Complement Component C1q Initiates Extrinsic Coagulation via gC1qR

Abstract

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.