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Introduction We have experienced a pandemic induced by the interaction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) structural proteins with innate structures. These interactions are especially prevalent for patients with underlying pathologies, such as cardiovascular diseases. However, there has been limited work to uncover the range of responses induced by SARS-CoV-2 structural proteins. Thus, our objective was to investigate how endothelial cell pro-thrombotic and pro-inflammatory responses are altered after exposure to SARS-CoV-2 spike, nucleocapsid, and membrane-envelope proteins. We hypothesized that after a short duration exposure, endothelial cells would have a heightened thrombotic and inflammatory potential. With longer exposures, this may lead to altered disease progression and the observed increased mortality and morbidity rates in patients with underlying vascular pathologies. Methods To test this hypothesis, human endothelial cells were exposed to SARS-CoV-2 structural proteins. After the exposure, the expression of thrombomodulin, PECAM-1, connexin-43, and gC1qR were assessed. In parallel, standard cell culture readouts were assessed to determine if these incubations altered cell growth and metabolism. Results and Conclusions We observed significant increases in thrombotic and inflammatory marker expression, with no change to the cell culture parameters (with the exception of a reduction in cell density in response to one SARS-CoV-2 structural protein). Importantly, these observations were dependent on the viral structural protein the cells were exposed to, suggesting that the interactions of SARS-CoV-2 with innate cells is complex and must be uncovered. Combined, this suggests that SARS-CoV-2 structural proteins can regulate inflammatory and thrombotic responses that underlie common pathologies observed during COVID-19.

Background Vascular diseases are highly associated with inflammation and thrombosis. Elucidating links between these two processes may provide a clearer understanding of these diseases, allowing for the design of more effective treatments. The activation of complement component 1 (C1) is a crucial contributor to innate immunity and is associated with significant concentrations of circulating C1q. Many pathological pathways initiate when C1q interacts with gC1qR. This interaction plays a major role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. However, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is the more physiologically relevant coagulation arm, has not been studied. We hypothesized that C1q binding to gC1qR enhances the expression of tissue factor (TF) in adventitial fibroblasts and vascular smooth muscle cells, the primary TF bearing cells in the body. Methods Using an enzyme‐linked immunosorbent assay approach, TF expression and the role of gC1qR was observed. Cells were conditioned for 1 h with C1q or a gC1qR blocker and C1q, to assess the role of gC1qR. Additionally, cell growth characteristics were monitored to assess changes in viability and metabolic activity. Results Our results indicate that the expression of TF increased significantly after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blockers and C1q exhibited no change in TF expression when compared with cells conditioned with the blocking antibodies alone. Our results show no significant differences in metabolic activity or cell viability under these conditions. Conclusions This indicates that gC1qR association with C1q induces TF expression and may initiate extrinsic coagulation. Overall, this data illustrates a role for C1q in the activation of extrinsic coagulation and that gC1qR activity may link inflammation and thrombosis.

Thrombosis and inflammation are prevalent during and characteristic of vascular diseases. Understanding the interplay of these processes would likely allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and ultimately drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. C1q interacting with gC1qR, one of its cell‐surface receptors, initiates many pathological pathways. There is considerable evidence that C1q/gC1qR plays a role in inflammatory processes observed during atherosclerosis and in initiating intrinsic coagulation. While this link between inflammation and thrombosis is important, the intrinsic cascade plays a minor role in physiological and pathological thrombosis. The effects of C1 and the role of C1q on extrinsic coagulation, which is more physiologically relevant, has not been studied. Extrinsic coagulation initiates upon vessel damage, which exposes blood to sub‐endothelial constituents, especially tissue factor. Adventitial fibroblasts and vascular smooth muscle cells are the primary tissue factor bearing cells. We hypothesized that C1q binding to gC1qR initiates this pathway through the enhanced expression of sub‐endothelial tissue factor (TF) in these cells. The objective of this study was to elucidate the role of C1q‐mediated TF expression. Using a solid‐phase ELISA, TF expression was observed on aortic adventitial fibroblasts (HAAFs) and aortic smooth muscle cells (HASMCs) under various conditions. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, anti‐gC1qR (60.11), or a combination of 60.11 and purified human C1q. After the exposure conditions, cells were incubated for one hour with one of the following primary antibodies: anti‐human gC1qR (74.5.2), anti‐human TF, or anti‐human CD54 (ICAM‐1). Following primary antibody incubation, cells were incubated with an appropriate alkaline phosphatase conjugated secondary antibody for one hour. PNPP was added to the cells and allowed to develop for 30 minutes. Absorbance was documented at 405 nm. Our results suggest that HAAF and HASMC expression of TF increased significantly by approximately 35% after incubation with C1q as compared with unconditioned cells. We also observed increased ICAM expression, 20% above baseline, in HASMCs. Cells conditioned with gC1qR blocking antibodies exhibited a marked decrease in TF and ICAM expression (by approximately 10%) when compared to cells conditioned with C1q alone. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammatory and thrombotic reactions that may occur during vascular diseases. This work illustrates a step in identifying common inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases and may provide new information regarding off‐target effects. Future work will determine if the expressed TF can support extrinsic coagulation activity and new therapeutics can be designed to mitigate vascular disease processes.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vascular diseases can be characterized by and are highly associated with inflammation and thrombosis. Understanding the relationship between these two processes would allow us to treat vascular diseases more effectively. Activation of complement component 1 (C1) is an important contributor to innate immunity and drives the lysis of pathogens. Upon C1 activation, significant concentrations of circulating C1q are observed. When C1q interacts with gC1qR, many pathological pathways initiate and C1q/gC1qR plays a role in inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. While this link between inflammation and thrombosis is important, the intrinsic cascade plays a minor role in physiological and pathological thrombosis. The effects of C1 and the role of C1q/gC1qR on extrinsic coagulation, which is more physiologically relevant, has not been studied. Extrinsic coagulation initiates upon vessel damage, exposing tissue factor (TF) to the blood. Adventitial fibroblasts and vascular smooth muscle cells are the primary TF bearing cells. We hypothesized that C1q binding to gC1qR enhances the expression of sub‐endothelial TF in these cells. The objective of this study was to elucidate the role of gC1qR‐mediated TF expression. Using a solid‐phase ELISA, TF expression was observed on aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. Further, a capture ELISA was used to assess released TF under these same conditions. Finally, a live/dead assay to assess cell viability and cell density, as well as an MTT assay to assess cell metabolic activity were performed to monitor cell culture conditions. Our results indicate that HAAF and HCASMC expression of TF increased significantly by approximately 35% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies exhibited a marked decrease in TF expression (by approximately 10‐20%) when compared to cells conditioned with C1q alone. Our results show no significant differences in metabolic activity or cell viability under these conditions; however, cell density decreased by approximately 25% when conditioned with C1q. Overall, this data illustrates a role for C1q in activation of extrinsic coagulation. Furthermore, our data suggests that gC1qR acts a mediator for both inflammation and thrombosis. This work serves as a meaningful step in identifying common inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF can support extrinsic coagulation activity.