Recombinant annexin 5

Plasma-EVs, aged RBC-EVs, or EV subtypes were stained with PKH26 Red Fluorescent Cell Linker Dye (Sigma-Aldrich). EVs were washed twice with 10% exosome-free FBS in RPMI to quench the unbound dye. Recombinant annexin V (BD Biosciences) or functional grade blocking monoclonal antibodies against phosphatidylserine (PS) (Millipore), CD36 (Stemcell Technologies), CD163, CD206 (BioLegend), TLR1 (Invivogen), TLR2, and TLR4 (BioLegend) were used at multiple concentrations (0.01–2.0 µg/mL) to block EV–monocyte binding. In some experiments, EVs were incubated with annexin V or anti-PS antibody. In other experiments PBMCs were incubated with monoclonal antibodies against CD36, CD163, CD206, TLR1, TLR2, or TLR4 in a 5% CO₂ incubator at 37°C for 1 h. PBMCs (500,000) were cultured with EVs, in a final volume of 0.5 mL for 24 h. PBMCs were stained with CD14-PerCP/Cy5.5 (BioLegend) and were fixed in 2% paraformaldehyde solution. PBMCs were subject to flow cytometry, and percent binding of monocytes to EVs was measured by gating on CD14⁺ cells.

Thrombin generation was measured in a Fluoroskan Ascent (ThermoFisher Scientific) as described by Lecompte et al [18 (link)]. Briefly, 80 μL plasma (Pool Norm) was mixed with 20 μL of 1 x 10⁴ EA.hy926 cells stimulated with histones for 4 h. Subsequently, 20 μL substrate (FluCa-Kit, Thrombinoscope BV, Maastricht, Netherlands) was added. The thrombin generation was measured using Thrombinoscope software (Thrombinoscope BV). In some experiments, antibodies against human TF (clone VD8, 30 μg/mL; American Diagnostica Inc., Stamford, CT, USA) and recombinant annexin V (10 μg/mL, BD Biosciences), anti-PDI antibody (clone RL90, 10 μg/mL; Abcam), glutathione (7.5 mM, Sigma-Aldrich), and quercetin (200 μM, Sigma-Aldrich) were used as inhibitors.

Primary cortical neurons were cultured for 11 DIV before starting treatment with either 20 nM Gas6 or 20 nM Gla-less Gas6. For 7 day long-term treatment, cortical neurons were treated starting at 11 DIV with supplement every other day when medium was half changed. For 1 day-short term treatment, 20 nM Gas6 were added only once to cultures at 16 DIV with or without pretreatment using 100 nM Purified Recombinant Annexin V (BD Pharmingen, #556416). Treatment with TBS buffer, the solvent for Gas6, were used as negative control. To visualize exposed phosphatidylserine on plasma membrane during neuron development in vitro, cortical neurons at 2, 5, 8, 12, 16, or 20 days in vitro (DIV) were incubated with pSIVA-IANBD (Novus Biologicals, NBP2-29382) diluted to 20 μl/ml and CellTracker Red CMTPX dye (Invitrogen C34552) at 0.5 μM in culture medium for 60 min at 37°C, 10% CO₂ before fixing with 4%PFA. To stain exposed phosphatidylserine after calcium ionophore A23187 treatment, cortical neurons at 18 DIV were treated with A23187 at concentration of 5 μM for 20 min in medium also containing pSIVA-IANBD diluted to 20 μl/ml and 0.5 mM CellTracker Red CMTPX dye before fixing with 4% PFA.