Release of human uPA-PLTs in vivo after infusion of not treated (NT) or scuPA-loaded CD34⁺ MKs in NSG mice. (A) Schematic representation of intrapulmonary generation in NSG mice of human PLTs from infused CD34⁺ MKs⁴⁹ that had or had not been incubated with exogenous uPA variant. (B) Day-12 CD34⁺ MKs (6 × 10⁶) that had or had not been loaded with Alexa488-scuPA (400 nM) for 24 hours were injected into NSG mice. At each time point, peripheral blood sample was withdrawn and stained with APC-human CD41 and BUV395-mouse CD41 antibodies to measure circulating human PLTs numbers relative to murine PLTs. MFI was measured using flow cytometry. Ordinate denotes percent human PLTs of the total number of human and mouse PLTs measured in the blood sample. CD34⁺ MKs (blue) or uPA-MKs (red) infused into mice. Mean ± 1 SD is shown. n = 8 (control MKs) and n = 6 (scuPA-MKs). (C) Same experiment as in panel B but scuPA retention was determined as the percent human PLTs that remained Alexa-488 scuPA positive at indicated time points. Mean ± 1 SD is shown. n = 6. (D) Studies of agonist responsiveness for the control MKs vs uPAT-loaded MKs. Flow cytometry studies of the control MKs vs uPAT-loaded MKs at 24 hours after adding uPAT (400 nM) to the medium. P-selectin exposure after activation of the control and uPAT-loaded MKs by human-specific, thrombin receptor activating peptide 6 (TRAP6; 50 μg/mL) was measured using fluorescein isothiocyanate (FITC)-conjugated anti-human CD41 and allophycocyanin (APC)-conjugated anti–P-selectin monoclonal antibody. Mean ± 1 SD is shown. n = 3 per arm. ∗P ≤ .0001 by 1-way ANOVA comparing TRAP responsiveness of NT-MKs vs uPAT-MKs. (E) Studies of agonist responsiveness for the PLTs released in vivo after infusion of NT-MKs vs scuPA-MKs. Flow cytometry studies 6 hours after infusion of the NT-MKs vs uPAT-MKs are shown. P-selectin exposure after activation of the non-treated (NT)-human (h) PLTs and uPAT-hPLTs by (TRAP6, 50 μg/mL) was measured in whole mouse blood using FITC anti-human CD41 and APC anti–P-selectin monoclonal antibody. Mean ± 1 SD is shown. n = 3 per arm. ∗P ≤ .0001 by 1-way ANOVA comparing TRAP responsiveness of NT-MK–derived PLTs vs uPAT-MK–derived PLTs.