LDL receptor-related protein 1 contributes to the clearance of the activated factor VII-antithrombin complex.

Essentials Factor VIIa is cleared principally as a complex with antithrombin. Enzyme/serpin complexes are preferred ligands for the scavenger-receptor LRP1. Factor VIIa/antithrombin but not factor VIIa alone is a ligand for LRP1. Macrophage-expressed LRP1 contributes to the clearance of factor VIIa/antithrombin. SUMMARY: Background Recent findings point to activated factor VII (FVIIa) being cleared predominantly (± 65% of the injected protein) as part of a complex with the serpin antithrombin. FVIIa-antithrombin complexes are targeted to hepatocytes and liver macrophages. Both cells lines abundantly express LDL receptor-related protein 1 (LRP1), a scavenger receptor mediating the clearance of protease-serpin complexes. Objectives To investigate whether FVIIa-antithrombin is a ligand for LRP1. Methods Binding of FVIIa and pre-formed FVIIa-antithrombin to purified LRP1 Fc-tagged cluster IV (rLRP1-cIV/Fc) and to human and murine macrophages was analyzed. FVIIa clearance was determined in macrophage LRP1 (macLRP1)-deficient mice. Results Solid-phase binding assays showed that FVIIa-antithrombin bound in a specific, dose-dependent and saturable manner to rLRP1-cIV/Fc. Competition experiments with human THP1 macrophages indicated that binding of FVIIa but not of FVIIa-antithrombin was reduced in the presence of annexin-V or anti-tissue factor antibodies, whereas binding of FVIIa-antithrombin but not FVIIa was inhibited by the LRP1-antagonist GST-RAP. Additional experiments revealed binding of both FVIIa and FVIIa-antithrombin to murine control macrophages. In contrast, no binding of FVIIa-antithrombin to macrophages derived from macLRP1-deficient mice could be detected. Clearance of FVIIa-antithrombin but not of active site-blocked FVIIa was delayed 1.5-fold (mean residence time of 3.3 ± 0.1 h versus 2.4 ± 0.2 h) in macLRP1-deficient mice. The circulatory presence of FVIIa was prolonged to a similar extent in macLRP1-deficient mice and in control mice. Conclusions Our data show that FVIIa-antithrombin but not FVIIa is a ligand for LRP1, and that LRP1 contributes to the clearance of FVIIa-antithrombin in vivo.

Shear stress-independent binding of von Willebrand factor-type 2B mutants p.R1306Q & p.V1316M to LRP1 explains their increased clearance.

BACKGROUND: von Willebrand factor (VWF) is cleared in a shear stress- and macrophage-dependent manner by LRP1. von Willebrand disease (VWD)-type 2B mutants are endocytosed more efficiently than wild-type (wt)-VWF by macrophages. OBJECTIVE: To investigate if VWD-type 2B mutations in the VWF A1-domain affect LRP1 binding and LRP1-dependent clearance. METHODS: Recombinant Fc-tagged A1 domain (A1-Fc, A2-Fc, A3-Fc) and full-length VWF (wt or mutants thereof) were tested for binding to LRP1 or a recombinant fragment thereof in a static immunosorbent assay. Mutant and wt-VWF were also compared for clearance in mice lacking macrophage LRP1 (macLRP1(-) ) and control mice (macLRP1(+) ). RESULTS: We found that A1-Fc but not A2-Fc or A3-Fc binds dose-dependently to LRP1. Binding of A1-Fc to LRP1 was markedly enhanced by the VWD-type 2B mutation p.V1316M. As expected, full-length wt-VWF was unable to bind LRP1 under static conditions unless ristocetin was added. In contrast, the presence of the p.V1316M or p.R1306Q mutation induced spontaneous binding to LRP1 without the need for ristocetin or shear stress. Both mutants were cleared more rapidly than wt-VWF in control macLRP1(+) mice. Surprisingly, deletion of macrophage LRP1 abrogated the increased clearance of the VWF/p.R1306Q and VWF/p.V1316M mutant. CONCLUSION: The VWF A1-domain contains a binding site for LRP1. Certain VWD-type 2B mutations relieve the need for shear stress to induce LRP1 binding. Enhanced LRP1 binding coincides with a reduced survival of VWF/p.R1306Q and VWF/p.V1316M. Our data provide a rationale for reduced VWF levels in at least some VWD-type 2B patients.

Clearance of von Willebrand factor.

