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The assembly of the enzyme-activated factor IX (FIXa) with its cofactor, activated factor VIII (FVIIIa) is a crucial event in the coagulation cascade. The absence or dysfunction of either enzyme or cofactor severely compromises hemostasis and causes hemophilia. FIXa is a notoriously inefficient enzyme that needs FVIIIa to drive its hemostatic potential, by a mechanism that has remained largely elusive to date. In this study, we employed hydrogen-deuterium exchange-mass spectrometry (HDX-MS) to investigate how FIXa responds to assembly with FVIIIa in the presence of phospholipids. This revealed a complex pattern of changes that partially overlaps with those changes that occur upon occupation of the substrate-binding site by an active site-directed inhibitor. Among the changes driven by both cofactor and substrate, HDX-MS highlighted several surface loops that have been implicated in allosteric networks in related coagulation enzymes. Inspection of FVIIIa-specific changes indicated that 3 helices are involved in FIXa-FVIIIa assembly. These are part of a basic interface that is also known as exosite II. Mutagenesis of basic residues herein, followed by functional studies, identified this interface as an extended FVIIIa-interactive patch. HDX-MS was also applied to recombinant FIXa variants that are associated with severe hemophilia B. This revealed that single amino acid substitutions can silence the extended network of FVIIIa-driven allosteric changes. We conclude that HDX-MS has the potential to visualize the functional impact of disease-associated mutations on enzyme-cofactor complexes in the hemostatic system., (© 2020 by The American Society of Hematology.)

In the complex with von Willebrand factor (VWF) factor VIII (FVIII) is protected from rapid clearance from circulation. Although it has been established that the FVIII binding site resides in the N-terminal D'-D3 domains of VWF, detailed information about the amino acid regions that contribute to FVIII binding is still lacking. In the present study, hydrogen-deuterium exchange mass spectrometry was employed to gain insight into the FVIII binding region on VWF. To this end, time-dependent deuterium incorporation was assessed in D'-D3 and the FVIII-D'-D3 complex. Data showed reduced deuterium incorporation in the D' region Arg782-Cys799 in the FVIII-D'-D3 complex compared to D'-D3. This implies that this region interacts with FVIII. Site-directed mutagenesis of the six charged amino acids in Arg782-Cys799 into alanine residues followed by surface plasmon resonance analysis and solid phase binding studies revealed that replacement of Asp796 affected FVIII binding. A marked decrease in FVIII binding was observed for the D'-D3 Glu787Ala variant. The same was observed for D'-D3 variants in which Asp796 and Glu787 were replaced by Asn796 and Gln787. Site-directed mutagenesis of Leu786, which together with Glu787 and Cys789 forms a short helical region in the crystal structure of D'-D3, also had a marked impact on FVIII binding. The combined results show that the amino acid region Arg782-Cys799 is part of a FVIII binding surface. Our study provides new insight into FVIII-VWF complex formation and defects therein that may be associated with bleeding caused by markedly reduced levels of FVIII., (Copyright© 2020 Ferrata Storti Foundation.)

The D'-D3 fragment of von Willebrand factor (VWF) can be divided into TIL'-E'-VWD3-C8_3-TIL3-E3 subdomains of which TIL'-E'-VWD3 comprises the main factor VIII (FVIII)-binding region. Yet, von Willebrand disease (VWD) Type 2 Normandy (2N) mutations, associated with impaired FVIII interaction, have been identified in C8_3-TIL3-E3. We now assessed the role of the VWF (sub)domains for FVIII binding using isolated D', D3 and monomeric C-terminal subdomain truncation variants of D'-D3. Competitive binding assays and surface plasmon resonance analysis revealed that D' requires the presence of D3 for effective interaction with FVIII. The isolated D3 domain, however, did not show any FVIII binding. Results indicated that the E3 subdomain is dispensable for FVIII binding. Subsequent deletion of the other subdomains from D3 resulted in a progressive decrease in FVIII-binding affinity. Chemical footprinting mass spectrometry suggested increased conformational changes at the N-terminal side of D3 upon subsequent subdomain deletions at the C-terminal side of the D3. A D'-D3 variant with a VWD type 2N mutation in VWD3 (D879N) or C8_3 (C1060R) also revealed conformational changes in D3, which were proportional to a decrease in FVIII-binding affinity. A D'-D3 variant with a putative VWD type 2N mutation in the E3 subdomain (C1225G) showed, however, normal binding. This implies that the designation VWD type 2N is incorrect for this variant. Results together imply that a structurally intact D3 in D'-D3 is indispensable for effective interaction between D' and FVIII explaining why specific mutations in D3 can impair FVIII binding., (© 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Low density lipoprotein receptor-related protein 1 (LRP1) is involved in the catabolism of many ligands, including factor VIII (FVIII) and alpha-2-macroglobulin (α2M). Transfer of FVIII to LRP1 is currently believed to be preceded by pre-concentration on the cell surface, by interacting with a so far unidentified component. In the present study, we used confocal microscopy and flow cytometry to compare endocytosis of FVIII and α2M using U87MG cells. The results show that α2M is rapidly internalized and does not compete for LRP1 mediated internalization of FVIII. FVIII endocytosis did not occur in the presence of receptor-associated-protein (RAP), but FVIII remained visible as a striated fluorescent pattern at the cell borders. In the presence of Von Willebrand Factor (VWF), no FVIII was observed on or within the cells, suggesting that VWF blocks interaction with both cell surface and LRP1. The same dual inhibition has previously been observed for FVIII C1 domain directed monoclonal antibody KM33. Elimination of the KM33 epitope by replacing FVIII C1 residues 2091-2095 and 2155-2160 for the homologues from factor V (FV), however, did not impair FVIII endocytosis. These membrane spikes alone were insufficient for cellular uptake, because FV was neither internalized by U87MG cells nor capable of effectively competing for FVIII endocytosis. These results show that FVIII endocytosis is driven by interaction with LRP1, but at the same time involves the spikes in the C1 domain that have been implicated in lipid binding., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function., (Copyright© Ferrata Storti Foundation.)

