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L. Bury et al.
Discussion
The formation of proplatelets by megakaryocytes is tightly regulated by the interaction with bone marrow matrix proteins. In fact, several molecules, such as type IV- collagen, fibrinogen, fibronectin or vWF, support platelet biogenesis while type I-collagen inhibits it, thus preventing the premature release of platelets in the bone marrow.13 Defective PPF as a cause of inherited thrombocytopenias has been reported in Bernard-Soulier syndrome6,7 and in type 2B-vWD,9,12 but so far no studies have assessed PPF in PT-vWD.
Here we show that vWF binds to PT-vWD megakary- ocytes at early stages of differentiation, causing a dysregu- lation of downstream signaling. This disturbs the suppres- sion of PPF by type-I collagen, leading to the ectopic release of platelets in the bone marrow. Moreover, we show that megakaryocytes form a reduced number of large platelets, and that circulating PT-vWD platelets have vWF bound to their surface. We also observed that this leads to their clear- ance, especially when circulating levels of vWF increase. These mechanisms contribute to generate the thrombocy- topenia typical of this disorder.
Differently from control megakaryocytes, which bind vWF only during PPF, PT-vWD megakaryocytes bind vWF at very early stages of differentiation. We show that, in vitro, differentiating megakaryocytes synthesize vWF and secrete it in the supernatant, with subsequent binding to mutant GPIba in PT-vWD. It is conceivable that this happens in vivo in the bone marrow, too.
Platelet-type von Willebrand disease megakaryocytes developed proplatelets with a reduced number of tips and slightly enlarged with respect to controls, in line with the mildly increased diameter of circulating platelets seen in patients.31 PPF requires a finely regulated cytoskeletal remodeling,13,22 and, given that GPIba interacts with several cytoskeletal proteins, it is likely that mutated GPIba leads to cytoskeletal perturbation with the formation of enlarged proplatelets.
Platelet-type von Willebrand disease megakaryocytes developed proplatelets on type I collagen, differently from control megakaryocytes. This abnormal behavior was reproduced with control megakaryocytes when vWF-GPIba binding was induced by ristocetin.
Platelet-type von Willebrand disease megakaryocytes also migrated through type I collagen-coated transwells more than controls, further showing a deranged interaction with collagen. A recent study showed normal response to type I collagen of platelets form TgG233V mice.32 However, in our experimental conditions, collagen was used as a sub- strate for adhesion of megakaryocytes and not in suspen- sion as inducer of platelet aggregation, and this may account for the difference.
We did not detect defects of the collagen receptors, rare or common genetic variants of GP6, ITGA2 and ITGB1 by flow cytometry or by sequencing. Therefore, we investigat- ed the signaling triggered by the interaction of megakary- ocyte a2β1 with collagen.14 We found decreased RhoA acti- vation (RhoA-GTP) and MLC2 phosphorylation, showing impaired a2β1–mediated signaling. Interestingly, a dysregu- lation of the RhoA pathway has been recently shown in type 2B-vWD, a condition associated with enhanced vWF- GPIba interaction.30 We also assessed SFK phosphorylation, given that GPVI-dependent SFK signaling inhibits PPF,15 and found it to be increased in resting PT-vWD megakary-
ocytes. Lyn, a SFK, exerts a dual function in platelets acting both as a positive and negative regulator of GPVI- and inte- grin-mediated activation. vWF binding to GPIb/V/IX trig- gers the association between the cytoplasmic tail of GPIba and Lyn with consequent phosphorylation of the latter16,33 and downregulation of integrin and GPVI signaling.34,35 Indeed, Lyn phosphorylation (p-Lyn) was evident in resting PT-vWD megakaryocytes and was also triggered by risto- cetin in control megakaryocytes. Thus, constitutive binding of vWF to GPIba in PT-vWD megakaryocytes causes Lyn phosphorylation, and p-Lyn in turn acts as a negative regu- lator of GPVI- and a2β1-triggered signaling,34-37 thus prevent- ing the inhibition of PPF by collagen (Online Supplementary Figure S10) with consequent ectopic PPF in the bone mar- row causing thrombocytopenia. In accordance with this, we found an increased number of platelets in human and mouse PT-vWD bone marrow.
We also provide the first direct evidence of the presence of circulating platelet/vWF complexes in PT-vWD, associat- ed in mice with a reduced platelet life span that exacerbates when the concentration of vWF in plasma is increased by DDAVP administration. These data confirm that the bind- ing of vWF to the platelet surface accelerates platelet clear- ance. Interestingly, not all PT-vWD platelets are bound to vWF, and this may partly account for the interindividual variability in platelet count and bleeding diathesis of PT- vWD patients1 and mice.32
Conditions enhancing vWF levels, such as infection, sur- gery, pregnancy, or the treatment with DDAVP typically decrease the platelet count in type 2B vWD and in PT-vWD patients.38,39 Our data support the recommendation that DDAVP administration may not be advisable for PT-vWD patients,40 although some patients do not experience throm- bocytopenia on treatment with DDAVP41.
In conclusion, we show for the first time that constitutive binding of vWF to mutated GPIba of PT-vWD megakary- ocytes triggers dysregulated intracellular signaling leading to the loss of the physiological inhibition of proplatelet for- mation on type I collagen, and thus to ectopic platelet release in the bone marrow. This, together with the forma- tion of a reduced number of large platelets by PT-vWD megakaryocytes and increased platelet clearance, causes thrombocytopenia. Our study clarifies the complexity of the mechanisms leading to thrombocytopenia in PT-vWD and may help to develop novel treatment options. In partic- ular, studies to investigate whether the inhibition of GPIba- vWF binding may, at least partially, revert thrombocytope- nia in PT-vWD are warranted.
Funding
This work was supported by a Telethon grant (GGP15063) to PG and by a grant from Cariplo Foundation (2013-0717) to AB.
Acknowledgments
The authors thank Prof. Jerry Ware (University of Arkansas, USA) and Dr. Maha Othman (Queen’s University, Canada) for the kind gift of the TgWT and TgG233V mice, Prof. Brunangelo Falini (University of Perugia, Italy) for providing human bone marrow biopsies, Dr. Barbara Bigerna (University of Perugia, Italy) for help with immunohistochemistry of platelets in murine bone mar- row, Dr. Anna Maria Mezzasoma and Dr. Emanuela Falcinelli for the measurement of VWF in plasma and cell culture medium, Dr. Francesca Milano and Dr. Giuseppe Guglielmini for help with confocal microscopy. The continued collaboration of our PT- VWD patient is gratefully acknowledged.
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haematologica | 2019; 104(7)