Ed Conway and Piyush Kapopara

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This week we profile a recent publication in the Journal of Thrombosis and Haemostasis from the laboratory of Dr. Ed Conway (pictured, left) from the UBC Centre for Blood Research, with Dr. Piyush Kapopara (right). Dr. Kapopara completed a postdoctoral fellowship in Dr. Conway’s lab and is now a Product Manager at Precision NanoSystems Inc.

Can you provide a brief overview of your lab’s current research focus?

I am a professor of Medicine and scientist at the Centre for Blood Research at UBC. We have 2 major lines of research in my lab. The first is to characterize molecular mechanisms that link the blood coagulation (clotting) system with innate immunity, aiming to identify novel therapeutic strategies for the many disorders associated with thrombosis and inflammation. This is particularly relevant to the current pandemic, as SARS-CoV-2 triggers a hyperacute thrombo-inflammatory response that causes multi-organ damage. Our second line of research is aimed at delineating the protective properties of cells that line and surround blood vessels. We are currently specifically focused on characterizing the structure-function correlates of a protein known as CD248 that is expressed on the surface of activated vascular smooth muscle cells, adipocytes and circulating leukocytes, and which promotes inflammation, cell proliferation, and fibrosis. By using molecular/cell biologic techniques and genetically modified mice, we are gaining new insights into how expression of this protein impacts on the risk of venous and arterial thrombosis, atherosclerosis, type 2 diabetes and cancer. 

What is the significance of the findings in this publication?

The blood clotting system is delicately balanced to protect us from excess bleeding or thrombosis. Under healthy conditions, coagulation system activation is low, allowing blood to flow freely throughout the vasculature.  But in response to an injury, the coagulation system rapidly becomes activated, switching into “high gear” in a localized manner, to limit bleeding, restrict infections, and to promote healing. The mechanisms that regulate this system to prevent unwanted bleeding or thrombosis, are complex and involve a dynamic interplay of multiple soluble and membrane bound factors, cofactors, and cells. The major trigger for clot formation is well known to be tissue factor (TF), a transmembrane glycoprotein that is expressed by perivascular cells (eg. vascular smooth muscle cells), stromal cells, adipocytes, circulating leukocytes and microvesicles. In health, the coagulation function of TF is encrypted. But in response to injury or cell stimulation, TF is decrypted and transformed to an active state, whereupon it binds to circulating coagulation factor VIIa (FVIIa) and factor X (FX), resulting in the almost immediate generation of the serine protease, FXa. This enzyme, FXa, then rapidly and efficiently initiates the so-called coagulation cascade, leading to generation of thrombin and formation of a fibrin clot. TF also has other properties, as excess expression has been implicated in a variety of inflammatory disorders, atherosclerosis, obesity and cancer. Moreover, pharmacologic or genetic reduction of TF expression in animal models, limits thrombosis, inflammation, obesity, and cancer. Defining the mechanisms by which TF is regulated, is therefore of major interest for the development of novel therapeutics.

CD248 is also a transmembrane glycoprotein, expressed at very low levels under healthy conditions, but upregulated in perivascular cells, stromal cells, adipocytes and leukocytes in response to injury and inflammation – all reminiscent of TF. Loss-of-function studies in mice by our group and others, revealed a role for CD248 in promoting cancer, inflammation (e.g., arthritis, atherosclerosis, obesity and type 2 diabetes) and fibrosis. In view of the overlapping cellular patterns of expression and inflammatory and pathobiological properties of TF and CD248, we considered that CD248 may work in parallel with TF to regulate coagulation. 

In this report, we showed that mice lacking CD248 are protected against injury-induced venous and arterial thrombosis. Using cultured vascular smooth muscle cells and leukocytes, we found that CD248 is in close proximity to TF and the TF-FVIIa-FX complex, and thus in a position to modulate its function. Indeed, activation of factor X by TF-FVIIa on the surface of cells that express CD248 was significantly increased as compared to cells that lack CD248. The mechanisms by which this occurs was revealed through the use of conformation-specific antibodies; we showed that CD248 induces allosteric changes in the TF-FVIIa-FX complex that likely positions FVIIa for more efficient activation of FX. These findings are consistent with the increased thrombosis that we observed in mice expressing CD248 as compared to the mice lacking CD248. 

This discovery of CD248 as a previously unrecognized cofactor for TF coagulant activity, the first of its kind, provides new understanding of the mechanisms by which TF activity is regulated and thus uncovers potential new therapeutic opportunities to treat and/or prevent thrombotic disorders.

What are the next steps for this research?

We have much exciting work to do toward translating our findings to value in the clinic. Highest on the list is to identify the precise structure(s) on CD248 that interact with the TF-FVIIa-FX complex, and to screen for compounds that interfere with the prothrombotic function of CD248. In parallel, we are exploring different approaches to suppress CD248 expression using antibodies and gene silencing techniques that will be tested in our mouse models of human disease. It is hoped that these approaches will yield new strategies to prevent and/or treat thrombotic disorders, as well as atherosclerosis, diabetes and likely other inflammatory diseases. 

This research was funded by: 

The Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Canada Foundations for Innovation, the Heart and Stroke Foundation of Canada, CanVECTOR, the Canada Research Chairs program,  and the National Institutes of Health.

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