Neutrophil extracellular traps (NETs) are a link between infection and coagulation in sepsis. The major structural component of NETs is nucleosomes, consisting of DNA and histones. NETs not only act as a scaffold to trap platelets, but NET components also promote coagulation and impair fibrinolysis. Thus, removal of extracellular DNA by DNases may be a potential therapeutic strategy for sepsis. Since heparin is used for thromboprophylaxis in sepsis and may also be a potential anti-sepsis therapy, we investigated the mechanisms by which various forms of heparins modulate DNase function.
There are two types of DNases in vivo: DNase I (produced by exocrine and endocrine glands) and DNase1L3 (secreted by immune cells). DNase I cleaves free DNA, whereas DNase1L3 preferentially cleaves DNA in complex with proteins such as histones. In this study, we investigated how DNase I and DNase1L3 activities are modulated by the following heparins: unfractionated heparin (UFH), enoxaparin (a low-molecular-weight heparin), Vasoflux (a low-molecular-weight, non-anticoagulant heparin), and fondaparinux (the pentasaccharide unit).
Using agarose gel experiments, we showed that UFH, enoxaparin, and Vasoflux enhance the ability of DNase I to digest DNA-histone complexes (presumably by displacing DNA from histones), whereas fondaparinux does not. These findings are consistent with the KD values of the binding of heparin variants to histones, with fondaparinux having >1000-fold lower affinity for histones compared to the other heparins. Taken together, our data suggests that the ability of heparin to enhance DNase I-mediated digestion of DNA-histone complexes is size-dependent and independent of the pentasaccharide region of heparin. With respect to DNase1L3, we observed that it is able to digest histone-bound DNA, and that all heparins, except fondaparinux, inhibited DNase1L3-mediated digestion of histone-bound DNA.
Next, we visualized the degradation of NETs by fluorescence microscopy. DNase I (± heparin variants) completely degraded NETs, presumably by digesting extracellular chromatin at histone-free linker regions, thereby releasing nucleosome units. DNase1L3 also degraded NETs, but not as effectively as DNase I, and was inhibited by all heparins except fondaparinux. Finally, we showed that DNase I levels are decreased and DNase1L3 levels are elevated in septic patients. Taken together, our findings demonstrate that heparin modulates the function of DNases, and that endogenous DNase levels are altered in sepsis pathophysiology. / Thesis / Master of Science (MSc) / Sepsis, a life-threatening condition due to hyperactivation of the immune system in response to infection, results in widespread inflammation and blood clotting. During sepsis, immune cells release sticky strands of DNA that block blood vessels and damage organs. Two different enzymes in the blood (DNase I and DNase1L3) can digest these DNA strands, and may represent a new class of anti-sepsis drugs. Our goal was to determine how heparins, commonly used blood thinners, alter the function of these enzymes. We found that (a) larger-sized heparins improved the activity of DNase I towards DNA-histone complexes and do not require any specific portion of heparin, (b) DNase I is more efficient than DNase1L3 in digesting DNA strands released from immune cells, and (c) levels of DNase I and DNase1L3 are altered in septic patients. Taken together, our studies provide new insights into how these enzymes function.
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/25753 |
| Date | January 2020 |
| Creators | Sohrabipour, Sahar |
| Contributors | Liaw, Patricia, Medical Sciences (Thrombosis & Haemostasis & Atherosclerosis) |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
| Type | Thesis |
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