Factor VIII Light Chain Research Articles

Binding Promiscuity of Therapeutic Factor VIII.

The binding promiscuity of proteins defines their ability to indiscriminately bind multiple unrelated molecules. Binding promiscuity is implicated, at least in part, in the off-target reactivity, nonspecific biodistribution, immunogenicity, and/or short half-life of potentially efficacious protein drugs, thus affecting their clinical use. In this review, we discuss the current evidence for the binding promiscuity of factor VIII (FVIII), a protein used for the treatment of hemophilia A, which displays poor pharmacokinetics, and elevated immunogenicity. We summarize the different canonical and noncanonical interactions that FVIII may establish in the circulation and that could be responsible for its therapeutic liabilities. We also provide information suggesting that the FVIII light chain, and especially its C1 and C2 domains, could play an important role in the binding promiscuity. We believe that the knowledge accumulated over years of FVIII usage could be exploited for the development of strategies to predict protein binding promiscuity and therefore anticipate drug efficacy and toxicity. This would open a mutational space to reduce the binding promiscuity of emerging protein drugs while conserving their therapeutic potency.

Influence of Inhibitor Characteristics on Response to Hemostatic and Immunosuppressive Treaments in Acquired Hemophilia Patients: The Ciha Project

INTRODUCTIONAcquired hemophilia (AH) is a disease induced by autoantibodies targeting coagulation factors, mostly factor VIII (FVIII). The disease manifests as bleeding episodes in unusual locations that usually prompt physicians to establish hemostatic and immunosuppressive treatments (IST) for hemorrhage cessation and inhibitor eradication respectively. The specificity of the so-called inhibitors varies among patients. We present herein the main findings of the CIHA project, which is a pilot study aimed to test the hypothesis that the inhibitor's features, i.e., its FVIII domain specificity, influences AH patient evolution and response to hemostatic and immunosuppressive therapies.METHODSThis is a prospective, descriptive, observational and non-interventional study. Between September 2011 and September 2013, 16 patients who had been diagnosed with AH caused by FVIII inhibitors were recruited, and their clinical and epidemiologic features were assessed. Inhibitors were titrated by the Bethesda method according to the Nijmegen modification. The inhibitor specificities were determined by Western blot: first, FVIII was cleaved by thombin in order to obtain the fragments corresponding to the light chain (LC), and the A1 and the A2 domains of the heavy chain. Once electrophoresed and transferred to a polyvinylidine difluoride membrane, this material was incubated with the patient´s plasma. Finally, an anti-human IgG antibody carrying a chemiluminiscent substrate was incorporated. The luminiscent pattern should be dependent on the molecular weight of the FVIII domains recognized by the inhibitor, allowing us to assess the inhibitor's specificity. In order to analyze the association between inhibitors' specificities and individual responses to therapies, sampling was performed at the time of diagnosis, 14 days afterwards, and, provided that remission was reached, 56 days after IST was withdrawn. When immunosuppression failed, a similar sampling pattern was applied after a second line of therapy was initiated.RESULTSThree patients died in the course of the study due to complications linked to their underlying pathologies related neither to the hemostatic treatment nor to the IST.According to the literature, inhibitors targeted mostly the LC (11 out of 16; 69%) or the A2 domain (8 out of 16; 50%). Two of them (12%) were directed against the A1 domain. It must be remarked that, in disagreement with the literature, the inhibitors targeted more than one domain in a non-negligible number of cases [5 out of 16 (31%): A2-LC, three cases; A1-A2-CL and A1-A2, one case each].Bypass agents, i.e., recombinant activated factor VII or activated prothrombin complex, were used for hemorrhage cessation. The inhibitor features did not influence the response to hemostatic treatment, since this efficiently stopped blood loss in all patients.The first line immunosuppressive drugs most commonly used were steroids (14 out of 16), alone or with concomitant cyclophosphamide (6 out of 14). Nine patients (56%) reached remission, and the rest of them required a second IST to eradicate the inhibitor. Interestingly, inhibitors targeted LC in 6 out of 7 (86%) patients that did not respond to the first IST. Conversely, inhibitors targeted LC only in 4 out of 9 (44%) patients who reached remission with the first line IST. Finally, 30 days after IST had started, inhibitor specificity changed in one of the IST responders, from multidomain targeting (A1-A2-LC) to LC-only targeting.CONCLUSIONSAntibody recognition of more than one FVIII domain is not uncommon in AH. More importantly, this pilot study suggests that inhibitors that interact with the LC of FVIII may induce a worse response to IST in AH patients. Since this finding may be clinically relevant, these results encourage us to carry out a larger study to confirm such observations. DisclosuresNo relevant conflicts of interest to declare.