Lysine residues are implicated in driving the ligand binding to the LDL receptor family. However, it has remained unclear how specificity is regulated. Using coagulation factor VIII as a model ligand, we now study the contribution of individual lysine residues in the interaction with the largest member of the LDL receptor family, low-density lipoprotein receptor-related protein (LRP1). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS) and SPR interaction analysis on a library of lysine replacement variants as two independent approaches, we demonstrate that the interaction between factor VIII (FVIII) and LRP1 occurs over an extended surface containing multiple lysine residues. None of the individual lysine residues account completely for LRP1 binding, suggesting an additive binding model. Together with structural docking studies, our data suggest that FVIII interacts with LRP1 via an extended surface of multiple lysine residues that starts at the bottom of the C1 domain and winds around the FVIII molecule., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

The C1 domain of factor VIII (FVIII) has been implicated in binding to multiple constituents, including phospholipids, von Willebrand factor, and low-density lipoprotein receptor-related protein (LRP). We have previously described a human monoclonal antibody called KM33 that blocks these interactions as well as cellular uptake by LRP-expressing cells. To unambiguously identify the apparent "hot spot" on FVIII to which this antibody binds, we have employed hydrogen-deuterium exchange mass spectrometry. The results showed that KM33 protects FVIII regions 2091-2104 and 2157-2162 from hydrogen-deuterium exchange. These comprise the two C1 domain spikes 2092-2093 and 2158-2159. Spike 2092-2093 has been demonstrated recently to contribute to assembly with lipid membranes with low phosphatidylserine (PS) content. Therefore, spike 2158-2159 might serve a similar role. This was assessed by replacement of Arg-2159 for Asn, which introduces a motif for N-linked glycosylation. Binding studies revealed that the purified, glycosylated R2159N variant had lost its interaction with antibody KM33 but retained substantial binding to von Willebrand factor and LRP. Cellular uptake of the R2159N variant was reduced both by LRP-expressing U87-MG cells and by human monocyte-derived dendritic cells. FVIII activity was virtually normal on membranes containing 15% PS but reduced at low PS content. These findings suggest that the C1 domain spikes 2092-2093 and 2158-2159 together modulate FVIII membrane assembly by a subtle, PS-dependent mechanism. These findings contribute evidence in favor of an increasingly important role of the C1 domain in FVIII biology.

