocking this interaction can clear vascular-associated pulmonary SARS-CoV-2.

Microfluidic devices have an established role in the study of platelets and coagulation factors in thrombosis, with potential diagnostic applications. However, few microfluidic devices have assessed the contribution of neutrophils to thrombus formation, despite increasing knowledge of neutrophils’ importance in cardiovascular thrombosis. We describe a thromboinflammation model which uses straight channels, lined with fixed human umbilical vein endothelial cells, after treatment with tumour necrosis factor-alpha. Re-calcified whole blood is perfused over the endothelium at venous and arterial shear rate. Neutrophil adhesion, platelet and fibrin thrombus formation, is measured over time by the addition of fluorescent antibodies to a whole blood sample. Fixed endothelium retains surface expression of adhesion molecules ICAM-1 and E-Selectin. Neutrophils adhere preferentially to platelet thrombi on the endothelium. Inhibitors of neutrophil adhesion and anti-inflammatory agents, such as isoquercetin, decrease neutrophil adhesion. Our model offers the advantage of the use of (1) fixed endothelium, (2) whole blood, instead of isolated neutrophils, and (3) a small amount of blood (1 mL). The characteristics of this thromboinflammation model provide the potential for further development for drug screening and point-of-care applications.