The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein

In the present study, the interaction between the endocytic receptor low de nsity lipoprotein receptor-related protein (LRP) and coagulation factor VII I (FVIII) was investigated. Using purified components, FVIII was found to b ind to LRP in a reversible and dose-dependent manner (K-d approximate to 60 nM). The interaction appeared to be specific because the LRP antagonist re ceptor-associated protein readily inhibited binding of FVIII to LRP (IC50 a pproximate to 1 nM). In addition, a 12-fold molar excess of the physiologic al carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of I-125-labeled FVIII by LRP-expressing cells (approximate to 8 fmol/10(5) cells after a 4.5-h incu bation) was reduced by approximately 70% in the presence of receptor-associ ated protein. LRP-directed antibodies inhibited degradation to a similar ex tent, indicating that LRP indeed contributes to binding and transport of FV III to the intracellular degradation pathway. Degradation of FVIII was comp letely inhibited by vWF, Because vWF binding by FVIII involves its light ch ain, LRP binding to this subunit was studied. In ligand blotting experiment s, binding of FVIII light chain to LRP could be visualized, More detailed a nalysis revealed that FVIII light chain interacts with LRP with moderate af finity (k(on) approximate to 5 x 10(4) M-1 s(-1); k(off) approximate to 2.5 x 10(-3) s(-1); K-d approximate to 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the inte raction with LRP. In contrast, no association of FVIII heavy chain to LRP c ould be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surfac e and to mediate its transport 60 the intracellular degradation pathway. FV III-LRP interaction involves the FVIII light chain, and FVIII-vWF complex f ormation plays a regulatory role in LRP binding. Our findings may explain t he beneficial effect of vWF on the in vivo survival of FVIII.