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In vitro uptake of recombinant factor VIIa by megakaryocytes with subsequent production of platelets containing functionally active drug

Recombinant activated factor VII (rFVIIa) has been recently shown to prevent spontaneous bleeding in inhibitorcomplicated haemophilia when administered once daily (Konkle et al, 2007; Young et al, 2012). The pro-haemostatic effect of rFVIIa prophylaxis is difficult to explain given its plasma half-life of 2 h. In the literature, four mechanisms explaining the prophylactic efficacy of once-daily rFVIIa administration have been proposed, including the requirement of much lower doses of rFVIIa to prevent than to treat bleeding, improvement of endothelial cell permeability, uptake of rFVIIa by platelets, and redistribution of rFVIIa into the extravascular space (Lisman & de Groot, 2015). Accumulation of rFVIIa in bone and joints may explain the prophylactic activity of rFVIIa. One study suggested that rFVIIa was taken up by megakaryocytes within the bone marrow (Gopalakrishnan et al, 2010). The present study tested the hypothesis that megakaryocytes that have taken up rFVIIa will produce rFVIIa-containing platelets, which may explain the prolonged haemostatic effect of rFVIIa in a prophylactic setting. We used the megakaryoblastic cell line MEG-01, which was cultured and differentiated by valproic acid as described earlier (Kirschbaum et al, 2015). After at least 10 d of differentiation, different concentrations of rFVIIa were added to the culture medium and after 2 h, MEG-01 cells were either harvested or stimulated to produce platelet-like particles (PLPs) by addition of 100 ng/ml recombinant human thrombopoietin (rTHPO; Life Technologies, Carlsbad, CA, USA) for 3 d. Cells or PLPs were harvested by centrifugation, washed and lysed by freeze-thawing the samples twice. We first showed that rFVIIa was dose-dependently taken up by MEG-01 cells (Fig 1A). After 3 d of rTHPO stimulation, hardly any rFVIIa was detected in the MEG-01 cells (Fig 1B). In contrast, the PLPs produced from these MEG-01 cells contained appreciable amounts of rFVIIa (Fig 1C), which suggests rFVIIa is selectively transferred to PLPs. Active selection of agents to be transferred to platelets in the process of megakaryocyte maturation has been previously demonstrated, as megakaryocytes transfer some, but not all, mRNA species examined to platelets (Cecchetti et al, 2011).