Edward Conway

Blood-contacting biomaterials are commonly and increasingly used in medical devices including for example, vascular grafts, coronary stents, prosthetic heart valves, and extracorporeal pumps. A major and persistent problem of exposing blood to a foreign biomaterial surface is that a thromboinflammatory response is triggered that may cause device failure and/or systemic inflammation and clotting. Several biomaterial coatings, particularly heparin, are currently used to prevent thromboinflammation, but these do not reliably work. The goal of my thesis was to develop a novel blood compatible surface coating that simultaneously prevents thrombosis and inflammation by immobilizing anticoagulant and anti-inflammatory molecules in a more functional manner, onto a universal surface coating. This thesis is divided into two parts. In the first part, I chemically modified unfractionated heparin, a potent anticoagulant, on one end such that it could be conjugated to the universal antifouling surface coating in an orientation that optimally and uniformly exposed its anticoagulant antithrombin binding site. The functional properties of this heparin coating were tested under static and dynamic conditions, and these verified that this novel heparin coating was protective against thrombosis. In the second part, I modified the cDNA encoding the naturally occurring anti-inflammatory lectin-like domain of thrombomodulin (TM-LLD), such that it could be expressed by cultured mammalian cells, purified by affinity chromatography, and then conjugated to the antifouling surface coating in an accessible orientation. The purified soluble TM-LLD exhibited anti-inflammatory properties, in that it dampened leukocyte adhesion and complement activation. However, its activity when bound to the surface coating has yet to be confirmed. While further studies are required to optimize the function and to test the stability of these immobilized molecules, this thesis has provided proof-of-concept that the potent anti-coagulant, heparin, and the anti-inflammatory TM-LLD, can be chemically modified for conjugation to the antifouling surface in an orientation to optimize function. Future work will include experiments to upscale production of the modified proteins, to verify their functions and stability under different conditions, and finally, to apply combinatorial approaches to simultaneously bind the molecules to the coating to yield a truly biocompatible, biomaterial surface that prevents both thrombosis and inflammation.

View record

Coagulation and complement are evolutionarily related, with several well-described mechanisms of cross-talk. Recently, it was established that polyphosphate (polyP) is a physiologic activator and promoter of coagulation. I hypothesized that polyP also plays a role in regulating complement, and thereby acts as an additional molecular bridge between coagulation and complement. Evidence to support this was provided by studies in bacteria, where defects in polyP synthesis and degradation alter its resistance to serum-mediated killing. In this thesis, I show that polyP suppresses total complement-mediated lytic activity and the terminal pathway, resulting in decreased lysis of foreign erythrocytes by the membrane attack complex (MAC). In contrast, monophosphate exhibits 10-fold less inhibition in total hemolytic activity, and has no effect on the terminal pathway. I also provide evidence that polyP destabilizes the C5b,6 complex to prevent functional MAC formation. Implications of the role of polyP in complement modulation are discussed.

View record

CD248 is a member of a family of transmembrane glycoproteins containing an N-terminal C-type lectin-like domain. This family includes thrombomodulin and CD93, proteins known to modulate immunity, cell proliferation and homeostasis. CD248 is expressed in perivascular and stromal cells, during embryonic development and post-natally during inflammation and cancer. In the mesenchymal compartment of most normal adult tissues, CD248 expression is not detectable. It is believed that CD248 is involved in cross-talk between endothelial cells and pericytes, thereby playing a role in growth, metastasis and angiogenesis associated with the development of tumours and inflammatory lesions. Intracellular signaling, mediated via the cytoplasmic domain of CD248, promotes tumour growth and inflammation. CD248 knock-out mice and mice expressing CD248 lacking the cytoplasmic domain were resistant to tumour growth and inflammatory arthritis. Although signaling pathways have not been delineated, examination of the cytoplasmic domain of CD248 reveals three highly conserved putative phosphorylation sites and a PDZ-binding motif. We hypothesised that these structural features are important for CD248 function.We generated a variety of murine CD248 pcDNA constructs that encode CD248 with mutations in the cytoplasmic domain. We confirmed that all the mutants were transcribed and translated. Mutant proteins were expressed on the cell surface, in a similar manner to wildtype CD248. Introduction of some mutant forms CD248 into cells caused CD248 to exhibit different intracellular localisation and induced changes in cellular morphology compared to wildtype. Limited functional studies demonstrated CD248-dependent alterations in cellular MMP-9 production. The findings underline an important role of CD248’s cytoplasmic domain in regulating cellular morphology and function that may impact its role in health and disease. Extracellular interacting partners for CD248 have been previously described. Searches for intracellular partners interacting with the cytoplasmic domain have been less successful. Our results strongly suggest that these exist. Co-immunoprecipitation studies have revealed several putative interacting proteins that set the stage for future confirmatory and functional analyses. The cytoplasmic domain of CD248 is important to study as it holds much promise as a therapeutic target for proliferative disorders. The information gathered in this project may be used to delineate clinically relevant CD248 signaling pathways.

View record