In vivo and in vitro processing of recombinant pro-von Willebrand factor.
Von Willebrand factor (vWF) is synthesized in endothelial cells as pre-pro-vWF and processed intracellularly to propeptide (vWFpp) and mature vWF. Recombinant pro-vWF when infused into animals can also be processed extracellularly in vivo. Within 1 h of infusion in a dog and mice the multimer pattern changed to that typically seen in mature vWF indicating that propeptide cleavage from unprocessed vWF occurs extracellularly in the circulation. Incubation of a recombinant pro-vWF preparation with canine and human vWF-deficient plasma induced a time-dependent decrease in pro-vWF antigen and an increase in vWFpp antigen without changing total vWF antigen or collagen-binding activity. Multimer analysis showed the gradual transformation of the pro-vWF multimers to mature vWF multimers and cleaved vWFpp was visualized on autoradiograms of SDS-polyacrylamide electrophoresis gels using (125)I-labeled pro-vWF. When recombinant pro-vWF was incubated with increasing amounts of purified thrombin, the extent of pro-vWF processing was dose dependent. The specific cleavage of vWFpp was confirmed by immunoblots using an anti-vWFpp antibody and by amino-terminal amino acid analysis. Hirudin preconditioning of vWF-deficient mice attenuated processing of infused recombinant pro-vWF suggesting that thrombin plays a part in the processing events in vivo.
Turecek
Peter L
PL
Baxter BioScience, Industriestrasse 67, 1220 Vienna, Austria.
Váradi
Katalin
K
Schlokat
Uwe
U
Pichler
Ludwig
L
Dorner
Friedrich
F
Schwarz
Hans Peter
HP
D000818:Animals; D006801:Humans; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000378:metabolism; D013917:Thrombin / Q000378:metabolism; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism*
D011498:Protein Precursors; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D013917:Thrombin