Quantitative deficiencies in von Willebrand factor (VWF) are associated with abnormal hemostasis that can manifest in bleeding or thrombotic complications. Consequently, many studies have endeavored to elucidate the mechanisms underlying the regulation of VWF plasma levels. This review focuses on the role of VWF clearance pathways. A summary of recent developments are provided, including results from genetic studies, the relationship between glycosylation and VWF clearance, the contribution of increased VWF clearance to the pathogenesis of von Willebrand disease and the identification of VWF clearance receptors. These different studies converge in their conclusion that VWF clearance is a complex phenomenon that involves multiple mechanisms. Deciphering how such different mechanisms coordinate their role in this process is but one of the remaining challenges. Nevertheless, a better insight into the complex clearance pathways of VWF may help us to better understand the clinical implications of aberrant clearance in the pathogenesis of von Willebrand disease and perhaps other disorders as well as aid in developing alternative therapeutic approaches.

von Willebrand factor: the old, the new and the unknown.

von Willebrand factor (VWF) is a protein best known from its critical role in hemostasis. Indeed, any dysfunction of VWF is associated with a severe bleeding tendency known as von Willebrand disease (VWD). Since the first description of the disease by Erich von Willebrand in 1926, remarkable progress has been made with regard to our understanding of the pathogenesis of this disease. The cloning of the gene encoding VWF has allowed numerous breakthroughs, and our knowledge of the epidemiology, genetics and molecular basis of VWD has been rapidly expanding since then. These studies have taught us that VWF is rather unique in terms of its multimeric structure and the unusual mechanisms regulating its participation in the hemostatic process. Moreover, it has become increasingly clear that VWF is a more all-round protein than originally thought, given its involvement in several pathologic processes beyond hemostasis. These include angiogenesis, cell proliferation, inflammation, and tumor cell survival. In the present article, an overview of advances concerning the various structural and functional aspects of VWF will be provided.

Immunoprotective effect of von Willebrand factor towards therapeutic factor VIII in experimental haemophilia A.

The development of inhibitory anti-factor VIII (FVIII) antibodies in patients with haemophilia A following replacement therapy is associated with several types of risk factors. Among these, the purity of FVIII concentrates, and in particular the presence of von Willebrand factor (VWF), was controversially proposed to influence the immunogenicity of exogenous FVIII. We re-assessed in vivo and in vitro the immuno-protective effect of VWF towards FVIII. The immuno-protective effect of VWF towards FVIII was investigated in vivo, in a model of haemophilia A. We studied the endocytosis of FVIII by murine bone marrow-derived dendritic cells and evaluated the capacity of VWF to block the internalization of FVIII. We characterized the relevance of VWF for the accumulation of FVIII in the marginal zone of the spleen, a secondary lymphoid organ where the immune response to therapeutically administered FVIII initiates. Our results confirm that VWF reduces the immunogenicity of FVIII in FVIII-deficient mice. Paradoxically, VWF is important for the accumulation of FVIII in the marginal zone of the spleen. We propose that VWF exerts at least two non-mutually exclusive immunoprotective roles towards FVIII in haemophilic mice: VWF prevents the endocytosis of FVIII by professional antigen-presenting cells by blocking the interaction of FVIII with as yet unidentified endocytic receptor(s). Hypothetically, VWF, by virtue of increasing the half-life of FVIII in the circulation, may allow an increased contact time with tolerogenic marginal zone B cells in the spleen.

Factor VIII and von Willebrand factor--too sweet for their own good.

SUMMARY: Although factor VIII (FVIII) and von Willebrand factor (VWF) are products of two distinct genes, they circulate in plasma as a tight non-covalent complex. Moreover, they both play a critical role in the haemostatic process, a fact that is illustrated by the severe bleeding tendency associated with the functional absence of either protein. FVIII is an essential cofactor for coagulation factor IX, while VWF is pertinent to the recruitment of platelets to the injured vessel wall under conditions of rapid flow. FVIII and VWF have in common that they are heavily glycosylated: full-length FVIII contains 20 N-linked and at least seven O-linked glycans, while VWF contains 12 N-linked and 10 O-linked glycans. Three decades of research have revealed that the carbohydrate structures of FVIII and VWF contribute to many of the steps that can be distinguished in the life-cycle of these proteins, including biosynthesis/secretion, function and clearance. In this review, several of these aspects will be discussed. In addition, the interaction of the FVIII/VWF complex with two families of carbohydrate-binding proteins, i.e. Galectins and Siglecs, and their potential physiological relevance will be discussed.