Lettuce Plants Expressing High Levels of Factor VIII Antigen in the Chloroplast for Oral Tolerance in Hemophilia a

Inhibitor formation is a serious complication in factor VIII (FVIII) replacement therapy for the X-linked bleeding disorder hemophilia A and occurs in 20-30% of patients. No prophylactic tolerance protocols exist. In previous proof-of-principle work, we demonstrated oral tolerance induction to FVIII in hemophilia A mice using frozen tobacco leaf cells expressing the heavy chain (HC) and C2 domain of human B domain-deleted (BDD) FVIII (Blood 124:1659-68, 2014). This approach is based on expression of heterologous proteins in the chloroplasts of green leaves and natural bioencapsulation provided by plant cell wall, so that the antigen is protected until its release in the small intestine. The antigens are expressed as fusions with the transmucosal carrier CTB proteins for effective targeting of the gut epithelium and translocation to the immune system. However, significant hurdles toward clinical feasibility remained that we now successfully addressed. Until now, methods to express large human proteins in chloroplasts of edible crop plants did not exist. Building on our recently established gene transfer technology for lettuce chloroplasts and identification of powerful posttranscriptional regulatory elements, we focused on the development of chloroplast genomics tools (e.g. bioinformatics tools for codon optimization and assays that identify sites of ribosome stalling), which ultimately allowed us to express high levels of FVIII antigen in edible crop plants that are suitable for clinical application. Remarkably, codon-optimization improved expression of FVIII heavy chain (HC) and light chain (LC) by15- to 42-fold. Hence, the entire BDD-FVIII sequence can be orally delivered, thereby covering the entire patient population regardless of individual CD4+ T cell epitope usage. A clinical grade manufacturing process is in place, and lyophilization of leaf cells generates material that is stable at ambient temperature for at least 2 years. Testing in hemophilia A mice showed effectiveness of low FVIII antigen doses upon delivery of lyophilized lettuce cells. Repeated oral gavage (twice/week for 2 months) of a mixture of CTB-HC and CTB-C2 suppressed subsequent inhibitor formation against intravenous FVIII (1IU, once/ week for 4 weeks) in hemophilia A mice as well as the original tobacco material, using very low doses of 0.15-0.5 ug/antigen/mouse. When using CTB-HC/CTB-LC combination, a dose of 1.5 ug was most optimal and suppressed inhibitor titers by ~1 log (avg. 4.7 BU/ml vs 44 BU/ml in controls; 50% of tolerized mice had 0-3 BU/ml, while 86% of controls developed 6-126 BU/ml, n=12-14, P <0.05). Translational oral tolerance studies in humans would benefit from cellular biomarkers. Our prior studies showed induction of two subsets of Treg that suppress antibody formation against FVIII, namely CD4+CD25+FoxP3+ Treg and LAP+CD4+CD25-FoxP3- Treg. The latter stain positive on the cell surface for LAP (latency-associated peptide) because of overexpression of TGF-b. Although we found increases in the frequencies of LAP+CD4+CD25-FoxP3- Treg in peripheral blood, a statistical significance was not always observed when compared to the blood of control mice that received intravenous FVIII but not oral tolerance. However, we found significant and consistent increases in the frequencies of circulating LAP-expressing CD4+CD25+FoxP3+ Treg in tolerized mice, suggesting that LAP may be a suitable cellular biomarker for plant-based oral tolerance induction. In conclusion, clinical candidates for oral tolerance to FVIII are now available in form of lettuce plants expressing immunogenic domains or the entire antigen. DisclosuresHerzog:Novo Nordisk: Research Funding; Applied Genetic Technologies Corporation: Consultancy; Spark Therapeutics: Patents & Royalties. Daniell:Novo Nordisk: Research Funding.

