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...

A binding site for von Willebrand factor (vWf) was previously localized to the carboxyl terminus of the C2 domain of the light chain (LCh) of factor VIII (fVIII). The acidic region of the LCh, residues 1649 -1689, also controls fVIII⅐vWf binding by an unknown mechanism. Although anti-acidic region monoclonal antibodies prevent formation of the fVIII⅐vWf complex, the direct involvement of the acidic region in this binding has not been demonstrated. By limited proteolysis of LCh with Staphylococcus aureus V8 protease, we prepared 14-and 63-kDa LCh fragments, which begin with fVIII residues 1672 and 1795, respectively. Using surface plasmon resonance to measure binding interactions, we demonstrated that the 14-kDa fragment binds to vWf, but its affinity for vWf (K d 72 nM) was 19-fold lower than that of LCh. This was not due to an altered conformation of the acidic region within the 14-kDa fragment, since its affinity for an anti-acidic region monoclonal antibody was similar to that of LCh. All LCh derivatives lacking the acidic region (thrombin-cleaved LCh, recombinant C2, and 63-kDa fragment) had also greatly reduced affinities for vWf (K d 564 -660 nM) compared with LCh (K d 3.8 nM). In addition, the similar affinities of these derivatives for vWf indicated that apart from its acidic region, the LCh contains no vWf binding site other than the one within C2. The reduced affinities of the LCh derivatives lacking the acidic region for monoclonal antibody NMC-VIII/5 (epitope, C2 residues 2170 -2327) indicated that removal of the acidic region leads to a conformational change within C2. This change is likely to affect the conformation of the vWf binding site in C2, which overlaps the epitope of NMC-VIII/5; therefore, the acidic region also appears to be required to maintain the optimal conformation of this vWf binding site. Our results demonstrate that the acidic region and the C2 domain are both directly involved in forming a high affinity binding site for vWf.

The A2 domain (residues 373-740) of human blood coagulation factor VIII (fVIII) contains a major epitope for inhibitory alloantibodies and autoantibodies. We took advantage of the differential reactivity of inhibitory antibodies with human and porcine fVIII and mapped a major determinant of the A2 epitope by using a series of active recombinant hybrid human/porcine fVIII molecules. Hybrids containing a substitution of porcine sequence at segment 410-508, 445-508, or 484-508 of the human A2 domain were not inhibited by a murine monoclonal antibody A2 inhibitory, mAb 413, whereas hybrids containing substitutions at 387-403, 387-444, and 387-468 were inhibited by mAb 413. This indicates that the segment bounded by Arg484 and Ile508 contains a major determinant of the A2 epitope. mAb 413 did not inhibit two more hybrids that contained porcine substitutions at residues 484-488 and 489-508, indicating that amino acid side chains on both sides of the Ser488-Arg489 bond within the Arg484-Ile508 segment contribute to the A2 epitope. The 484-508, 484-488, and 489-508 porcine substitution hybrids displayed decreased inhibition by A2 inhibitors from four patient plasmas, suggesting that there is little variation in the structure of the A2 epitope in the inhibitor population.

Apolipoprotein E (apoE) associates with lipoproteins and mediates their interaction with members of the LDL receptor family. ApoE exists as three common isoforms that have important distinct functional and biological properties. Two apoE isoforms, apoE3 and apoE4, are recognized by the LDL receptor, whereas apoE2 binds poorly to this receptor and is associated with type III hyperlipidemia. In addition, the apoE4 isoform is associated with the common late-onset familial and sporadic forms of Alzheimer's disease. Although the interaction of apoE with the LDL receptor is well characterized, the specificity of other members of this receptor family for apoE is poorly understood. In the current investigation, we have characterized the binding of apoE to the VLDL receptor and the LDL receptorrelated protein (LRP). Our results indicate that like the LDL receptor, LRP prefers lipid-bound forms of apoE, but in contrast to the LDL receptor, both LRP and the VLDL receptor recognize all apoE isoforms. Interestingly, the VLDL receptor does not require the association of apoE with lipid for optimal recognition and avidly binds lipid-free apoE. It is likely that this receptor-dependent specificity for various apoE isoforms and for lipid-free versus lipid-bound forms of apoE is physiologically significant and is connected to distinct functions for these receptors. Apolipoprotein E (apoE) is a 34 kDa protein that plays an important role in lipoprotein metabolism by association with lipoprotein particles and with members of the LDL receptor family (1, 2). ApoE contains a 22 kDa N-terminal domain (residues 1-191) that is recognized by receptors and a 10 kDa C-terminal domain (residues 222-299) that has high affinity for lipid and is responsible for the association of apoE with lipoproteins (3, 4). Three major isoforms of apoE exist in the population and differ by cysteine and arginine at residues 112 and 158. The most common isoform, apoE3, contains cysteine and arginine at these positions, respectively, whereas apoE2 contains cysteine at both positions and apoE4 contains arginine at both positions (5). These substitutions have important biological consequences. First, the various apoE isoforms are differentially recognized by the LDL receptor. Thus, apoE3 and apoE4 readily bind to the LDL receptor, whereas apoE2 binds poorly to the LDL receptor and is associated with type III hyperlipidemia (6). Second, the APOE-4 allele is associated with the common late-onset familial and sporadic forms of Alzheimer's disease (AD) (7,8). The biochemical mechanism by which the APOE-4 allele increases the risk of AD is unknown, but several possibilities have been proposed (9-11), including differential functions of apoE isoforms upon interaction with members of the LDL receptor family (9).The LDL receptor family includes the LDL receptor, the LDL receptor-related protein (LRP), LRP1b, megalin (or LRP-2), the VLDL receptor, and apoE receptor 2 (for Abbreviations: AD, Alzheimer's disease; apoE, apolipoprotein E; LRP, low density lipoprotein...

Blood coagulation in vivo is a spatially nonuniform, multistage process: coagulation factors from plasma bind to tissue factor (TF)-expressing cells, become activated, dissociate, and diffuse into plasma to form enzymatic complexes on the membranes of activated platelets. We studied spatial regulation of coagulation using two approaches: 1), an in vitro experimental model of clot formation in a thin layer of plasma activated by a monolayer of TF-expressing cells; and 2), a computer simulation model. Clotting in factor VIII- and factor XI-deficient plasmas was initiated normally, but further clot elongation was impaired in factor VIII- and, at later stages, in factor XI-deficient plasma. The data indicated that clot elongation was regulated by factor Xa formation by intrinsic tenase, whereas factor IXa was formed by extrinsic tenase on activating cells and diffused into plasma, thus sustaining clot growth. Far from the activating cells, additional factor IXa was produced by factor XIa. Exogenously added TF had no effect on the clot growth rate, suggesting that plasma TF does not contribute significantly to the clot propagation process in a reaction-diffusion system without flow. Addition of thrombomodulin at 3-100 nM caused dose-dependent termination of clot elongation with a final clot size of 2-0.2 mm. These results identify roles of specific coagulation pathways at different stages of spatial clot formation (initiation, elongation, and termination) and provide a possible basis for their therapeutic targeting.