A von Willebrand factor fragment containing the D'D3 domains is sufficient to stabilize coagulation factor VIII in mice.
Plasma factor VIII (FVIII) and von Willebrand factor (VWF) circulate together as a complex. We identify VWF fragments sufficient for FVIII stabilization in vivo and show that hepatic expression of the VWF D'D3 domains (S764-P1247), either as a monomer or a dimer, is sufficient to raise FVIII levels in Vwf(-/-) mice from a baseline of ∼5% to 10%, to ∼50% to 100%. These results demonstrate that a fragment containing only ∼20% of the VWF sequence is sufficient to support FVIII stability in vivo. Expression of the VWF D'D3 fragment fused at its C terminus to the Fc segment of immunoglobulin G1 results in markedly enhanced survival in the circulation (t1/2 > 7 days), concomitant with elevated plasma FVIII levels (>25% at 7 days) in Vwf(-/-) mice. Although the VWF D'D3-Fc chimera also exhibits markedly prolonged survival when transfused into FVIII-deficient mice, the cotransfused FVIII is rapidly cleared. Kinetic binding studies show that VWF propeptide processing of VWF D'D3 fragments is required for optimal FVIII affinity. The reduced affinity of VWF D'D3 and VWF D'D3-Fc for FVIII suggests that the shortened FVIII survival in FVIII-deficient mice transfused with FVIII and VWF D'D3/D'D3-Fc is due to ineffective competition of these fragments with endogenous VWF for FVIII binding.
Life Sciences Institute, University of Michigan.
Andrew
A
Yee
Life Sciences Institute, University of Michigan.
Robert D
RD
Gildersleeve
Department of Pediatrics.
Shufang
S
Gu
Life Sciences Institute, University of Michigan.
Colin A
CA
Kretz
Life Sciences Institute, University of Michigan.
Beth M
BM
McGee
Life Sciences Institute, University of Michigan.
Keisha M
KM
Carr
Department of Pediatrics, Department of Pathology.
Steven W
SW
Pipe
Life Sciences Institute, University of Michigan, Department of Pediatrics, Howard Hughes Medical Institute, Department of Internal Medicine, and Department of Human Genetics, University of Michigan Medical School, Ann Arbor, MI.
David
D
Ginsburg
D000818:Animals; D005169:Factor VIII / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D057507:Mice, 129 Strain; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014841:von Willebrand Factor / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G; D046912:Multiprotein Complexes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014841:von Willebrand Factor; D005169:Factor VIII