A Spacer Sequence Inserted between the A1 and A2 Domains of Factor VIII Overcomes the Requirement for Proteolytic Cleavage at Arg 372 in the Generation of Cofactor Activity

Factor VIII (FVIII) with a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) is a procofactor and precursor for the anti-hemophilic cofactor protein, FVIIIa. Following the intracellular processing within the B domain, secreted FVIII circulates as a heterodimer with variably sized (90K-200K) heavy chain (A1-a1-A2-a2-B) and an 80K light chain (a3-A3-C1-C2). Proteolytic activation of FVIII by thrombin that yields heterotrimeric FVIIIa (A1-a1/A2-a2/A3-C1-C2), the cofactor for intrinsic tenase, involves cleavage of three peptide bonds between Arg372-Ser373, Arg740-Ser741, and Arg1689-Ser1690. Cleavage at Arg740 removes the B-domain, and cleavage at Arg1689 removes the a3-acidic region and releases FVIII from vWF, its carrier protein, and exposes membrane binding sites within the FVIII light chain. Cleavage at Arg372 separates A1-a1 and A2-a2 domains and is implicated in the cofactor-dependent recognition and enhancement in the rate of factor X (FX) activation by intrinsic tenase. Subsequently, the separated A2-a2 domain dissociates spontaneously from the heterotrimeric FVIIIa resulting in the rapid loss of cofactor activity. We speculated that the requirement for cleavage at Arg372 might be obviated by the insertion of an optimized linker sequence between A1-a1 and A2-a2 domains on an uncleavable Gln372 backbone. To investigate this possibility, we prepared cDNA constructs of B-domain deleted FVIII variants; FVIII wild-type (FVIIIWT), FVIII372Q, and FVIII372Q followed by a rigid (Ala-Pro)5 linker sequence (FVIII372Q-AP5). All three FVIII constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Selected stable clones were further expanded to collect 15L of expression media over 5-day period, and recombinant FVIII variants were purified using a three-step chromatographic approach. These FVIII variants were studied using SDS-PAGE, western blotting, aPTT assays, thrombin generation assay (TGA) and purified assays to assess kinetics of FX activation and spontaneous loss of cofactor activity. In contrast to FVIIIWT, FVIII372Q and FVIII372Q-AP5 were completely resistant to cleavage at Gln372 by thrombin, yielding bands corresponding to A1-a1-A2-a2 (90K) and A3-C1-C2 (73K). In one stage aPTT assays, FVIII372Q showed prolonged clotting times with specific activity in the range of 200-400 U/mg, while FVIIIWT and FVIII372Q-AP5 displayed comparable clotting times with specific activities ranging between 8000-10000 U/mg and 4500-5500 U/mg, respectively. In TGA initiated with either 0.1 pM tissue factor or 1 pM factor XIa, both FVIIIWT and FVIII372Q-AP5 displayed similar TGA profiles. In steady state kinetic studies of FX activation using limiting concentrations of factor IXa, saturating concentrations of FVIII variants pretreated with thrombin, membranes and increasing concentrations of FX, the cofactor function of thrombin-cleaved FVIII372Q was severely impaired. However, despite lack of cleavage at Gln372 in FVIII372Q-AP5, catalytic efficiency for FX activation by intrinsic tenase assembled by this variant was comparable to that seen with FVIIIaWT. At the physiological concentration of FX, the initial velocity for Xa formation (v/E) for intrinsic tenase assembled with FVIIIa372Q-AP5 was within a factor of 2 of that observed with FVIIIaWT while the rate observed with FVIIIa372Q was &amp;gt;10-fold lower. Following rapid activation with thrombin, loss of cofactor function was significantly slower for FVIIIa372Q-AP5(t1/2 ~ 10 min) compared to FVIIIaWT (t1/2 ~ 2 min). Our findings indicate that the requirement for cleavage at Arg372 for the development of full FVIIIa cofactor function can be overcome by modulating the A1-A2 connector with an optimized linker sequence. Failure to yield an infinitely stable cofactor in the case of FVIIIa372Q-AP5 suggests that cleavage at Arg372 does not solely explain the spontaneous loss of FVIIIa cofactor function. Disclosures Krishnaswamy: Bayer: Research Funding.

Expression and assembly of largest foreign protein in chloroplasts: oral delivery of human FVIII made in lettuce chloroplasts robustly suppresses inhibitor formation in haemophilia A mice.

SummaryInhibitor formation is a serious complication of factor VIII (FVIII) replacement therapy for the X‐linked bleeding disorder haemophilia A and occurs in 20%–30% of patients. No prophylactic tolerance protocol currently exists. Although we reported oral tolerance induction using FVIII domains expressed in tobacco chloroplasts, significant challenges in clinical advancement include expression of the full‐length CTB‐FVIII sequence to cover the entire patient population, regardless of individual CD4+ T‐cell epitope responses. Codon optimization of FVIII heavy chain (HC) and light chain (LC) increased expression 15‐ to 42‐fold higher than the native human genes. Homoplasmic lettuce lines expressed CTB fusion proteins of FVIII‐HC (99.3 kDa), LC (91.8 kDa), C2 (31 kDa) or single chain (SC, 178.2 kDa) up to 3622, 263, 3321 and 852 μg/g in lyophilized plant cells, when grown in a cGMP hydroponic facility (Fraunhofer). CTB‐FVIII‐SC is the largest foreign protein expressed in chloroplasts; despite a large pentamer size (891 kDa), assembly, folding and disulphide bonds were maintained upon lyophilization and long‐term storage as revealed by GM1‐ganglioside receptor binding assays. Repeated oral gavages (twice/week for 2 months) of CTB‐FVIII‐HC/CTB‐FVIII‐LC reduced inhibitor titres ~10‐fold (average 44 BU/mL to 4.7 BU/mL) in haemophilia A mice. Most importantly, increase in the frequency of circulating LAP‐expressing CD4+ CD25+FoxP3+ Treg in tolerized mice could be used as an important cellular biomarker in human clinical trials for plant‐based oral tolerance induction. In conclusion, this study reports the first clinical candidate for oral tolerance induction that is urgently needed to protect haemophilia A patients receiving FVIII injections.

Neither Factor VIII Nor the Putative F8B Protein Is Detectable in Human Peripheral Blood Mononuclear Cells

Factor VIII (FVIII) is encoded by 26 exons comprising the F8 gene. The F8 mRNA transcript encodes the full-length FVIII protein, which consists of 2332 amino acids. A CpG island within intron 22 of F8 includes an initiation site for a second, shorter transcript termed F8B. F8B contains a short exon spliced in-frame to F8 exons 23-26, and this mRNA transcript is expressed in multiple human tissues. The putative protein encoded by this gene corresponds to 8 amino acids encoded by the exon within F8 intron 22, followed by the FVIII C2 domain sequence. Earlier studies have unambiguously demonstrated FVIII expression in human endothelial cells, including liver and lung. Animal model studies have indicated FVIII is expressed, possibly exclusively, in endothelial cells. Other recent reports have presented evidence for FVIII and F8B protein expression in human peripheral blood mononuclear cells (PBMCs). In this study, we utilized several methods to test the hypothesis that FVIII and/or the putative F8B protein is expressed in human PBMCs.Frozen PBMCs from healthy human subjects were thawed and then washed 5-10X to ensure removal of plasma-derived FVIII. Samples containing 50 million PBMCs were lysed in buffer containing protease inhibitors, centrifuged, and the supernatants analyzed by immunoprecipitation (IP) followed by SDS-PAGE and Western blots. The IP experiments utilized both polyclonal anti-FVIII antibodies and cocktails of monoclonal antibodies (mAbs) specific for different FVIII domains, and Western blots used several combinations of primary and secondary antibodies. Negative controls consisted of BHK cell lysates, and positive controls were purified FVIII and lysates of BHK cells transduced with a plasmid encoding FVIII. Western blots carried out with sensitive chemiluminescence detection identified the expected bands in the positive control lanes. Bands at expected positions for FVIII or F8B proteins (~240kDa, 75 kDa, 25-30 kDa) visualized by Coomassie-blue staining of lysates analyzed by SDS-PAGE were cut from the gels, subjected to trypsin digestion and then analyzed by mass spectrometry (LC-MS). FVIII-derived peptides were detected only in the positive control samples.Intracellular staining (ICS) of permeabilized PBMCs was carried out using mAbs specific for the FVIII A1, A2, C1, and C2 domains and a mAb specific for the FVIII light chain. The mAbs were covalently labeled with Alexa-Fluor 488 to avoid possible nonspecific binding of labeled secondary antibodies. PBMCs from (1) normal control subjects; (2) a severe hemophilia A subject with an intron-22 inversion mutation and (3) a severe hemophilia A subject with a deletion of F8 exons 3-6 were analyzed. All 5 anti-FVIII mAbs detected FVIII protein in FVIII-expressing BHK cells, while none detected FVIII in BHK cells or in any of the human PBMC samples. Our results are consistent with several recent studies reporting proteomics analysis of human PBMCs, which have identified no FVIII peptides or proteins. We conclude that if FVIII or F8B proteins are expressed in human PBMCs, their concentration is below the detection limits of these assays. DisclosuresNo relevant conflicts of interest to declare.