The disappearing act of factor VIII.

Factor VIII (FVIII) is a plasma protein critical to the haemostatic system. This notion is illustrated by the severe bleeding disorder that is associated with its functional absence, known as haemophilia A. In addition, several epidemiological studies have revealed an association between the presence of elevated levels of FVIII and thrombotic complications. In view of its relation to thrombotic and haemorrhagic disorders, it is not surprising that FVIII has gained wide attention from the research community in the previous decades. This research has led to a better understanding of not only the structural, functional and physiological aspects of this intriguing protein, but also of the pathogenesis of haemostatic defects associated with FVIII. In the present review, focus will be on the interaction between FVIII and surface receptors that are able to capture FVIII. These interactions are of importance for FVIII, as they may affect both function and survival of FVIII.

Splenic marginal zone antigen-presenting cells are critical for the primary allo-immune response to therapeutic factor VIII in hemophilia A.

BACKGROUND: Alloimmune responses to intravenously administered protein therapeutics are the most common cause of failure of replacement therapy in patients with defective levels of endogenous proteins. Such a situation is encountered in some patients with hemophilia A, who develop inhibitory anti-factor (F)VIII alloantibodies after administration of FVIII to treat hemorrhages. OBJECTIVES: The nature of the secondary lymphoid organs involved in the initiation of immune responses to human therapeutic has not been studied. We therefore investigated this in the case of FVIII, a self-derived exogenous protein therapeutic. METHODS: The distribution of intravenously administered FVIII was followed after FVIII-deficient mice were injected with radiolabeled FVIII and using immunohistochemistry. The role of the spleen and antigen-presenting cells (APC) in the onset of the anti-FVIII immune response was analyzed upon splenectomy or treatment of the mice with APC-depleting compounds. RESULTS: FVIII preferentially accumulated in the spleen at the level of metallophilic macrophages in the marginal zone (MZ). Surgical removal of the spleen or selective in vivo depletion of macrophages and CD11c-positive CD8 alpha-negative dendritic cells resulted in a drastic reduction in anti-FVIII immune responses. CONCLUSIONS: Using FVIII-deficient mice as a model for patients with hemophilia A, and human pro-coagulant FVIII as a model for immunogenic self-derived protein therapeutics, our results highlight the importance of the spleen and MZ APCs in the initiation of immune responses to protein therapeutics. Identification of the receptors implicated in retention of protein therapeutics in the MZ may pave the way towards novel strategies aimed at reducing their immunogenicity.

Mouse models of von Willebrand disease.

von Willebrand disease (VWD), caused by quantitative or qualitative abnormalities in von Willebrand factor (VWF) is considered the most common inherited bleeding disorder in humans. Mild and severe quantitative defects in VWF cause VWD types 1 and 3 respectively, whereas qualitative abnormalities induce VWD type 2. VWD has also been diagnosed in a number of animal species such as dogs, pigs, cats and horses, as a result of naturally occurring mutations. More recently, murine models have drawn a great deal of attention. Their small size along with their well-defined genetic background makes them ideal tools to study the in vivo function of VWF. The most commonly used model is the VWF-deficient mouse engineered through homologous recombination. However, models resulting from changes in modifier genes indirectly affecting VWF have also been described. These various models have proven very useful in elucidating some aspects of VWF biology not easily addressed through in vitro approaches.

Molecular characterization of Iranian patients with type 3 von Willebrand disease.

von Willebrand's disease (VWD) type 3 is a rare but severe autosomal-recessive inherited bleeding disorder with a prevalence higher in certain locations where consanguineous marriages are relatively frequent. The genetic defects causing recessive type 3 VWD in 10 unrelated families from Iran have been investigated and the genetic heterogeneity among these patients was evaluated. All exons and their flanking regions of von Willebrand factor gene were amplified by PCR and sequenced using specific primers. Eight patients were fully characterized at the molecular level. Six different gene alterations were identified. All the mutations caused null alleles, three being nonsense mutations (Q104X, Q793X and E1981X), two possible splice site mutations (2443-1G>C and 1110-1G>A) and one small deletion (3237delA). Three of them have not been described previously. Most patients were born from consanguineous marriages and all were homozygous for their mutations. The results confirm that molecular defects in type 3 VWD are heterogeneous with mutations arising randomly within the entire gene.

Characterization of the interaction between von Willebrand factor and osteoprotegerin.

