American Society of Hematology

Figure 5.

$TF activity is exclusively found in SARS-CoV-2 and EV-containing chromatography fractions. (A) β-propiolactone–inactivated SARS-CoV-2 obtained from human A549 (ACE2) cell infection was concentrated by Ultrafiltration (10 kDa filter columns) as size-exclusion chromatography dilutes samples up to 10 times. Celltracker-DR–positive (Cell+) particles (EVs/virus) and protein content before and after sample ultrafiltration were quantified by flow cytometry (left y-axis, 3 technical replicates) and BCA assay (right y-axis, 2 technical replicates), respectively. The concentrated sample was then loaded on an SEC column, and fractions of 0.5 mL after the void volume were collected (see cartoon created with Biorender). (B) Celltracker-DR–positive particles (EVs/virus) and protein content of each SEC fraction were quantified by FACS (left y-axis, 3 technical replicates) and BCA assay (right y-axis, 2 technical replicates), respectively. (C) Celltracker-DR–positive and spike-positive (samples labeled by mouse IgG1 anti-spike SARS-CoV-2, fluorescein-conjugated) particles (EV/virus) and SARS-CoV-2 RNA content (left or right y-axis, respectively) were quantified in each SEC fraction. (D) Left y-axis. TF activity was assessed by TF/FVII-mediated conversion of FX to FXa and subsequent conversion of FXa substrate. The specific TF activity indicated is calculated as total TF activity measured in the presence of control Ig minus the total TF activity measured in the presence of anti-TF antibody HTF-1. Samples were preincubated for 15 minutes with control Ig or HTF-1. The numbers above the bars indicate the estimated TF activity in pM. Measured in 3 technical replicates. (D) Right y-axis, wt C57BL/6J mouse platelet stimulation (n = 6) with SEC fractions (1:25 vol/vol) in the presence of pooled, recalcified (10 mM CaCl2) mouse PFP (0.5%). The amount of CaCl2 was twofold higher than in previous platelet stimulations as phosphate contained in SEC buffer (PBS) may chelate free calcium. Data are represented as mean ± SD. Statistical analysis: (D) one-way ANOVA followed by Dunnett’s multiple comparison test. ****P < .0001.$

TF activity is exclusively found in SARS-CoV-2 and EV-containing chromatography fractions. (A) β-propiolactone–inactivated SARS-CoV-2 obtained from human A549 (ACE2) cell infection was concentrated by Ultrafiltration (10 kDa filter columns) as size-exclusion chromatography dilutes samples up to 10 times. Celltracker-DR–positive (Cell+) particles (EVs/virus) and protein content before and after sample ultrafiltration were quantified by flow cytometry (left y-axis, 3 technical replicates) and BCA assay (right y-axis, 2 technical replicates), respectively. The concentrated sample was then loaded on an SEC column, and fractions of 0.5 mL after the void volume were collected (see cartoon created with Biorender). (B) Celltracker-DR–positive particles (EVs/virus) and protein content of each SEC fraction were quantified by FACS (left y-axis, 3 technical replicates) and BCA assay (right y-axis, 2 technical replicates), respectively. (C) Celltracker-DR–positive and spike-positive (samples labeled by mouse IgG1 anti-spike SARS-CoV-2, fluorescein-conjugated) particles (EV/virus) and SARS-CoV-2 RNA content (left or right y-axis, respectively) were quantified in each SEC fraction. (D) Left y-axis. TF activity was assessed by TF/FVII-mediated conversion of FX to FXa and subsequent conversion of FXa substrate. The specific TF activity indicated is calculated as total TF activity measured in the presence of control Ig minus the total TF activity measured in the presence of anti-TF antibody HTF-1. Samples were preincubated for 15 minutes with control Ig or HTF-1. The numbers above the bars indicate the estimated TF activity in pM. Measured in 3 technical replicates. (D) Right y-axis, wt C57BL/6J mouse platelet stimulation (n = 6) with SEC fractions (1:25 vol/vol) in the presence of pooled, recalcified (10 mM CaCl2) mouse PFP (0.5%). The amount of CaCl₂ was twofold higher than in previous platelet stimulations as phosphate contained in SEC buffer (PBS) may chelate free calcium. Data are represented as mean ± SD. Statistical analysis: (D) one-way ANOVA followed by Dunnett’s multiple comparison test. ****P < .0001.

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