Anti-FVIII Autoantibodies Share Similar Characteristics In Acquired Hemophilia A Patients With and Without An Associated Disease

IntroductionAcquired hemophilia A (AHA) is an autoimmune disease caused by the development of inhibitory autoantibodies against factor VIII (FVIII) resulting in severe hemorrhages. Associated pathologies, such as autoimmune disease, malignancy and pregnancy are observed in approximately 50% of patients. AimTo elucidate the relationship between an underlying disease, the bleeding tendency and patients immunological profile the characteristics of anti-FVIII autoantibodies in AHA patients with (n=6) and without an underlying disease (n=9) were determined. Patients and MethodsThe median age of this cohort (n=15) was 71 years with two-thirds older than 70 years. Treatment parameters were analysed and patients were classified according to there bleeding tendency into a mild, moderate and severe phenotype. FVIII domain specificity of anti-FVIII autoantibodies was analysed in ELISA by binding to (i) FVIII fragments (heavy (HC) and light chain (LC), A2 and C2 domain) and (ii) single human domain hybrid human/porcine FVIII proteins. The amount of FVIII-specific IgGs was measured by ELISA and their relative contribution to the total amount of anti-FVIII IgG was calculated from standard curves for FVIII-specific IgG1, IgG2, IgG3, and IgG4. ResultsAll but one patient were treated with bypassing agents including activated FVII, activated prothrombin complex concentrate or porcine FVIII. All patients received immunosuppressive treatments. 14/15 achieved initial complete remission with 6 patients experiencing another episode of inhibitors. Characteristics of anti-FVIII autoantibodies in AHA patients with or without an underlying disease were similar: FVIII-specific autoantibodies targeted primarily the FVIII LC with a dominance of epitopes located C2 domain compared to the C1 domain. FVIII-specific antibodies belonged to the subclasses IgG1, IgG2, and IgG4. The individual IgG subclass levels did not correlate with the total amount of anti-FVIII antibodies or inhibitory anti-FVIII antibodies in Bethesda units. IgG1 and 2 vs IgG4 levels did not correlate with bleeding tendency. Patients with a mild bleeding phenotype only recognized the C2 domain, whereas other patients had antibodies against C2 and or other domains. Although lower levels of FVIII activity (FVIII:C) were observed in disease-associated AHA patients with a median FVIII:C of 0.5% (range, 0-6%) compared to 1.5% (range, 0-10%) in idiopathic AHA patients, this difference was not statistically significant. FVIII:C levels and FVIII inhibitor titers at clinical presentation did not correlate to the severity of bleeds: the median FVIII:C level in patients who had strong bleeds was 0.5% (range, 0-10%), moderate bleeds 3.6% (range, 0-6%), and mild bleeds 1.2% (range, 1-6%). The FVIII inhibitor titer at presentation was similar in patients who had mild, moderate and severe bleeding tendency with a median of 35 BU/mL (range, 29-55 BU/mL), 49.5 BU/mL (range, 9-156 BU/mL), and 17.3 BU/mL (range, 2.2-614 BU/mL), respectively. ConclusionThe presented data challenges the view from other small cohorts that differential immunological profiles exist between disease-associated and idiopathic AHA patients. Data on the influence of epitopes and IgG subclasses on outcome in AHA patients remains conflicting and needs further study. Disclosures:No relevant conflicts of interest to declare.

A3 Domain Region 1803–1818 Contributes to the Stability of Activated Factor VIII and Includes a Binding Site for Activated Factor IX

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.

