Platelet production and megakaryocytopoiesis in culture. (A) Platelet production from cultured CD34+ MKs obtained from 3 different HDs (empty histograms) used as controls and from 3 patients with VWD2B: 1 patient with mutation R1306W (light gray histograms) and 2 patients with mutation V1316M (dark gray histograms). Relative number of platelets at day 7 of treatment with TPO (left) or TPO and VWF (right). The number of platelets was calculated by flow cytometry on a CD41+/calcein am+-labeled population analyzed in combination with a known number of calibration beads. Data from 2 independent experiments (means ± SD; *P < .05 HD versus each mutant VWF as well as HD and each mutant VWF exposed to VWF versus TPO treatment only). The statistical analyses according to Bonferroni test were grouped as follows. In Group 1, all samples were calculated against TPO-treated HDs. In this group, the TPO-treated VWD2B samples show statistical significance; endogenous VWD2B inhibits platelet production. In Group 2, samples from VWD2B were calculated against TPO-treated VWD2B. In this group, samples treated with normal exogenous VWF are significantly different compared with TPO treatment; normal exogenous VWF normalizes platelet production from VWD2B CD34+ cells. (B) Morphologic studies of the formation of proplatelets as seen by confocal microscopy of MK cultures labeled with an anti-αIIbβ3 (green) antibody and by DAPI (blue). In panels Bi and Bii, the progressive disruption of MK cytoplasm and formation of proplatelets can be seen; MKs appeared with long pseudopods decorated with small blisters corresponding to proplatelets. In panel Biii, the very long pseudopods have a bead-like appearance. For the VWD2B MKs, instead of thin pseudopods, the cytoplasm forms thick protusions (Biv-ix) with interwoven membranes (vii, ix). In panel Bx, 3 distinct but adjacent proplatelets are to be seen. (C) Examination of culture MKs from controls and patients by EM. On the left side (Ci,Ciii), we illustrate the morphology of control MKs, and in panels Cii and Civ, the morphology of VWD2B V1316M MKs. (Ci) Homogeneous presence of granules and DMS. (Cii) All the granules (G) are concentrated in one part of the MK cytoplasm; most of the DMS is also seen in this same area, suggesting a defect in the process of partitioning the components. (Ciii) Shown at a higher magnification is the cytoplasm of another control mature MK, where small platelet territories are well delimited by abundant and well-organized DMS. (Civ) Shown at a high magnification is part of a mature VWD2B MK where an abnormal large platelet territory (arrows) surrounded by DMS can be seen. Globally abnormal distribution of DMS suggests a defect in the organization of membranes during maturation. (Cv-vi) Newly produced platelets from the R1306W MK. (Cv) the platelets are heterogeneous in size, not separated/or agglutinated, and the distribution of granules is heterogeneous. (Cvi) Illustration of a giant platelet as found in the circulation.
