In the present study, we found that catabolism of coagulation factor VIII (fVIII) is mediated by the low density lipoprotein receptor-related protein (LPR), a liver multiligand endocytic receptor. In a solid phase assay, fVIII was shown to bind to LRP (K d 116 nM). The specificity was confirmed by a complete inhibition of fVIII/ LRP binding by 39-kDa receptor-associated protein (RAP), an antagonist of all LRP ligands. The region of fVIII involved in its binding to LRP was localized within the A2 domain residues 484 -509, based on the ability of the isolated A2 domain and the synthetic A2 domain peptide 484 -509 to prevent fVIII interaction with LRP. Since vWf did not inhibit fVIII binding to LRP, we proposed that LRP receptor may internalize fVIII from its complex with vWf. Consistent with this hypothesis, mouse embryonic fibroblasts that express LRP, but not fibroblasts genetically deficient in LRP, were able to catabolize 125 I-fVIII complexed with vWf, which was not internalized by the cells. These processes could be inhibited by RAP and A2 subunit of fVIII, indicating that cellular internalization and degradation were mediated by interaction of the A2 domain of fVIII with LRP. In vivo studies of 125 I-fVIII⅐vWf complex clearance in mice demonstrated that RAP completely inhibited the fast phase of the biphasic 125 I-fVIII clearance that is responsible for removal of 60% of fVIII from circulation. Inhibition of the RAP-sensitive phase prolonged the half-life of 125 I-fVIII in circulation by 3.3-fold, indicating that LRP receptor plays an important role in fVIII clearance.The plasma glycoprotein factor VIII (fVIII) 1 functions as a cofactor for factor IXa in the factor X activation enzyme complex in the intrinsic pathway of blood coagulation, and its level is decreased or the protein is nonfunctional in patients with hemophilia A. The fVIII protein consists of a homologous A and C domains and a unique B domain which are arranged in the order A1-A2-B-A3-C1-C2 (1). It is processed to a series of Me 2ϩ -linked heterodimers produced by cleavage at the B-A3 junction (2), generating a light chain (LCh) which consists of an acidic region and A3, C1, and C2 domains and a heavy chain (HCh) which consists of the A1, A2, and B domains (Fig. 1).Transplantational studies both in animals and humans demonstrated that liver hepatocytes are the major fVIII-producing cells (3, 4). Immediately after release into circulation, fVIII binds with a high affinity (K d Ͻ 0.5 nM (5, 6)) to its carrier protein vWf to form a tight, noncovalent complex. The binding to vWf is required for maintenance of a normal fVIII level in circulation, since vWf stabilizes association of the LCh and HCh (7). This prevents fVIII from binding to phospholipid membranes (8), activation by factor Xa (9), and protein Ccatalyzed inactivation (10). vWf comprises a series of high molecular mass, disulfide-bonded multimers with molecular mass values as high as 2 ϫ 10 7 Da (11) and circulates in plasma at 10 g/ml or 50 nM assuming a molecular mass of 270 kDa for vW...

PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex.