Figure 2.
Differential expression of activation markers on megakaryocyte and platelet extracellular vesicles. EVs were harvested from MKs and platelets and characterized via nanoparticle tracking analysis (NTA), electron microscopy, and flow cytometry. (A-B) Murine BM-derived MK (Mu BM), murine fetal liver-derived MK (Mu FL), and human CD34+-derived MKs (CD34+) produced between ∼500 and 1500 EVs per cell, after 24 hours in culture, with an average size of ∼200 nm. (C) Immunogold labeling confirmed the majority of EVs expressed CD41 (60% to 80%), with CD34+-derived EVs expressing more gold particles per EV than their murine counterparts. (D-F) Isolated human platelets were stimulated with either thrombin (0.1 U/mL) and collagen (1 µg/mL) (traditional platelet agonists) or LPS (inflammatory agonist) to generate platelet-derived EVs (PEVs). (D-E) NTA analysis showed thrombin/collagen (Thr/Col) activation generated ∼3 EVs/cell and LPS stimulation generated an average of ∼1 EV/cell, with an average size of 100 to 200 nm. (F) Immunogold labeling showed >80% of EVs generated expressed CD41; at least 50 particles were quantified manually by 2 blinded reviewers per n, per condition. Flow cytometry was used to determine the presence of P-selectin (CD62P) and phosphatidyl serine (Annexin V, Ann5). Calcium ionophore-stimulated platelet EVs (Iono) were used as a positive control for CD62P+, Ann5+ staining and antibody-alone controls (Ab) were used to control for background fluorescence and antibody aggregates. EV preparations were incubated with anti-CD41, anti-CD62P, and Annexin V (10 µg/mL) and analyzed by flow cytometry. Representative examples of (G) flow cytometry gating strategy and for (H-I) sample and negative controls. Megamix beads of known size were used to gauge approximate size of particles. Strict fluorescence minus 1 controls were used to determine negative population spread and set gates. Red bars are MkEVs and blue bars are PEVs percentage of CD41+ EVs, which are (J) negative for both CD62P and Annexin V expression, (K) bind CD62P but not Annexin V, (L) bind Annexin V but not CD62P, and (M) bind both Annexin V and CD62P. Data are mean ± SEM of 3 to 4 independent experiments.

Differential expression of activation markers on megakaryocyte and platelet extracellular vesicles. EVs were harvested from MKs and platelets and characterized via nanoparticle tracking analysis (NTA), electron microscopy, and flow cytometry. (A-B) Murine BM-derived MK (Mu BM), murine fetal liver-derived MK (Mu FL), and human CD34+-derived MKs (CD34+) produced between ∼500 and 1500 EVs per cell, after 24 hours in culture, with an average size of ∼200 nm. (C) Immunogold labeling confirmed the majority of EVs expressed CD41 (60% to 80%), with CD34+-derived EVs expressing more gold particles per EV than their murine counterparts. (D-F) Isolated human platelets were stimulated with either thrombin (0.1 U/mL) and collagen (1 µg/mL) (traditional platelet agonists) or LPS (inflammatory agonist) to generate platelet-derived EVs (PEVs). (D-E) NTA analysis showed thrombin/collagen (Thr/Col) activation generated ∼3 EVs/cell and LPS stimulation generated an average of ∼1 EV/cell, with an average size of 100 to 200 nm. (F) Immunogold labeling showed >80% of EVs generated expressed CD41; at least 50 particles were quantified manually by 2 blinded reviewers per n, per condition. Flow cytometry was used to determine the presence of P-selectin (CD62P) and phosphatidyl serine (Annexin V, Ann5). Calcium ionophore-stimulated platelet EVs (Iono) were used as a positive control for CD62P+, Ann5+ staining and antibody-alone controls (Ab) were used to control for background fluorescence and antibody aggregates. EV preparations were incubated with anti-CD41, anti-CD62P, and Annexin V (10 µg/mL) and analyzed by flow cytometry. Representative examples of (G) flow cytometry gating strategy and for (H-I) sample and negative controls. Megamix beads of known size were used to gauge approximate size of particles. Strict fluorescence minus 1 controls were used to determine negative population spread and set gates. Red bars are MkEVs and blue bars are PEVs percentage of CD41+ EVs, which are (J) negative for both CD62P and Annexin V expression, (K) bind CD62P but not Annexin V, (L) bind Annexin V but not CD62P, and (M) bind both Annexin V and CD62P. Data are mean ± SEM of 3 to 4 independent experiments.

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