Isolation of Silica-free DNA and Studies of its Effects on Organ Damage in Mice
ISTH Academy. Medeiros S. Jul 10, 2019; 273867; OC 71.1 Topic: Contact Pathway
Sarah Medeiros
Sarah Medeiros
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OC 71.1

Isolation of Silica-free DNA and Studies of its Effects on Organ Damage in Mice

S.K. Medeiros1,2, N. Zafar2, N. Sharma1,2, D. Dwivedi1,2, J. Zhou1,2, P.Y. Kim1,2, P.C. Liaw1,2
1McMaster University, Hamilton, Canada, 2Thrombosis and Atherosclerosis Research Institute, Hamilton, Canada

Main Topic: Coagulation and Anticoagulation
Category: Contact Pathway

Background: Neutrophil extracellular traps (NETs) are an important interface between innate immunity and coagulation. The major structural components of NETs are nucleosomes (DNA-histone complexes). Although nucleosomes do not modulate coagulation, there are conditions where DNA and histones dissociate from each other in circulation (e.g. in the presence of heparan sulfate or therapeutic heparin). Previous studies have shown that DNA (when not in complex with histones) activates coagulation and impairs fibrinolysis. However, these effects have been questioned due to silica contamination when DNA is isolated using common silica-based kits.
Aims:
(1) To identify and characterize alternative methods of isolating silica-free DNA and
(2) to determine the impact of elevated cell-free DNA (CFDNA) concentrations in circulation in mice.
Methods: DNA was isolated from whole blood using the QIAGEN PAXgene Blood DNA kit (PAX-DNA), and QuickGene DNA kit (DBL-DNA). The procoagulant properties of DNA were assessed by thrombin generation and plasma clotting assays. C57Bl/6 mice (8-14 weeks) were intravenously administered 200uL of PAX-DNA (200ug) or saline. Blood and organs were collected at 0, 15, 30, and 60 minutes post-injection. CFDNA was quantified from plasma at each time-point. Lungs, livers, and kidneys were stained with H&E and blindly scored for thrombus formation.
Results: PAX-DNA and DBL-DNA yielded silica-free DNA that increased thrombin generation and accelerated clotting in a dose-dependent manner, which was neutralized by DNaseI. The circulating half-life of PAX-DNA based on CFDNA quantitation in mice was ~10min. Lung and liver harvested post-DNA injection possessed thrombi/emboli, as evidenced by the presence of fibrin and platelet aggregation.
Conclusions: We identified and characterized two methods of isolating silica-free DNA. These studies are the first to investigate the effects of silica-free DNA in vivo. These studies may shed light into the functional role of elevated CFDNA in the pathogenesis of thrombosis in vascular diseases.

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