Since platelets and megakaryocytes were identified under microscopes, hematopoietic factors acting directly or indirectly on megakaryopoisis has been reported. Thrombopoietin (TPO) was identified as a primary factor for platelet production. After that time, TPO -receptor agonists have been approved recently for the treatment of thrombocytopenia. However there are still many points to be clarified as to their particular cellular development. In the course of a study of the molecular function of TPO and its receptor c-Mpl, some missing pieces of the puzzle derived from earlier findings should be revisited. The regulation of TPO levels in the circulation is reasonably explained in most deceases by a “sponge” model, which means a dynamic equilibrium of the binding amount of TPO to total c-Mpl mass on megakaryocytes and platelets. However the possible regulation by other mechanisms including proteolytic events that cleaves TPO to generate active truncated forms, and the induction of TPO expression in bone marrow are unex plained sufficiently, in contrast to its constitutive expression in the liver. From another perspective, molecular interaction of TPO and c-Mpl has not been completely elucidated. Although phosphorylation of c-Mpl for the ensuing TPO signaling requires optimally oriented Mpl dimers, the feature of c-Mpl dimerization is poorly understood. Our single-molecule fluorescence imaging revealed an equilibration of monomers, dimers and polymers of c-Mpl on the cell surface prior to their bindings to TPO, and then c-Mpl dimers were stabilized by TPO binding. Furthermore, subsequent internalization and degradation by ubiquitin pathway were directly observed. The knowledge of the life history of TPO and c-Mpl molecules gradually begins to widen.

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Heparin is often administered along with pulsed steroid therapy, for preventing thrombosis. However, the effects of steroids on blood coagulability remain to be clarified. To clarify the thrombogenicity of steroids, we measured the thrombogenicity using the thrombin generation test (TGT) before and after administration of pulsed methylprednisolone (m-PSL) injections to rabbits. Among the TGT parameters, the lag time was shortened (0.8±0.11 times) and the ETP (1.39±0.31 times) and peak (1.91±0.69 times) were increased in the pulsed m-PSL group as compared to the control group, indicating a marked increase of the thrombin generation potential (TGP) . Plasma factor VII activity (FVII : C) was increased (1.68±0.47 times) in the pulsed m-PSL group. The increased TGP could be reproduced in vitro by the addition of recombinant activated FVII, but not by that of m-PSL. The increased TGP clearly reflected blood hypercoagulability, caused mainly by the elevated FVII : C induced by the drug. However, neither the soluble fibrin (SF) nor D-dimer levels were increased, indicating the thrombosis was not induced by the steroid therapy alone. Therefore, we propose that heparin use during steroid pulse therapy may be clinically appropriate when thrombotic risk factors are associated with the underlying disease.

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[in Japanese]

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