Thromboplastin d

Exosomes were isolated using the Exosome Precipitation Solution (EXOQ20A-1, SBI, Mountain View, CA) as previously mentioned (Wang et al., 2017 (link)). Briefly, 300 μL of plasma was thawed on ice and centrifuged at 3,000 g for 10 min to remove possible residual cell debris. After incubating with thromboplastin D (Thermo, Middletown, CA) at 37°C for 15 min and centrifuging, the 250 μL of supernatant was aspirated to a new tube and mixed with 65 μL of ExoQuick Solution. To digest free and lipoprotein-binding RNAs outside of small extracellular vesicles, RNaseA (Sigma, St. Louis, MO) was added to the mixture at a final concentration of 10 μg/mL. After keeping the mixture at 4°C overnight, murine RNase inhibitor (NEB, Ipswich, MA) was added at 150 units/mL before precipitation of exosomes by centrifuging at 1,500 g for 30 min. The exosome pellet was dissolved in 50 μL of phosphate-buffered saline (PBS) and subjected to RNA extraction immediately.

Plasma was collected at euthanasia, prior to perfusion and stored at −80°C. Plasma exosomes were isolated according to the method of exosome precipitation (Fiandaca et al., 2015 (link)). Briefly, 250 μl of plasma was spun at 3000 × g for 15 min then the supernatant with added protease inhibitor cocktail (Roche Applied Sciences, Inc.) was incubated with 100 μl thromboplastin-D (Thermo Scientific, Inc.) at room temperature for 60 min. After spinning at 13,500 rpm for 5 min, the supernatant with added protease inhibitor cocktail was mixed with 63 μl of ExoQuick^TM-TC exosome precipitation solution (System Biosciences, Inc.) and incubated at 4°C for 60 min. The samples were again spun at 1500 × g for 30 min, were then removed and the supernatant was spun again for 5 min. The pellets were re-suspended in 1 × PBS with H₂O/protein inhibitor. Then the isolated exosomes were purified by Exo-spin^TM column (Cell Guidance Systems, Inc.) according to the manufacturer’s instructions. After purification, exosomes were run on reduced 6–15% Tris-Glycine/SDS gel and western blot was run using an anti-Aqp-4 (Novus Biologicals, Littleton, CO, United States) antibody.

ExoQuick (Systems Biosciences) was used for plasma exosome isolation according to the manufacturer’s instructions. Plasma supernatant was aspirated and labeled “exosome depleted.” The precipitate was washed, re-suspended in 75 µl of PBS, and labeled “exosome enriched.” Exosome fractions used for visualization and Dynabead immunoprecipitation were derived from plasma pre-treated with Thromboplastin-D (ThermoScientific), an anti-coagulant, prior to ExoQuick precipitation to remove aggregating fibrins and fibrinogens. Briefly, 100 µL aliquots of the plasma were warmed to 37°C, treated with an equal volume of Thromboplastin-D, and incubated for 15 min. Fibrins and fibrinogens were pelleted from the plasma following centrifugation at 10,000 rpm for 5 min. The supernatant was fractionated with ExoQuick as described above.

Pentamidine isethionate was purchased from Millipore-Sigma (St. Louis, MO, USA). Reagents for clotting assays including thromboplastin-D, APTT reagent, and thrombin time reagent were all from Fisher Scientific (Pittsburgh, PA, USA). Chemicals used to prepare enzyme assay buffers were from Millipore-Sigma or Fisher Scientific. N,N–dimethylcasein, dansylcadaverine, and dithiothreitol were also from Millipore-Sigma. All types of plasmas were purchased from George King Bio-Medical, Inc. (Overland Park, KS, USA). Coagulation enzymes including thrombin, factor Xa (FXa), factor XIa (FXIa), and factor XIIIa (FXIIIa) were from Haematologic Technologies, Inc. (Essex Junction, VT, USA). Digestive enzymes including trypsin and chymotrypsin were from Millipore-Sigma. Neutrophil elastase was from Elastin Products Company (Owensville, MO, USA). Chromogenic substrates: Spectrozyme TH, Spectrozyme FXa, and Spectrozyme PL, were obtained from Biomedica-Diagnostics (Windsor, NS, Canada). FXIa chromogenic substrate (S-2366) and trypsin substrate (S-2222) were obtained from Diapharma (West Chester, OH, USA). Chromogenic substrate for chymotrypsin (N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and that for neutrophil elastase (S-1384) were from Millipore-Sigma.

Anhydrous
organic solvents (dichloromethane, hexanes, and ethyl acetate) were
obtained from Fisher Scientific (Pittsburgh, PA) and used as they
were received. 1-Ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDCI),
4-dimethylaminopyridine (DMAP), 1-hydroxy benzotriazole (HOBt), and
molecules 11–18 were obtained from
Milipore-Sigma (Burlington, MA). For analytical TLC, UNIPLATETM silica
gel GHLF 250 μm precoated plates from ANALTECH, Newark, DE,
were employed. Column chromatography utilized Sigma-Aldrich’s
silica gel (200–400 mesh, 60 Å). All reactions were conducted
in oven-dried glassware. Flash chromatography was carried out using
Teledyne ISCO’s Combiflash RF system and disposable normal
silica cartridges with a particle size of 30–50 μm, mesh
size of 230–400, and pore size of 60 Å. The flow rate
of the mobile phase was in the range of 18–35 mL/min, and mobile
phase gradients of ethyl acetate/hexanes were used to elute inhibitors.
Human plasmas were purchased from George King Bio-Medical, Inc. (Overland
Park, KS). Reagents for clotting assays, including APTT reagent, thromboplastin
D, and CaCl₂ solution, were all from Fisher Scientific.
UFHs were from Milipore-Sigma, whereas argatroban HCl, rivaroxaban,
and C6B7 were from Fisher Scientific.

Plasma was isolated from HANDLS participants as previously described^{11 (link)}. Plasma (0.4 mL) was thawed on ice and EVs were isolated using ExoQuick™ Exosome Precipitation Solution (System Bioscience Inc.) with some modifications from the manufacturer’s protocol. This isolation procedure provided more reproducible data than differential ultracentrifugation or size exclusion columns^{11 (link)} and also allowed for the processing of a large number of human samples.
Plasma was first treated with 0.2 mL Thromboplastin D (Cat#:100354; Fisher Scientific, Inc.), incubated at room temperature for 30 minutes and then 0.3 mL of Dulbecco’s phosphate buffered saline (DBPS) supplemented with protease and phosphatase inhibitors was added. Samples were centrifugation at 3000 × g for 20 minutes at 4 °C. The supernatants were collected and mixed with ExoQuick™ (252 μl), incubated for one hour at 4 °C and then centrifuged at 1500 × g at 4 °C for 20 minutes. The supernatant was removed and saved for analysis as the EV-depleted plasma fraction. The EV pellet was resuspended in 0.5 mL of nanopure water supplemented with protease and phosphatase inhibitors. An aliquot of the EVs was diluted in DPBS at 1:300 dilution for NTA. A 0.1 mL aliquot of EVs were lysed in MPER at a 1:3 dilution for ELISAs and immunoblotting. All samples were stored at −80 °C until further analysis.