C1q/gC1qR‐Mediated Activation of Extrinsic Coagulation

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. C1q has an affinity for gC1qR, a cell surface receptor molecule that is commonly found on cell types associated with cardiovascular disease development, including vascular smooth muscle cells and adventitial fibroblasts. When C1q interacts with gC1qR, many pathological pathways initiate including the inflammation observed during atherosclerosis and the initiation of intrinsic coagulation. The intrinsic cascade plays a minor role in physiological and pathological thrombosis as compared with the extrinsic cascade; however, the effects of C1 and the role of C1q/gC1qR on extrinsic coagulation have not been studied. We hypothesized that C1q association with gC1qR would increase the amount of sub‐endothelial tissue factor expressed by both aortic adventitial fibroblasts (HAAFs) and coronary artery smooth muscle cells (HCASMCs). We investigated these cells as they are the primary TF bearing cells. Further, we hypothesized that the newly expressed TF would bind Factor VII (FVII), which is a crucial step in the initiation of the extrinsic coagulation cascade. The objective of this study was to elucidate the role of C1q/gC1qR‐mediated TF expression and its ability to bind FVII. Using a solid‐phase ELISA, TF expression was observed on both HAAFs and HCASMCs. Cells were conditioned for one hour with either platelet poor plasma (PPP), purified human C1q, LPS, or a combination of anti‐gC1qR antibodies (60.11 or 74.5.2 clones) and purified human C1q, PPP, or LPS. A capture ELISA approach was used to evaluate the binding of FVII to TF following exposure after the same conditions. Further, a capture ELISA was used to assess released TF. 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 15% after incubation with C1q as compared with unconditioned cells. Cells conditioned with gC1qR blocking antibodies prior to conditioning with C1q exhibited no change in TF expression when compared to unconditioned cells. Additionally, a 20‐30% increase in bound FVII was observed in both cell types when conditioned with C1q as compared to unconditioned cells. 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 a convergence of inflammatory and thrombotic pathways that can be therapeutically targeted to mitigate vascular diseases. Future work will determine if the expressed TF and the binding of FVII can support downstream extrinsic coagulation activity. Thanks to NIH for supporting this research.