Platelet production from HD CD34⁺ cell cultures. Relative number of platelets at day 7 of treatment with TPO or TPO and VWF (A). The effects of fibrinogen (FG) and fibronectin (FN) and different inhibitors on platelet production in the presence or absence of VWF is shown. The number of platelets was calculated by flow cytometry on a CD41⁺/calcein am⁺-labeled population; a representative dot plot is shown (B) analyzed in combination with a known number of calibration beads. (C) Time-course kinetics of platelet production after 7 and 14 days of treatment with TPO (control) or TPO and VWF. Data from 4 independent experiments are shown. The statistical analyses according to Bonferroni test were grouped as follows: In Group 1, samples from A to G were all calculated against A. In this group, only sample D showed statistical significance (means ± SD; *P < .05 vs TPO treatment); VWF promotes platelet production. In Group 2, samples from A to G were all calculated against D. In this group, samples A, B, and E were significantly different; GPIba inhibition selectively counteracts VWF stimulation (means ± SD; #P < .05 vs TPO and VWF treatment). In Group 3, samples A, D, and H were all calculated against A and D. In this group, the sample H showed statistical significance; recombinant mutated VWF strongly promotes platelet production from normal CD34⁺ cells (means ± SD; *P < .05 vs TPO treatment; ^#P < .05 versus TPO and VWF treatment).