A recent chemical footprinting study in our laboratory suggested that region 1803-1818 might contribute to A2 domain retention in activated factor VIII (FVIIIa). This site has also been implicated to interact with activated factor IX (FIXa). Asn-1810 further comprises an N-linked glycan, which seems incompatible with a role of the amino acids 1803-1818 for FIXa or A2 domain binding. In the present study, FVIIIa stability and FIXa binding were evaluated in a FVIII-N1810C variant, and two FVIII variants in which residues 1803-1810 and 1811-1818 are replaced by the corresponding residues of factor V (FV). Enzyme kinetic studies showed that only FVIII/FV 1811-1818 has a decreased apparent binding affinity for FIXa. Flow cytometry analysis indicated that fluorescent FIXa exhibits impaired complex formation with only FVIII/FV 1811-1818 on lipospheres. Site-directed mutagenesis revealed that Phe-1816 contributes to the interaction with FIXa. To evaluate FVIIIa stability, the FVIII/FV chimeras were activated by thrombin, and the decline in cofactor function was followed over time. FVIII/FV 1803-1810 and FVIII/FV 1811-1818 but not FVIII-N1810C showed a decreased FVIIIa half-life. However, when the FVIII variants were activated in presence of FIXa, only FVIII/FV 1811-1818 demonstrated an enhanced decline in cofactor function. Surface plasmon resonance analysis revealed that the FVIII variants K1813A/K1818A, E1811A, and F1816A exhibit enhanced dissociation after activation. The results together demonstrate that the glycan at 1810 is not involved in FVIII cofactor function, and that Phe-1816 of region 1811-1818 contributes to FIXa binding. Both regions 1803-1810 and 1811-1818 contribute to FVIIIa stability.

Complex formation between coagulation factor VIII (FVIII) and von Willebrand factor (VWF) is of critical importance to protect FVIII from rapid in vivo clearance and degradation. We have now employed a chemical footprinting approach to identify regions on VWF involved in FVIII binding. To this end, lysine amino acid residues of VWF were chemically modified in the presence of FVIII or activated FVIII, which does not bind VWF. Nano-LC-MS analysis showed that the lysine residues of almost all identified VWF peptides were not differentially modified upon incubation of VWF with FVIII or activated FVIII. However, Lys-773 of peptide Ser-766-Leu-774 was protected from chemical modification in the presence of FVIII. In addition, peptide Ser-764-Arg-782, which comprises the first 19 amino acid residues of mature VWF, showed a differential modification of both Lys-773 and the α-amino group of Ser-764. To verify the role of Lys-773 and the N-terminal Ser-764 in FVIII binding, we employed VWF variants in which either Lys-773 or Ser-764 was replaced with Ala. Surface plasmon resonance analysis and competition studies revealed that VWF(K773A) exhibited reduced binding to FVIII and the FVIII light chain, which harbors the VWF-binding site. In contrast, VWF(S764A) revealed more effective binding to FVIII and the FVIII light chain compared with WT VWF. The results of our study show that the N terminus of VWF is critical for the interaction with FVIII and that Ser-764 and Lys-773 have opposite roles in the binding mechanism.

In endothelial cells, von Willebrand factor (VWF) multimers are packaged into tubules that direct biogenesis of elongated Weibel-Palade bodies (WPBs). WPB release results in unfurling of VWF tubules and assembly into strings that serve to recruit platelets. By confocal microscopy, we have previously observed a rounded morphology of WPBs in blood outgrowth endothelial cells transduced to express factor VIII (FVIII). Using correlative light-electron microscopy and tomography, we now demonstrate that FVIII-containing WPBs have disorganized, short VWF tubules. Whereas normal FVIII and FVIII Y1680F interfered with formation of ultra-large VWF multimers, release of the WPBs resulted in VWF strings of equal length as those from nontransduced blood outgrowth endothelial cells. After release, both WPB-derived FVIII and FVIII Y1680F remained bound to VWF strings, which however had largely lost their ability to recruit platelets. Strings from nontransduced cells, however, were capable of simultaneously recruiting exogenous FVIII and platelets. These findings suggest that the interaction of FVIII with VWF during WPB formation is independent of Y1680, is maintained after WPB release in FVIII-covered VWF strings, and impairs recruitment of platelets. Apparently, intra-cellular and extracellular assembly of FVIII-VWF complex involves distinct mechanisms, which differ with regard to their implications for platelet binding to released VWF strings.