BACKGROUND AND OBJECTIVE: Osteoprotegerin (OPG), a member of the tumor necrosis-factor receptor superfamily, plays an important role in bone remodeling and is also involved in vascular diseases. OPG is physically associated with von Willebrand factor (VWF), a glycoprotein involved in primary hemostasis, within the Weibel-Palade bodies (WPBs) of endothelial cells and in plasma. The present study aimed to elucidate the molecular mechanisms underlying the interaction between OPG and VWF. METHODS AND RESULTS: In a solid-phase binding assay, VWF was able to bind specifically to OPG in a calcium-dependent manner. This interaction displayed strong pH dependence with optimal binding occurring at pH 6.5 and was severely impaired by chloride-ion concentrations above 40 mm. Using a series of purified VWF derivatives the functional site that supports VWF interaction with OPG was localized on its Al domain. Fluorescence microscopy on human umbilical vein endothelial cells showed co-localization of VWF and OPG in WPBs. When secretion was induced, OPG remained associated with VWF in extracellular patches of release under biochemical conditions found in blood plasma. CONCLUSIONS: Our observations demonstrate the existence of an interactive site for OPG within the VWF A1-domain. This study established that the optimal biochemical parameters allowing a complex formation between VWF and OPG are those thought to prevail in the trans-Golgi network. These conditions would allow VWF to act as a cargo targeting OPG to WPBs. Finally, blood environments appear suitable to preserve the complex, which may participate in vascular injury, arterial calcification and inflammation.

Functional mapping of anti-factor IX inhibitors developed in patients with severe hemophilia B.

Development of inhibitory antibodies is a serious complication of treatment with repeated factor IX infusions in a minority of patients with hemophilia B. Such antibodies detected in 8 patients have been characterized. Typing studies revealed that patients' immune response toward factor IX is highly heterogeneous and involves immunoglobulin G (IgG) antibodies, preferentially IgG1 and IgG4. The preservation of the sequence and the 3-dimensional orientation of the amino acids constituting one epitope are highly important for the assembly of an antibody-antigen complex. To localize the epitopes on the factor IX molecule, an original approach was designed using a set of factor X chimeras carrying regions of factor IX. Results showed that some patients' antibodies were directed against both the domain containing the gamma-carboxy glutamic acid residues (Gla domain) and the protease domain of factor IX. In contrast, no binding was observed to the epidermal growth factor-like domains or to the activation peptide. Functional characterization showed that the purified IgG from patients' serum inhibited the factor VIIIa-dependent activation of factor X. Moreover, patients' IgG directed against the Gla domain inhibited the binding of factor IX to phospholipids as well as the binding of factor VIII light chain to factor IXa. These data demonstrate that inhibitors appearing in patients with severe hemophilia B display specificity against restricted functional domains of factor IX.

Surface loop 199-204 in blood coagulation factor IX is a cofactor-dependent site involved in macromolecular substrate interaction.

In factor IX residues 199-204 encompass one of six surface loops bordering its substrate-binding groove. To investigate the contribution of this loop to human factor IX function, a series of chimeric factor IX variants was constructed, in which residues 199-204 were replaced by the corresponding sequence of factor VII, factor X, or prothrombin. The immunopurified and activated chimeras were indistinguishable from normal factor IXa in hydrolyzing a small synthetic substrate, indicating that this region is not involved in the interaction with substrate residues on the N-terminal side of the scissile bond. In contrast, replacement of loop 199-204 resulted in a 5-25-fold reduction in reactivity toward the macromolecular substrate factor X. This reduction was due to a combination of increased K(m) and reduced k(cat). In the presence of factor VIIIa the impaired reactivity toward factor X was largely restored for all factor IXa variants, resulting in a more pronounced stimulation by factor VIIIa compared with normal factor IXa (3 to 5 x 10(4)-fold versus 5 x 10(3)-fold). Inhibition by antithrombin was only slightly affected for the factor IXa variant with the prothrombin loop sequence, whereas factor IXa variants containing the analogous residues of factor VII or factor X were virtually insensitive to antithrombin inhibition. In the presence of heparin, however, all chimeric factor IXa variants formed complexes with antithrombin. Thus the cofactors heparin and factor VIIIa have in common that they both alleviate the deleterious effects of mutations in the factor IX loop 199-204. Collectively, our data demonstrate that loop 199-204 plays an important role in the interaction of factor IXa with macromolecular substrates.

Blood coagulation factor IX residues Glu78 and Arg94 provide a link between both epidermal growth factor-like domains that is crucial in the interaction with factor VIII light chain.

