Tissue dynamics of von Willebrand factor characterized by a novel fluorescent protein–von Willebrand factor chimera

Abstract

BackgroundTissue dynamics of von Willebrand factor (VWF) that are vital to its biological function have not been fully characterized. ObjectiveTo develop a new fluorescent protein‐‐VWF chimera (FP‐VWF) that has similar hematologic function to wild‐type VWF and use it to monitor the tissue dynamics of VWF distribution. MethodsGenotyping, platelet counting, tail bleeding time assay, agarose gels, western blot, platelet aggregation, proteolytic analysis, and ELISA were applied in characterizing the function of FP‐VWF; fluorescence spectrometer and confocal fluorescence microscope were used to monitor the plasma and tissue distribution of FP‐VWF. ResultsThe transgenic mice that carry the FP‐VWF retain hematologic activity of VWF with plasma levels of FP‐VWF reduced by 50% and there are reduced high molecular weight FP‐VWF multimers compared to the wild‐type mice. The GPIb‐binding and ADAMTS‐13 (A Disintegrin and Metalloprotease with ThrombSpondin type 1 motif, member 13) proteolytic efficiency of FP‐VWF are similar to wild‐type VWF. The tissue distribution of FP‐VWF was probed directly through its intrinsic fluorescence at normal or stimulated status, which indicated that the medicine‐stimulated endogenous FP‐VWF seems primarily released from the aorta and cleared in the spleen. Similar results were observed in non‐fluorescent mice through a standard immunofluorescence approach. The fluorescence signals of FP‐VWF were also similar to the standard dye‐based approach in detecting the FeCl3‐induced blood clotting in vivo. ConclusionsTogether, these results suggest that this novel FP‐VWF chimera is valuable in probing the tissue dynamics of VWF in quite a few biological and pharmaceutical applications.