Factor VIII (FVIII) catabolism has been demonstrated to involve LDL receptor-related protein (LRP). We have established that antibody fragment KM33 inhibits cofactor function of FVIII by interacting with the membrane binding region 2092-2093 of the C1 domain. As KM33 also inhibits LRP-dependent uptake of FVIII, we now assessed the role of region 2092-2093 for LRP-dependent endocytosis. For this purpose, we employed functional fluorescent FVIII-YFP or -GFP derivatives and U87MG cells which express high levels of LRP. Confocal microscopy studies and flow cytometry analysis combined with siRNA technology showed that the fluorescent FVIII derivatives are indeed effectively internalized by U87MG cells in a LRP-dependent manner. Competition experiments employing an antagonist of the LDL receptor family members revealed that there is a cell surface binding event for FVIII, which is independent of LRP. Cell surface binding proved to be less effective for the FVIII-YFP variants K2092A, F2093A and K2092A/F2093A. Surface plasmon resonance analysis showed that these substitutions affect LRP binding as well. Finally, flow cytometry analysis revealed a major reduction of endocytic uptake of these FVIII-YFP variants. Our results demonstrate that C1 domain residues 2092-2093 are of major importance for FVIII endocytosis by contributing to cell surface binding and receptor binding., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Activation of T-helper cells is dependent upon the appropriate presentation of antigen-derived peptides on MHC class II molecules expressed on antigen presenting cells. In the current study we explored the repertoire of peptides presented on MHC class II molecules on human monocyte derived dendritic cells (moDCs) from four HLA-typed healthy donors. MHC class II-bound peptides could be routinely recovered from small cultures containing 5 × 10(6) cells. A fraction of the identified peptides were derived from proteins localized in the plasma membrane, endosomes, and lysosomes, but the majority of peptides that were presented on MHC class II originate from other organelles. Subsequently, we studied the antigen-specific peptide repertoire after endocytosis of a soluble antigen. Blood coagulation factor VIII (FVIII) was chosen as the antigen since our current knowledge on MHC class II presented peptides derived from this immunogenic therapeutic protein is limited. Analysis of the total repertoire of MHC class II-associated peptides revealed that per individual sample 20-50 FVIII-derived peptides were presented on FVIII-pulsed moDCs. Repertoires of FVIII-derived peptides eluted from moDCs derived from a panel of four HLA typed donors revealed that some MHC class II-presented FVIII peptides were presented by multiple donors, whereas the presentation of other FVIII peptides was donor-specific. In total 32 different core peptides were presented on FVIII-pulsed moDCs from four HLA-typed donors. Together our findings provide an unbiased approach to identify peptides that are presented by MHC class II on antigen-loaded moDCs from individual donors.

Background: Point mutations resulting in reduced factor VIII (FVIII) binding to von Willebrand factor (VWF) are an important cause of mild/moderate hemophilia A. Treatment includes desmopressin infusion, which concomitantly increases VWF and FVIII plasma levels, apparently from storage pools containing both proteins. The source of these VWF/FVIII co-storage pools and the mechanism of granule biogenesis are not fully understood., Methodology/principal Findings: We studied intracellular trafficking of FVIII variants implicated in mild/moderate hemophilia A together with VWF in HEK293 cells and primary endothelial cells. The role of VWF binding was addressed using FVIII variants displaying reduced VWF interaction. Binding studies using purified FVIII proteins revealed moderate (Arg2150His, Del2201, Pro2300Ser) to severe (Tyr1680Phe, Ser2119Tyr) VWF binding defects. Expression studies in HEK293 cells and primary endothelial cells revealed that all FVIII variants were present within VWF-containing organelles. Quantitative studies showed that the relative amount of FVIII storage was independent of various mutations. Substantial amounts of FVIII variants are co-stored in VWF-containing storage organelles, presumably by virtue of their ability to interact with VWF at low pH., Conclusions: Our data suggest that the potential of FVIII co-storage with VWF is not affected in mild/moderate hemophilia A caused by reduced FVIII/VWF interaction in the circulation. These data support the hypothesis that Weibel-Palade bodies comprise the desmopressin-releasable FVIII storage pool in vivo.

Binding of factor VIII to membranes containing phosphatidyl-L-serine (Ptd-L-Ser) is mediated, in part, by a motif localized to the C2 domain. We evaluated a putative membrane-binding role of the C1 domain using an anti-C1 antibody fragment, KM33(scFv), and factor VIII mutants with an altered KM33 epitope. We prepared a dual mutant Lys2092/Phe2093 --> Ala/Ala (fVIII(YFP 2092/93)) and 2 single mutants Lys2092 --> Ala and Phe2093 --> Ala. KM33(scFv) inhibited binding of fluorescein-labeled factor VIII to synthetic membranes and inhibited at least 95% of factor Xase activity. fVIII(YFP 2092/93) had 3-fold lower affinity for membranes containing 15% Ptd-L-Ser but more than 10-fold reduction in affinity for membranes with 4% Ptd-L-Ser. In a microtiter plate, KM33(scFv) was additive with an anti-C2 antibody for blocking binding to vesicles of 15% Ptd-L-Ser, whereas either antibody blocked binding to vesicles of 4% Ptd-L-Ser. KM33(scFv) inhibited binding to platelets and fVIII(YFP 2092/93) had reduced binding to A23187-stimulated platelets. fVIII(YFP 2092) exhibited normal activity at various Ptd-L-Ser concentrations, whereas fVIII(YFP 2093) showed a reduction of activity with Ptd-L-Ser less than 12%. fVIII(YFP 2092/93) had a greater reduction of activity than either single mutant. These results indicate that Lys 2092 and Phe 2093 are elements of a membrane-binding motif on the factor VIII C1 domain.

