Figure 1.
S100A8/A9 accelerates the recruitment of annexin-V–positive platelets and fibrin generation at a venous shear rate. (A-C) Plasma levels of S100A8/A9 were measured by ELISA in healthy donors and patients with uncomplicated and complicated (ICU survivors/nonsurvivors) COVID-19 over 7 consecutive days after inclusion in the study. (A) S100A8/A9 levels in the plasma of healthy donors (control, n = 10), uncomplicated COVID-19 patients (n = 48), ICU survivors (n = 26), and ICU nonsurvivors (n = 13) on the first day of patient inclusion in the study. (B-C) S100A8/A9 levels in the plasma over 7 consecutive days after patient recruitment. (D-G) Representative immunofluorescence staining of lung autopsies obtained from COVID-19 patients or age-matched control. Images were captured using Epi fluorescence microscope and slide scanner Axioscan Z1. (D) Platelet CD42b and heterodimeric S100A8/A9 staining in lung parenchyma and microcirculation in COVID-19 and control lung autopsies. (E) Staining for S100A8/A9, MPO, and nuclei (DAPI) of a large vessel in the lung of a patient with COVID-19 with thrombotic complications. (F) Staining for platelet CD42b, fibrin, and nuclei (DAPI) in large thrombi of a patient with COVID-19. (G) S100A9, CD42b, and fibrin staining in COVID-19 lung. Arrow shows platelet-fibrin microaggregates. (H) Whole blood under recalcified conditions was perfused at a venous shear rate (100 s−1) over S100A8/A9 (40 μg/mL), collagen (100 μg/mL), or combination of S100A8/A9 and collagen-coated chambers. The presence of PS-positive platelets and fibrin were assessed using annexin-V-Alexa Fluor-647 and FITC-anti-fibrinogen/fibrin antibody, respectively. (H) Representative images taken at 15 minutes. (I) Quantification of annexin-V signal (3 minutes) (n = 5) and (J) fibrin signal time course (0-15 minutes) (n = 5) using predefined semiautomated scripts in Fiji as detailed in the supplemental Methods (available on the Blood website). Results are shown as mean ± standard error of the mean (SEM). The statistical significance was analyzed using two-way ANOVA with Tukey’s multiple comparison test between all groups. ∗P < .05, ∗∗P < .005, ∗∗∗P < .001, ∗∗∗∗P < .0001.

S100A8/A9 accelerates the recruitment of annexin-V–positive platelets and fibrin generation at a venous shear rate. (A-C) Plasma levels of S100A8/A9 were measured by ELISA in healthy donors and patients with uncomplicated and complicated (ICU survivors/nonsurvivors) COVID-19 over 7 consecutive days after inclusion in the study. (A) S100A8/A9 levels in the plasma of healthy donors (control, n = 10), uncomplicated COVID-19 patients (n = 48), ICU survivors (n = 26), and ICU nonsurvivors (n = 13) on the first day of patient inclusion in the study. (B-C) S100A8/A9 levels in the plasma over 7 consecutive days after patient recruitment. (D-G) Representative immunofluorescence staining of lung autopsies obtained from COVID-19 patients or age-matched control. Images were captured using Epi fluorescence microscope and slide scanner Axioscan Z1. (D) Platelet CD42b and heterodimeric S100A8/A9 staining in lung parenchyma and microcirculation in COVID-19 and control lung autopsies. (E) Staining for S100A8/A9, MPO, and nuclei (DAPI) of a large vessel in the lung of a patient with COVID-19 with thrombotic complications. (F) Staining for platelet CD42b, fibrin, and nuclei (DAPI) in large thrombi of a patient with COVID-19. (G) S100A9, CD42b, and fibrin staining in COVID-19 lung. Arrow shows platelet-fibrin microaggregates. (H) Whole blood under recalcified conditions was perfused at a venous shear rate (100 s−1) over S100A8/A9 (40 μg/mL), collagen (100 μg/mL), or combination of S100A8/A9 and collagen-coated chambers. The presence of PS-positive platelets and fibrin were assessed using annexin-V-Alexa Fluor-647 and FITC-anti-fibrinogen/fibrin antibody, respectively. (H) Representative images taken at 15 minutes. (I) Quantification of annexin-V signal (3 minutes) (n = 5) and (J) fibrin signal time course (0-15 minutes) (n = 5) using predefined semiautomated scripts in Fiji as detailed in the supplemental Methods (available on the Blood website). Results are shown as mean ± standard error of the mean (SEM). The statistical significance was analyzed using two-way ANOVA with Tukey’s multiple comparison test between all groups. ∗P < .05, ∗∗P < .005, ∗∗∗P < .001, ∗∗∗∗P < .0001.

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