The role of shear in agonist-induced PS exposure and microvesiculation in platelets. (A) Typical flow cytometry histograms of washed mouse and human platelets stimulated with thrombin (mouse, 0.05 U/mL; human, 1 U/mL), collagen (mouse, 2.5 µg/mL; human, 6 µg/mL), or thrombin plus collagen, subjected to shear (6000 seconds⁻¹) or not, and stained with fluorescent Annexin V. (B) Quantification of A (mean ± standard error of the mean [SEM], n = 3 for mouse; n = 6 for human). (C-D) Flow cytometry analyses of platelets treated with or without thrombin (mouse, 0.2 U/mL; human, 10 U/mL), subjected to shear and labeled with fluorescent Annexin V; 0.5- (black arrow) and 0.9-µm (gray arrow) standard beads mark particle sizes, and blue lines passing through the size standards define the region between 0.5 and 0.9 µm. (C) SSC vs FSC. (D) FSC vs Annexin V fluorescence. MVs are defined as having sizes below 1 µm in diameter by ISTH consensus. (E) Washed mouse (n = 3) and human (n = 4) platelets were stimulated with 0.1 and 1 U/mL thrombin, respectively, or unstimulated, and subjected to increasing levels of shear. Annexin V binding, and MVs were analyzed using flow cytometry (mean ± SEM). (F-G) Resting or A23187 (100 nM)-stimulated mouse platelets were subjected to shear or not and analyzed for Annexin V binding and MV release. (F) Typical SSC vs FSC density plots and histogram of Annexin V binding. (G) Quantification of Annexin V binding and microvesiculation (mean ± SEM, n = 3). For all data, * and *** represent statistical significance of P < .05 and .001, respectively.