Background: Gene therapy provides an attractive alternative for protein replacement therapy in hemophilia A patients. Recent studies have shown the potential benefit of directing factor (F)VIII gene delivery to cells that also express its natural carrier protein von Willebrand factor (VWF). In this study, we explored the feasibility of blood outgrowth endothelial cells as a cellular FVIII delivery device with particular reference to long-term production levels, intracellular storage in Weibel-Palade bodies and agonist-induced regulated secretion., Design and Methods: Human blood outgrowth endothelial cells were isolated from peripheral blood collected from healthy donors, transduced at passage 5 using a lentiviral vector encoding human B-domain deleted FVIII-GFP and characterized by flow cytometry and confocal microscopy., Results: Blood outgrowth endothelial cells displayed typical endothelial morphology and expressed the endothelial-specific marker VWF. Following transduction with a lentivirus encoding FVIII-GFP, 80% of transduced blood outgrowth endothelial cells expressed FVIII-GFP. Levels of FVIII-GFP positive cells declined slowly upon prolonged culturing. Transduced blood outgrowth endothelial cells expressed 1.6+/-1.0 pmol/1 x 10(6) cells/24h FVIII. Morphological analysis demonstrated that FVIII-GFP was stored in Weibel-Palade bodies together with VWF and P-selectin. FVIII levels were only slightly increased following agonist-induced stimulation, whereas a 6- to 8-fold increase of VWF levels was observed. Subcellular fractionation revealed that 15-22% of FVIII antigen was present within the dense fraction containing Weibel-Palade bodies., Conclusions: We conclude that blood outgrowth endothelial cells, by virtue of their ability to store a significant portion of synthesized FVIII-GFP in Weibel-Palade bodies, provide an attractive cellular on-demand delivery device for gene therapy of hemophilia A.

Weibel-Palade bodies (WPBs) are the endothelial storage organelles that are formed upon von Willebrand factor (VWF) expression. Apart from VWF, WPBs contain a variety of hemostatic and inflammatory proteins. Some of these are thought to be targeted to WPBs by directly interacting with VWF in the secretory pathway. Previous studies have demonstrated that coexpression of factor VIII (FVIII) with VWF results in costorage of both proteins. However, whether cotrafficking is driven by intracellular FVIII-VWF assembly has remained unclear. We now have addressed this issue using recombinant VWF type 2N variants that are known to display reduced FVIII binding in the circulation. Binding studies using purified fluorescent FVIII and VWF type 2N variants revealed FVIII binding defects varying from moderate (Arg854Gln, Cys1060Arg) to severe (Arg763Gly, Thr791Met, Arg816Trp). Upon expression in HEK293 cells, all VWF variants induced formation of WPB-like organelles that were able to recruit P-selectin, as well as FVIII. WPBs containing FVIII did not display their typical elongated shape, suggesting that FVIII affects the organization of VWF tubules therein. The finding that VWF type 2N variants are still capable of cotargeting FVIII to storage granules implies that trafficking of WPB cargo proteins does not necessarily require high-affinity assembly with VWF.

The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated alpha2-macroglobulin (alpha2M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3-10) and cluster IV of 11 repeats (CR21-31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding alpha2M* and RAP. We now evaluated the binding to RAP, alpha2M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4-8 of cluster II and within region CR24-28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5-6, CR26-27), combinations of three repeats showed most efficient binding to FIXa (CR6-8, CR26-28), FVIII light chain (CR5-7, CR6-8, CR24-26), and alpha2M* (CR4-6, CR24-26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands.

