von Willebrand factor mediates protection of factor VIII from activated protein C-catalyzed inactivation.
Factor VIII, a cofactor of the intrinsic clotting pathway, is proteolytically inactivated by the vitamin K-dependent serine protease, activated protein C in a reaction requiring Ca2+ and a phospholipid surface. Factor VIII was inactivated 15 times faster than factor VIII in complex with either von Willebrand factor (vWf) or the large homodimeric fragment, SPIII (vWf residues 1-1365). Free factor VIII or factor VIII in complex with a smaller fragment, SPIII-T4 (vWf residues 1-272), were inactivated at the same rate, suggesting that this effect was dependent upon the size of factor VIII-vWf complex rather than changes in factor VIII brought about by occupancy of the vWf-binding site. Thrombin cleavage of the factor VIII light chain to remove the vWf-binding site eliminated the protective effects of vWf. In the absence of phospholipid, high levels of the protease inactivated both free and vWf-bound factor VIII at equivalent rates. Using the same conditions, isolated heavy chains and the heavy chains of factor VIII were proteolyzed at similar rates. Taken together, these results suggested that, in the absence of phospholipid, inactivation of factor VIII is independent of factor VIII light chain and further suggest that vWf did not mask susceptible cleavage sites in the cofactor. Solution studies employing fluorescence energy transfer using coumarin-labeled factor VIII (fluorescence donor) and synthetic phospholipid vesicles labeled with octadecyl rhodamine (fluorescence acceptor) indicated saturable binding and equivalent extents of donor fluorescence quenching for factor VIII alone or when complexed with SPIII-T4. However, complexing of factor VIII with either vWf or SPIII eliminated its binding to the phospholipid. Since a phospholipid surface is required for efficient catalysis by the protease, these results suggest that vWf protects factor VIII by inhibiting cofactor-phospholipid interactions.
Fay
P J
PJ
Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Coumans
J V
JV
Walker
F J
FJ
D004591:Electrophoresis, Polyacrylamide Gel; D005169:Factor VIII / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010446:Peptide Fragments / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010980:Platelet Membrane Glycoproteins; D011486:Protein C / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism*
D010446:Peptide Fragments; D010743:Phospholipids; D010980:Platelet Membrane Glycoproteins; D011486:Protein C; D011956:Receptors, Cell Surface; D014841:von Willebrand Factor; C028254:von Willebrand factor receptor; D005169:Factor VIII