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L. Bury et al.
sure to high shear rates.10,11 Megakaryocytes from type 2B- vWD patients form a reduced number of abnormally large proplatelets,9,12 explaining the macrothrombocytopenia associated with this condition and suggesting that an enhanced GPIba-vWF interaction may alter PPF. It is, therefore, conceivable that defective PPF may contribute to thrombocytopenia in PT-vWD.
occasions) and of 15 healthy controls. The obtained cells were then induced to differentiate into megakaryocytes in StemSpan serum free expansion medium (SFEM) supplemented with human recombinant stem cell factor (SCF) (25 ng/mL) and thrombopoi- etin (TPO) (10 ng/mL) for seven days and TPO alone for the fol- lowing seven days, as previously described.5-7,21,22 All subjects gave their informed consent and all studies were carried out according to the principles of the Declaration of Helsinki. Murine megakary- ocytes were cultured from bone marrow cells flushed from mouse femurs in Dulbecco’s modified eagle’s medium (DMEM) supple- mented with 10% fetal calf serum (FCS) and recombinant murine TPO (10 ng/mL) for four days, as previously described.22 For details see the Online Supplementary Appendix.
Megakaryocyte spreading and proplatelet formation
Spreading and proplatelet formation in human and murine megakaryocytes were evaluated by immunofluorescence, as pre- viously described.5-7,21,22 For details see the Online Supplementary Appendix.
von Willebrand factor binding to megakaryocytes
Exogenous vWF was not added to the medium in any of the experiments. vWF binding to human and murine megakaryocytes was assessed by confocal microscopy5 and by flow cytometry.20,23 For details see the Online Supplementary Appendix. vWF secretion by megakaryocytes was measured in cell culture supernatants at days 3, 7 and 14 of cell differentiation using an ELISA kit (Asserachrom VWF:Ag, Stago Italia, Milan, Italy).
Megakaryocyte intracellular signaling triggered by type I collagen
Megakaryocytes were plated for 16 hours (h) in 12-well plates pre-coated with 25 μg/mL of type I collagen or 1% BSA and then lysed in HEPES-glycerol lysis buffer (HEPES 50 mM, 10% glycerol, 1% Triton X-100, MgCl21.5 mM, EGTA 1 mM, 1% protease inhibitors). RhoA activity (RhoA-GTP), the phosphorylation of MLC2, of Src-family kinases (SFK) and of Lyn were assessed by Western blotting.24 For details see the Online Supplementary Appendix.
Megakaryocyte migration assay
Megakaryocyte migration assay was performed as described25 in transwell migration chambers (8 μm, Millipore) coated or not with 25 μg/mL type I collagen, and cells were counted by flow cytometry.26 Results were expressed as chemotaxis index (CI).27,28 For details see the Online Supplementary Appendix.
Bone marrow histology
Immunostaining of platelets was carried out in ten sections of human bone marrow from the PT-vWD patient, from three patients with immune thrombocytopenia (ITP), and from three controls and ten sections of murine bone marrow from femurs and tibiae of 3 TgWT and 3 TgG233V mice.27,28 For details see the Online Supplementary Appendix.
The same experiments were repeated by administering to mice desmopressin (DDAVP) (0.3 μg/kg) by subcutaneous injection immediately after intravenous injection of the DyLight 488-conju-
Besides GPIba, other megakaryocyte receptors for adhesive proteins play an important role in the regulation of PPF.13 In particular, the interaction of a2β1 and GPVI with type I collagen inhibits PPF, in this way preventing ectopic platelet release in the bone marrow endosteal niche. The interaction of megakaryocyte a2β1 with type I collagen activates the Rho-ROCK pathway which induces the phosphorylation of myosin light chain 2 (MLC2),14 thus inhibiting PPF, while GPVI triggers inhibitory signaling mediated by Src family kinases (SFK),15 a family of kinases acting on an array of downstream effectors, including adaptor, enzyme, and cytoskeletal proteins, that collec- tively co-ordinate cytoskeletal remodeling.16
The loss of physiological suppression of PPF by type I collagen, and consequently the ectopic release of platelets in the bone marrow, have been reported to cause throm- bocytopenia in WAS-/- mice (a model of Wiskott-Aldrich syndrome)17 and in patients with MYH9-RD,18 two inher- ited disorders characterized by reduced platelet number.
We studied proplatelet formation using human and murine PT-vWD megakaryocytes and show that vWF is bound to megakaryocyte surface GPIba at early differen- tiation stages. We also show that megakaryocytes form a reduced number of large platelets compared to control megakaryocytes. Moreover, suppression of proplatelet formation by type I collagen is impaired and associates with abnormalities of the intracellular signaling pathways triggered by collagen, involved in the suppression of pro- platelet formation. An enhanced number of free platelets were consistently observed in the bone marrow of PT- vWD mice. Increased clearance of platelet/vWF complex- es is also evident, and it contributes to reduce the platelet count, especially in stress conditions in which circulating vWF levels are increased.
Methods
Throughout the manuscript the terms control and PT-vWD will refer to human megakaryocytes/platelets and the terms TgWT and TgG233V to murine megakaryocytes/platelets.
This study was approved by the ethics committee Comitato Etico Aziende Sanitarie (CEAS) of the Region of Umbria (approval number 2663/15).
Animals
Human and murine megakaryocyte cultures
To obtain human megakaryocytes, CD45+ or alternatively CD34+ cells were separated from peripheral blood of a PT-vWD patient carrying the Met239Val mutation20 (studied on 12 different
The generation of mice expressing the human GPIba transgene carrying the G233V variant in homozygous form (TgG233V) and of control mice expressing a wild-type human GPIba transgene (TgWT) has been previously described.3,19 These animals express a human GPIba transgene and no mouse GPIba, and both TgG233V and TgWT have been consistently backcrossed with C57BL/6J ani- mals.
Measurement of platelet life span in mice
Mice were injected intravenously with 0.5 μg/g body weight of an anti-GPIX mAb (Emfret Analytics, Eibelstadt, Germany) conju- gated with DyLight 488 (Life Technologies, Italia) and the percent- age of residual fluorescent platelets was assessed for five days by flow cytometry as described.4,26 Blood was obtained from tail tip to cuts in tubes containing 4% sodium citrate.
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haematologica | 2019; 104(7)