The light chain of activated factor IX (FIXa) is involved in a number of functional properties, including FIXa enzymatic activity. This suggests the existence of a functional link between the FIXa light chain and the catalytic domain. The FIXa structure includes a few putative interactions between EGF2 and the protease domain. The role thereof has been addressed in this study. Recombinant FIX variants FIX-N92A, FIX-N92H, FIX-Y295A, and FIX-F299A were produced in 293 cells. After activation, the purified mutants were analyzed for a variety of functional parameters. None of these substitutions had a major effect on the interaction with antithrombin or the cleavage of the chromogenic substrate CH(3)SO(2)-d-CHG-Gly-Arg-p-nitroanilide. All FIXa mutants, however, exhibited a reduced level of factor X (FX) activation. Defective proteolytic activity occurred both in the absence and in the presence of activated factor VIII (FVIIIa). All mutants also exhibited a reduced level of FX activation in the absence of phospholipids. This suggests that putative interdomain contacts involving residues Asn(92), Tyr(295), and Phe(299) affect reactivity toward FX. Detailed kinetic studies in the presence of phospholipids and FVIIIa revealed substrate inhibition, particularly for mutants FIXa-N92A and FIXa-N92H. Surface plasmon resonance demonstrated that the same replacements weaken the association with the isolated factor VIII (FVIII) A2 domain and the FVIII light chain. This implies a defect in the formation of the FX-activating complex that is membrane-independent. We conclude that contacts between EGF2 and the protease domain of FIXa are crucial for FIXa enzymatic activity and for the assembly of the FX-activating complex.

Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.

Low-density lipoprotein (LDL) receptor (LDLR) and LDLR-related protein (LRP) are members of the LDLR family of endocytic receptors. LRP recognizes a wide spectrum of structurally and functionally unrelated ligands, including coagulation factor VIII (FVIII). In contrast, the ligand specificity of LDLR is restricted to apolipoproteins E and B-100. Ligand binding to the LDLR family is inhibited by receptor-associated protein (RAP). We have previously reported that, apart from LRP, other RAP-sensitive mechanisms contribute to the regulation of FVIII in vivo. In the present study, we showed that the extracellular ligand-binding domain of LDLR interacts with FVIII in vitro and that binding was inhibited by RAP. The physiologic relevance of the FVIII-LDLR interaction was addressed using mouse models of LDLR or hepatic LRP deficiency. In the absence of hepatic LRP, LDLR played a dominant role in the regulation and clearance of FVIII in vivo. Furthermore, FVIII clearance was accelerated after adenovirus-mediated gene transfer of LDLR. The role of LDLR in FVIII catabolism was not secondary to increased plasma lipoproteins or to changes in lipoprotein profiles. We propose that LDLR acts in concert with LRP in regulating plasma levels of FVIII in vivo. This represents a previously unrecognized link between LDLR and hemostasis.

Factor VIII binds to phospholipid membranes through the C2 domain (S2173-Y2332). Residues M2199, F2200, L2251, L2252, V2223, W2313 and V2314 at the tips of beta-hairpins and loops are thought to contribute to phospholipid membrane binding. Similarly, residues in the C2 domain of the homologous protein factor V forma phospholipid binding site, but residues in the A3 and C1 domains are also thought to contribute to membrane binding. Phage display technology was previously used to isolate factor VIII light chain specific single-chain variable domain fragments (scFv) from patients with factor VIII inhibitors. Phospholipid vesicles inhibited the binding of factor VIII to scFvs WR1 and WR16 (epitope : E2181-M2199) with half saturation values of 23 and 47 muM respectively. The single point mutant F2200A factor VIII light chain bound to WR1 and WR16 with a much lower affinity than wild type protein suggesting that residue F2200 is also included in the epitopes of these scFvs. Binding of factor VIII to C2-specific scFvs WR13 and EL14 (epitope : K2207-M2321) was not inhibited by phospholipid vesicles. Consistent with this, F2200A factor VIII light chain bound to these scFvs with the same affinity as the wild type protein. However, phospholipid vesicles also inhibited the binding of factor VIII to the A3-C1-specific scFvs KM36 (epitope : Q1778-D1840) and KM38 (epitope : S1690-N1777 and/or V1841-N2172) with half saturation values of 84 and 165 microM, respectively, suggesting that the A3 and/or C1 domains may contribute to membrane binding of the cofactor.

Low-density lipoprotein receptor-related protein (LRP) contributes to factor VIII (FVIII) catabolism in vivo. Besides LRP, FVIII also interacts with very low-density lipoprotein receptor (VLDLR) in vitro. We investigated the physiological role of VLDLR in FVIII catabolism, using knockout mouse models for VLDLR and LRP, alone and in combination. VLDLR(-/-) mice displayed normal plasma FVIII, whereas VLDLR(-/-) LRP(-) double-knockout mice had slightly increased FVIII compared with LRP-deficient mice. Remarkably, VLDLR deficiency slightly accelerated FVIII clearance. Adenovirus-mediated overexpression of VLDLR did not lower plasma FVIII in LRP-deficient mice. We conclude that VLDLR does not act in concert with LRP in FVIII clearance in vivo.

