Proteolytic cleavage of factor VIII heavy chain is required to expose the binding-site for low-density lipoprotein receptor-related protein within the A2 domain.
BACKGROUND
Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that contributes to the clearance of coagulation factor (F) VIII from the circulation. Previously, we have demonstrated that region Glu(1811)-Lys(1818) within FVIII light chain constitutes an important binding region for this receptor. We have further found that FVIII light chain and intact FVIII are indistinguishable in their LRP-binding affinities. In apparent contrast to these observations, a second LRP-binding region has been identified within A2 domain region Arg(484)-Phe(509) of FVIII heavy chain.


OBJECTIVE
In this study, we addressed the relative contribution of FVIII heavy chain in binding LRP.


RESULTS
Surface plasmon resonance analysis unexpectedly showed that FVIII heavy chain poorly associated to the receptor. The binding to LRP was, however, markedly enhanced upon cleavage of the heavy chain by thrombin. The A2 domain, purified from thrombin-activated FVIII, also showed efficient binding to LRP. Competition studies employing a recombinant antibody fragment demonstrated that region Arg(484)-Phe(509) mediates the enhanced LRP binding after thrombin cleavage.


CONCLUSIONS
We propose that LRP binding of non-activated FVIII is mediated via the FVIII light chain while in activated FVIII both the heavy and light chain contribute to LRP binding.
Bovenschen
N
N
Department of Plasma Proteins, Sanquin Research, Plesmanlaan 125, 1066 CX Amsterdam, The Netherlands.
van Stempvoort
G
G
Voorberg
J
J
Mertens
K
K
Meijer
A B
AB
D001665:Binding Sites; D005169:Factor VIII / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry / Q000378:metabolism*; D010447:Peptide Hydrolases / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000378:metabolism; D020349:Surface Plasmon Resonance
D026503:Low Density Lipoprotein Receptor-Related Protein-1; D021122:Protein Subunits; D005169:Factor VIII; D010447:Peptide Hydrolases