Human platelets issued from CD34+ cells in NOD/SCID PB can be activated by CRP and TRAP. (A) PB from representative untransplanted (i, n ≥ 3) and 3 × 106 CD34+-transplanted mouse (ii-iii, n ≥ 3) were incubated without or with 0.5 μg/mL CRP or 40μM TRAP for 10 minutes, 2 (ii) or 8 (iii) weeks after transplantation. An increase in P-selectin and PAC1 binding was observed, indicative of activated human platelets. Numbers in quadrants indicate percentage of cells in each. (B) The population of human platelets PAC1+/CD62P+ after 0.5 μg/mL CRP (■) or 40μM TRAP (▩) stimulation was expressed in percentage of the entire population of human platelets (CD61+) detected in NOD/SCID mice 2 or 8 weeks after transplantation (n ≥ 5; mean ± SEM); **.005 < P < .05. (C) A clear correlation was observed between the percentage of human platelets detected with anti–human CD61-PE and with PAC1 or anti–human CD62P after stimulation with 0.5 μg/mL CRP (■) or 40μM TRAP (○; n ≥ 7; r > 0.711; P < .001). Data include percentages of activated platelets at all time points except 2 weeks after transplantation. (D) Representative flow cytometric analysis of PB cells (n = 3) from the same transplanted mouse as in Figure 2Aiii. PB was incubated without agonist (i) or with 0.5 μg/mL CRP (ii), 5 μg/mL CRP (iii,v), or 40μM TRAP (iv), and subsequently stained with moAbs anti–murine Wug.E9-FITC and anti–human CD62P-PE. Activated human platelets (hCD62P+), murine platelets (mCD62P+), and human/murine platelets (hCD62P+-mCD62P+) are represented in green, blue, and yellow, respectively. (v) Each population was visualized according to its forward (FSC_LOG) and side scatter (SS_LOG).