It has been established that low-density lipoprotein receptor-related protein (LRP) is involved in the cellular uptake and degradation of coagulation factor VIII (FVIII) in vitro. To address the physiologic role of LRP in regulating plasma FVIII in vivo, we used cre/loxP-mediated conditional LRP- deficient mice (MX1cre(+)LRP(flox/flox)). Upon inactivation of the LRP gene, MX1cre(+)LRP(flox/flox) mice had significantly higher plasma FVIII as compared with control LRP(flox/flox) mice (3.4 and 2.0 U/mL, respectively; P <.001). Elevated plasma FVIII levels in MX1cre(+)LRP(flox/flox) mice coincided with increased plasma von Willebrand factor (VWF) (2.0 and 1.6 U/mL for MX1cre(+)LRP(flox/flox) and control LRP(flox/flox) mice, respectively; P <.05). Elevation of plasma FVIII and VWF persisted for at least 6 weeks after inactivation of the LRP gene. Upon comparing plasma FVIII and VWF in individual mice, we observed an increase of the FVIII/VWF ratio in MX1cre(+)LRP(flox/flox) mice as compared with control LRP(flox/flox) mice. Administration of either a vasopressin analog or an endotoxin resulted in increased plasma VWF, but not FVIII. In clearance experiments, MX1cre(+)LRP(flox/flox) mice displayed a 1.5-fold prolongation of FVIII mean residence time. Adenovirus-mediated overexpression of the 39-kDa receptor-associated protein (RAP) in normal mice resulted in a 3.5-fold increase of plasma FVIII. These data confirm that the regulation of plasma FVIII in vivo involves a RAP-sensitive mechanism. Surprisingly, plasma FVIII in MX1cre(+)LRP(flox/flox) mice increased 2-fold after RAP gene transfer. We propose that RAP-sensitive determinants other than hepatic LRP contribute to the regulation of plasma FVIII in vivo.

Low-density lipoprotein receptor-related protein (LRP) is an endocytic receptor that binds multiple distinct ligands, including blood coagulation factor VIII (FVIII). FVIII is a heterodimeric multidomain protein that consists of a heavy chain (domains A1, a1, A2, a2, and B) and a light chain (domains a3, A3, C1, and C2). Both chains contribute to high-affinity interaction with LRP. One LRP-interactive region has previously been located in the C2 domain, but its affinity is low in comparison with that of the entire FVIII light chain. We now have compared a variety of FVIII light chain derivatives with the light chain of its homolog FVa for LRP binding. In surface plasmon resonance studies employing LRP cluster II, the FVa and FVIII light chains proved different in that only FVIII displayed high-affinity binding. Because the FVIII a3-A3-C1 fragment was effective in associating with LRP, this region was explored for structural elements that are exposed but not conserved in FV. Competition studies using synthetic peptides suggested that LRP binding involves the FVIII-specific region Lys(1804)-Ala(1834) in the A3 domain. In line with this observation, LRP binding was inhibited by a recombinant antibody fragment that specifically binds to the FVIII sequence Glu(1811)-Lys(1818). The role of this sequence in LRP binding was further tested using a FVIII/FV chimera in which sequence Glu(1811)-Lys(1818) was replaced with the corresponding sequence of FV. Although this chimera still displayed residual binding to LRP cluster II, its affinity was reduced. This suggests that multiple sites in FVIII contribute to high-affinity LRP binding, one of which is the FVIII A3 domain region Glu(1811)-Lys(1818). This suggests that LRP binding to the FVIII A3 domain involves the same structural elements that also contribute to the assembly of FVIII with FIXa.

Most plasmas from patients with inhibitors contain antibodies that are reactive with the C2 domain of factor VIII. Previously, we have shown that the variable heavy chain (V(H)) regions of antibodies to the C2 domain are encoded by the closely related germline gene segments DP-10, DP-14, and DP-88, which all belong to the V(H)1 gene family. Here, we report on the isolation and characterization of additional anti-C2 antibodies that are derived from V(H) gene segments DP-88 and DP-5. Competition experiments using murine monoclonal antibodies CLB-CAg 117 and ESH4 demonstrated that antibodies derived from DP-5 and DP-88 bound to different sites within the C2 domain. Epitope mapping studies using a series of factor VIII/factor V hybrids revealed that residues 2223 to 2332 of factor VIII are required for binding of the DP-10-, DP-14-, and DP-88-encoded antibodies. In contrast, binding of the DP-5-encoded antibodies required residues in both the amino- and carboxy-terminus of the C2 domain. Inspection of the reactivity of the antibodies with a series of human/porcine hybrids yielded similar data. Binding of antibodies derived from germline gene segments DP-10, DP-14, and DP-88 was unaffected by replacement of residues 2181 to 2243 of human factor VIII for the porcine sequence, whereas binding of the DP-5-encoded antibodies was abrogated by this replacement. Together these data indicate that antibodies assembled from V(H) gene segments DP-5 and the closely related germline gene segments DP-10, DP-14, and DP-88 target 2 distinct antigenic sites in the C2 domain of factor VIII.

