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

Abstract

Background: Tissue dynamics of von Willebrand factor (VWF) that are vital to its biological function have not been fully characterized. Objective: To 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. Methods: Genotyping, 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. Results: The 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. Conclusions: Together, 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.