The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII.
von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.
Genetics Institute, Cambridge, Massachusetts 02140.
R J
RJ
Wise
A J
AJ
Dorner
M
M
Krane
D D
DD
Pittman
R J
RJ
Kaufman
D000818:Animals; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D004220:Disulfides / Q000032:analysis; D004795:Enzyme Stability; D005169:Factor VIII / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010974:Platelet Aggregation / Q000187:drug effects; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism / Q000494:pharmacology; D012310:Ristocetin / Q000494:pharmacology; D014162:Transfection; D014841:von Willebrand Factor / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology
D004220:Disulfides; D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; D014841:von Willebrand Factor; D012310:Ristocetin; D005169:Factor VIII