Apparently quiescent, nonapoptotic endothelial cells mediate the activation of factor X by activated factor IX in the presence of its cofactor, activated factor VIII. In a previous study, we reported that during the activation of factor X, the interaction of the cofactor with the endothelial cell membrane clearly differs from the interaction of the cofactor with artificial lipid membranes. In the present study, we identified the peptide domain of factor VIII involved in the assembly of the enzyme-cofactor complex on the endothelial cell surface. With the use of monoclonal antibodies against different peptide sequences on the factor VIII light chain, it was observed that the lipid-binding region of the C2 domain on the factor VIII light chain mediates the assembly of the factor X-activating complex on the endothelial cell surface. In addition, a synthetic peptide that constitutes region Ala2318-Tyr2332 of the C2 domain and that is known for its ability to inhibit the binding of factor VIII to artificial lipid membranes also showed inhibition of the cofactor activity of factor VIII on endothelial cells. Thus, the carboxy-terminal part of the factor VIII light chain not only contains sites involved in lipid binding but also contains sites involved in complex assembly on the endothelial cell membrane.

Despite recent studies, the organization of coagulation factor VIII (FVIII) on a phospholipid (PL) membrane is not known in detail. Thus, 2-dimensional (2D) crystals of human FVIII lacking the B domain were prepared for electron microscopy onto negatively charged PL monolayers. The 3-dimensional (3D) density map of the PL-bound FVIII protein was calculated at 1.5 nm. Existing atomic data and models for FVIII domains were fitted unambiguously within the 3D density map of the molecule. FVIII domains arrangement followed a compact spiral organization with the A3 domains in close association with the C1 and C2 domains near the PL surface. Viewed toward the membrane the A domains' heterotrimer is oriented side-on with the pseudo-3-fold axis almost parallel to the PL surface and A1 fully covering C1. The C2 domain is partially overlapped by the A2 domain of an adjacent molecule in the 2D crystal, favoring close packing. Viewed parallel to the membrane, C2 is slightly inclined to the PL surface covering an area of 12 nm(2). Four C2 loops are embedded within the lipid monolayer at about 0.7 to 1.0 nm depth. C1 forms almost a right angle with C2, its long axis nearly parallel to the membrane. The proposed structure for membrane-bound FVIII results from modeling of the FVIII domains within a 3D density map obtained from electron crystallography and accords with the main biochemical and structural information known to date. A model is proposed for FVIIIa and factor IXa assembly within the membrane-bound factor X-activating complex. (Blood. 2002;99:1215-1223)

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region LysT13-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X, phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3±0.3×106 M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713–1637) factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713–1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713–1637)-factor VIII is clearly reduced. Furthermore, the second-order rate constant (0.2±0.1×106 M-1 s-1) of des-(713–1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713–1637)-factor VIII light chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des (713–1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis. [ABSTRACT FROM AUTHOR]

Abstract: The low-density lipoprotein receptor-related protein (LRP) binds a range of proteins including receptor associated protein (RAP), activated α2-macroglobulin (α2M*), factor IXa (FIXa), and factor VIII (FVIII) light chain. The binding is mediated by the complement-type repeats, which are clustered in four distinct regions within LRP. Cluster II of 8 repeats (CR3–10) and cluster IV of 11 repeats (CR21–31) have been implicated in ligand-binding. Previous studies have aimed to identify the cluster II repeats involved in binding α2M* and RAP. We now evaluated the binding to RAP, α2M*, FIXa and FVIII light chain of triplicate repeat-fragments of not only clusters II but also of cluster IV. Employing surface plasmon resonance analysis, we found that most efficient ligand-binding was displayed by the repeats within region CR4–8 of cluster II and within region CR24–28 of cluster IV. Whereas the binding to RAP could be attributed to two consecutive repeats (CR5–6, CR26–27), combinations of three repeats showed most efficient binding to FIXa (CR6–8, CR26–28), FVIII light chain (CR5–7, CR6–8, CR24–26), and α2M* (CR4–6, CR24–26). The results imply that there is an internal functional duplication of complement-type repeats within LRP resulting in two clusters that bind the same ligands. [Copyright &y& Elsevier]