Abstract
It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s−1) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf −/− mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf −/− and Vwf +/+ blood, exposed to VWF at 1800 s−1 in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf−/− mice than from Vwf+/+ mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf−/− mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s−1 on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.
Highlights
Von Willebrand factor (VWF) is a multimeric protein synthesized by endothelial cells and stored in Weibel Palade bodies, from which it is released into the circulation [1]
The use of a microfluidic system at a high shear rate allowed proplatelets and platelets to be captured on VWF by keeping them in a reversibly adherent condition, and enabled morphological transitions of proplatelets to be observed (Video S2), as well as the direct release of a small discoid platelet from a translocating proplatelet shown on a sequence indicated by the simultaneous presence of an arrow and an arrowhead (Video S1).These results suggested that blood of both Vwf +/+ and Vwf 2/2 mice contained a mixture of proplatelets and terminally processed discoid platelets
To examine the possible role of VWF in this terminal phase of thrombopoiesis, we used a Vwf 2/2 mouse model characterized by a total absence of VWF
Summary
Von Willebrand factor (VWF) is a multimeric protein synthesized by endothelial cells and stored in Weibel Palade bodies, from which it is released into the circulation [1]. A striking feature of GPIb-mediated platelet rolling on VWF is that it occurs at high shear rates: VWF seems to be unique among adhesive proteins, in that it undergoes a conformational change when exposed to high shear rates [6]. There is clear evidence that only a fraction of GPIb-binding sites of freshly secreted VWF on endothelial cells are occupied, leaving ample possibilities for rapid regulatory mechanisms to increase the part of available binding sites for VWF [7]. One such mechanism is an increase in hemodynamic shear forces
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