Recently, we established that mutations at calcium-binding sites within the first epidermal growth factor (EGF)-like domain of activated factor IX affect its interaction with factor VIIIa (Lenting, P. J., Christophe, O. D., ter Maat, H., Rees, D. J. G., and Mertens, K. (1996) J. Biol. Chem. 271, 25332-25337). In the present study, we have investigated the functional role of residue Glu78, which is not involved in calcium binding. Glu78 is also located in the first EGF-like domain and, when mutated to Lys, is associated with severe hemophilia B. Because Glu78 is conserved in related vitamin K-dependent proteins, it is difficult to understand how a mutation at this position is associated with factor IX-specific function. In this study, we addressed the hypothesis that Glu78 exerts its biological activity by interacting with another residue. One candidate was found to be the second EGF-like domain residue, Arg94, which is also associated with severe hemophilia B when mutated. We constructed a series of mutants that included mutations at position 78 alone (Glu78 to Lys/Glu78 to Asp) or at both positions 78 and 94 (Glu78 to Lys and Arg94 to Asp). The functional parameters of immunopurified and activated mutants were compared with normal activated factor IX. Mutants were indistinguishable from normal factor IXa in cleaving the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide or activating factor X in the absence of factor VIIIa. In contrast, in the presence of factor VIIIa, factor IXa Glu78 to Asp and factor IXa Glu78 to Lys/Arg94 to Asp were stimulated to the same extent as normal factor IXa, whereas factor IXa Glu78 to Lys was markedly less stimulated (140-fold versus 2,000-fold). This suggests that residues 78 and 94 should carry an opposite charge for a normal interaction of factor IXa to factor VIIIa. This hypothesis was confirmed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy (Ser558-Gln565) or light chain (Glu1811-Lys1818) and in direct binding studies. We propose that residues 78 and 94 link both EGF-like domains and thereby maintain the integrity of the factor VIII light chain binding site.

Ca2+ binding to the first epidermal growth factor-like domain of human blood coagulation factor IX promotes enzyme activity and factor VIII light chain binding.

Ca2+ binding to the first epidermal growth factor (EGF)-like domain of factor IX is known to be required for biological activity, but the mechanism by which Ca2+ contributes to factor IX function has remained unclear. We have studied recombinant factor IX mutants which lack Ca2+ binding to the first EGF-like domain, due to a replacement of Asp64 by Glu, Lys, or Val. The purified mutants (factors IX D64E, D64K, and D64V), were compared to plasma-derived and recombinant wild-type factor IX with regard to a number of metal-ion dependent functional parameters. In the presence of Mg2+, the activated mutants were indistinguishable from normal factor IXa in hydrolyzing the synthetic substrate CH3-SO2-Leu-Gly-Arg-p-nitroanilide. Replacing Mg2+ by Ca2+ further stimulated the activity of normal factor IXa but not of mutant factor IXa. In factor VIII-independent factor X activation, factor IXa D64K and D64E displayed reduced catalytic activity compared to normal factor IXa (apparent kcat/Km approximately 1, 2, and 4 x 10(3) M-1 s-1, respectively). In the presence of factor VIIIa, factor X activation rates by normal and mutant factor IXa were stimulated by factor VIIIa to a different extent ( approximately700- and 200-fold, respectively), indicating that Asp64 replacements affect the interaction with factor VIIIa. This possibility was addressed in inhibition studies employing synthetic peptides comprising the factor IXa-binding motifs of factor VIII heavy or light chains. Whereas the heavy chain peptide (Ser558-Gln565) inhibited factor VIII-dependent factor X activation by normal and mutant factor IXa with similar efficiency, the light chain peptide (Lys1804-Lys1818) inhibited normal factor IXa 2-3-fold more efficiently than did mutant factor IXa. This indicates that the reduced response to factor VIIIa may be due to impaired binding of mutant factor IXa to the factor VIII light chain. This was further explored in direct binding studies. In the presence of Mg2+, normal and mutant factor IXa were similar in binding to the factor VIII light chain. However, in the presence of Ca2+, factor IXa mutants were less efficient than normal factor IXa, which was illustrated by a 4-5-fold lower affinity than normal factor IXa for factor VIII light chain. Collectively, our data demonstrate that a number of factor IXa functions, including enzymatic activity and assembly into the factor IXa-factor VIIIa complex, are dependent on Ca2+ binding to the first EGF-like domain of factor IX.