The binding sites for the very low density lipoprotein receptor and low-density lipoprotein receptor-related protein are shared within coagulation factor VIII.

Coagulation factor VIII (FVIII) is a ligand for two members of the low-density lipoprotein receptor family, low-density lipoprotein receptor-related protein (LRP) and low-density lipoprotein receptor, which cooperate in regulating clearance of FVIII from the circulation. This study was aimed to explore the mechanism of interaction of FVIII with very low density lipoprotein receptor (VLDLR), another member of the family, and map receptor-binding sites. Binding of plasma-derived FVIII and its fragments to recombinant soluble ectodomain of VLDLR (sVLDLR) was studied in solid-phase and surface plasmon resonance assays. Full-length FVIII and its light chain bound to sVLDLR with similar affinities (KD = 114 +/- 14 and 95 +/- 11 nmol/l, respectively); in contrast, exposure of high-affinity VLDLR-binding site within the heavy chain (KD = 30 +/- 2 nmol/l) required proteolytic cleavage by thrombin. The VLDLR-binding sites within heavy and light chains were mapped to the A2 domain residues 484-509 and the A3-C1 fragment, based on the inhibitory effects of anti-A2 monoclonal antibody 413 and anti-A3-C1 antibody fragment scFv KM33, respectively, previously shown to inhibit FVIII/LRP interaction. Soluble ligand-binding fragment of VLDLR inhibited activation of factor X by the intrinsic Xase in purified system. In cell culture, a higher Xase activity was associated with wild-type human embryonic kidney cells compared with transfected cells that express VLDLR on the cell surface. We conclude that the binding sites for VLDLR and LRP within FVIII overlap and the A2 site becomes exposed upon physiological activation of FVIII. A functional role of FVIII/VLDLR interaction may be related to regulation of intrinsic Xase activity.

Localization of the low-density lipoprotein receptor-related protein regions involved in binding to the A2 domain of coagulation factor VIII.

Catabolism of coagulation factor VIII (FVIII) is mediated by low-density lipoprotein receptor-related protein (LRP). The ligand-binding sites of LRP are formed by complement-type repeats (CR), and CR clusters II and IV bind most of LRP ligands. FVIII contains two major LRP-binding sites located in the A2 and A3 domains. This study was aimed to identify specific complement-type repeats of LRP involved in interaction with the A2 site and to probe their functional importance in A2 catabolism. We generated individual LRP clusters II, III and IV, along with nine overlapping CR triplets encompassing clusters II and IV in a baculovirus expression system and studied their interaction with isolated A2. In surface plasmon resonance (SPR) assay, A2 bound to clusters II and IV with KDs 22 and 39 nM, respectively, and to the majority of CR triplets with affinities in the range of KDs 25-90 nM. Similar affinities were determined for A2 interaction with a panel of CR doublets overlapping cluster II (CR 3-4, 4-5, 5-6, 6-7 and 7-8). These LRP fragments inhibited the binding of 125I-A2 to LRP in solid-phase assay, LRP-mediated internalization of 125I-A2 in cell culture and 125I-A2 clearance from the mouse circulation. Point mutations of critical A2 residues of the LRPbinding site resulted in differential reduction or abolishment of its binding to LRP fragments. We conclude that A2 interacts with LRP via multiple binding sites spanning CR 3-8 in cluster II and CR 23-29 in cluster IV, and the minimal A2-binding unit of LRP is formed by two adjacent CR.

Development of a peptidomimetic ligand for efficient isolation and purification of factor VIII via affinity chromatography.

Hemophilia A, one of the most severe bleeding disorders, results from an inherited deficiency of factor VIII (FVIII) function. Treatment by injection of FVIII has been a common procedure for decades. Nevertheless, the production and purification of FVIII remains a challenging task. Current procedures using immunoaffinity chromatography are expensive and suffer from the instability of the applied antibody ligands, which elute along with the product and contaminate it. Recently, FVIII was purified by use of octapeptide ligands, but their low protease-resistance limits their application. We here report the systematic rational and combinatorial optimization procedure that allowed us to transfer the octapeptide ligands into a small peptidomimetic. This compound is the smallest ligand known for separation of such a large protein (330 kDa). It not only binds and purifies FVIII with high efficiency but also is stable, protease-resistant, and cheap to produce in preparative scale. Hence it offers a valuable alternative to antibody-based purification procedures.

Role of the B domain in proteolytic inactivation of activated coagulation factor VIII by activated protein C and activated factor X.

Hereditary deficiency of factor VIII (FVIII), haemophilia A, is treated by plasma-derived FVIII (pd-FVIII) or recombinant FVIII (rFVIII) infusions. B-domain-deleted FVIII (BDD-rFVIII), although generally safe and effective, was less effective than pd-FVIII in prophylaxis -- evidenced by a 2.5-fold higher bleeding incidence. Assessment of BDD-rFVIII activity in chromogenic and one-stage clotting assays gives up to 50% difference in activity values. As earlier studies demonstrated identical activation and cofactor activity of BDD-rFVIII and pd-FVIII, we decided to study susceptibility of thrombin-activated pd-FVIII, full-length rFVIII and BDD-rFVIII to proteolytic inactivation by activated protein C (APC) and activated factor X (FXa) in a purified system. Proteolysis was monitored by Western blot using monoclonal antibodies C5 and R8B12 specific for the A1 and A2 domains, respectively. Inactivation was monitored by measuring the residual cofactor activity of FVIII forms in a one-stage clotting assay. Proteolysis of A1 and A2 domains of activated BDD-rFVIII proceeded 11 or 13 times faster than that of pd-FVIII or full-length rFVIII. Inactivation of activated BDD-rFVIII was two to three times faster by APC and five to six times faster by FXa. We suggest that differences in proteolytic inactivation may contribute to differences between BDD-rFVIII and pd-FVIII in assaying and in clinical use.

Identification of coagulation factor VIII A2 domain residues forming the binding epitope for low-density lipoprotein receptor-related protein.

Regulation of the coagulation factor VIII (fVIII) level in circulation involves a hepatic receptor low-density lipoprotein receptor-related protein (LRP). One of two major LRP binding sites in fVIII is located within the A2 domain (A2), likely exposed within the fVIII complex with von Willebrand factor and contributing to regulation of fVIII via LRP. This work aimed to identify A2 residues forming its LRP-binding site, previously shown to involve residues 484-509. Isolated A2 was subjected to alanine-scanning mutagenesis followed by expression of a set of mutants in a baculovirus system. In competition and surface plasmon resonance assays, affinities of A2 mutants K466A, R471A, R484A, S488A, R489A, R490A, H497A, and K499A for LRP were found to be decreased by 2-4-fold. This correlated with 1.3-1.5-fold decreases in the degree of LRP-mediated internalization of the mutants in cell culture. Combining these mutations into pairs led to cumulative effects, i.e., 7-13-fold decrease in affinity for LRP and 1.6-2.2-fold decrease in the degree of LRP-mediated internalization in cell culture. We conclude that the residues mentioned above play a key role in formation of the A2 binding epitope for LRP. Experiments in mice revealed an approximately 4.5 times shorter half-life for A2 in the circulation in comparison with that of fVIII. The half-lives of A2 mutant R471A/R484A or A2 co-injected with receptor-associated protein, a classical ligand of LRP, were prolonged by approximately 1.9 and approximately 3.5 times, respectively, compared to that of A2. This further confirms the importance of the mutated residues for interaction of A2 with LRP and suggests the existence of an LRP-dependent mechanism for removing A2 as a product of dissociation of activated fVIII from the circulation.

Inhibitors in hemophilia A: mechanisms of inhibition, management and perspectives.

Factor VIII (FVIII) replacement therapy remains the mainstay in hemophilia A care. The major complication of replacement therapy is formation of antibodies, which inhibit FVIII activity, thus dramatically reducing treatment efficiency. The present review summarizes the accumulated knowledge on epitopes of FVIII inhibitors and mechanisms of their inhibitory effects. FVIII inhibitors most frequently target the A2, C2 and A3 domains of FVIII and interfere with important interactions of FVIII at various stages of its functional pathway; a class of FVIII inhibitors inactivates FVIII by proteolysis. We discuss therapeutic approaches currently used for treatment of hemophilia A patients with inhibitors and analyze the factors that influence the outcome. The choice between options should depend on the level of inhibitors and consideration of efficacy, safety, and availability of particular regimens. Advances of basic science open avenues for alternative targeted, specific and long-lasting treatments, such as the use of peptide decoys for blocking FVIII inhibitors, bypassing them with human/porcine FVIII hybrids, neutralizing FVIII-reactive CD4 T cells with anti-clonotypic antibodies, or inducing immune tolerance to FVIII with the use of universal CD4 epitopes or by genetic approaches.

Epitope mapping of polyclonal clotting factor VIII-inhibitory antibodies using phage display.

Clotting factor VIII (fVIII)-inhibitory antibodies represent a major problem in the treatment of haemophilia A. To understand the inactivation mechanisms and to pave the way towards modifications of recombinant clotting factors that reduce their immunogenicity, the exact localization of immunodominant epitopes is required. Here, a random peptide phage display library was employed to identify epitopes of polyclonal fVIII antibodies isolated from patient's plasma by affinity chromatography. FVIII-binding specificity and inhibitory activity of the isolated fVIII antibodies were confirmed by ELISA and Bethesda assays. Phage selection on the individual samples yielded several phages which were displaced from binding to the respective antibody preparation by fVIII. Their homology with amino acid motifs of human fVIII and immunoprecipitation results with radioactively labelled fVIII fragments suggested putative epitopes in the A1, A2 and C1 domains of fVIII for one and in the C2 domain for another patient. Synthetic peptides corresponding to the A2, C1 and C2 domain epitopes blocked antibody binding to fVIII and partially neutralized the inhibitory activity of the respective plasma in Bethesda assays. These results provide the proof of principle that random peptide libraries can be used for the mapping of epitopes in a polyclonal antibody preparation.

Coagulation pathways in atherothrombosis.

Atherosclerosis is a disease recognized as the main cause of death in industrial countries. The current paradigm establishes thrombosis to be the major reason for complications of atherosclerosis, such as myocardial infarction and stroke, and the major factor responsible for atherosclerosis-related mortality. Development of adequate treatment of patients with risk of atherothrombosis requires the comprehensive understanding of mechanisms underlying coagulation processes at the site of atherosclerotic lesion. The present review discusses contribution of the extrinsic and intrinsic pathways of blood coagulation in thrombogenicity of atherosclerotic plaque and factors determining the overall procoagulant/anticoagulant balance.