Mapping the Binding Region on the Low Density Lipoprotein Receptor for Blood Coagulation Factor VIII

Low density lipoprotein receptor (LDLR) was shown to mediate clearance of blood coagulation factor VIII (FVIII) from the circulation. To elucidate the mechanism of interaction of LDLR and FVIII, our objective was to identify the region of the receptor necessary for binding FVIII. Using surface plasmon resonance, we found that LDLR exodomain and its cluster of complement-type repeats (CRs) bind FVIII in the same mode. This indicated that the LDLR site for FVIII is located within the LDLR cluster. Similar results were obtained for another ligand of LDLR, α-2-macroglobulin receptor-associated protein (RAP), a common ligand of receptors from the LDLR family. We further generated a set of recombinant fragments of the LDLR cluster and assessed their structural integrity by binding to RAP and by circular dichroism. A number of fragments overlapping CR.2-5 of the cluster were positive for binding RAP and FVIII. The specificity of these interactions was tested by site-directed mutagenesis of conserved tryptophans within the LDLR fragments. For FVIII, the specificity was also tested using a single-chain variable antibody fragment directed against the FVIII light chain as a competitor. Both cases resulted in decreased binding, thus confirming its specificity. The mutagenic study also showed an importance of the conserved tryptophans in LDLR for both ligands, and the competitive binding results showed an involvement of the light chain of FVIII in its interaction with LDLR. In conclusion, the region of CR.2-5 of LDLR was defined as the binding site for FVIII and RAP.

Inter-chain disulfide bond improved protein trans-splicing increases plasma coagulation activity in C57BL/6 mice following portal vein FVIII gene delivery by dual vectors

Protein trans-splicing based dual-vector factor VIII (FVIII) gene delivery is adversely affected by less efficiency of protein splicing. We sought to increase the amount of spliced FVIII protein and plasma coagulation activity in dual-vector FVIII transgene in mice by means of strengthening the interaction of inteins, protein splicing elements, thereby facilitating protein trans-splicing. Dual-vector delivery of the FVIII gene in cultured cells showed that replacement of Met226 in the heavy chain and Asp1828 in the light chain with Cys residues could facilitate inter-chain disulfide linking and improve protein trans-splicing, increasing the levels of spliced FVIII protein. In this study, C57BL/6 mice were coadministered dual vectors of intein-fused human FVIII heavy chain and light chain with Cys mutations via portal vein injection into the liver. Forty-eight hours post-injection, plasma was collected and analyzed for FVIII antigen concentration and coagulation activity. These mice showed increased circulating FVIII heavy chain polypeptide (442 ± 151 ng m(L-1) vs. 305 ± 103 ng mL(-1)) and coagulation activity (1.46 ± 0.37 IU m(-1) vs. 0.85 ± 0.23 IU mL(-1)) compared with control mice co-administered dual vectors expressing the heavy and light chains of wild-type FVIII. Moreover, coagulation activity was similar to that of mice receiving a single vector expressing FVIII (1.79 ± 0.59 IU mL(-1)). These findings indicate that improving protein trans-splicing by inter-chain disulfide bonding is a promising approach for increasing the efficacy of dual-vector based FVIII gene transfer.

Distinct Roles of Ser-764 and Lys-773 at the N Terminus of von Willebrand Factor in Complex Assembly with Coagulation Factor VIII

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.

Domain Organization of Membrane-Bound Factor VIII

Factor VIII (FVIII) is the blood coagulation protein which when defective or deficient causes for hemophilia A, a severe hereditary bleeding disorder. Activated FVIII (FVIIIa) is the co-factor to the serine protease Factor IXa (FIXa) within the membrane-bound Tenase complex, responsible for amplifying its proteolytic activity more than 100,000 times, necessary for normal blood clotting. FVIII is composed of two non-covalently linked peptide chains: a light chain holding the membrane interaction sites and a heavy chain holding the main FIXa interaction sites. The interplay between the light and heavy chains in the membrane-bound state is critical for FVIII biological efficiency. Here, we present our cryo-electron microscopy and structure analysis studies of human FVIII light chain (LC), as helically assembled onto negatively charged single lipid bilayer nanotubes (LNT). The resolved FVIII-LC membrane-bound structure at 20 Å, supports aspects of our previously proposed FVIII structure from membrane-bound two-dimensional (2D) crystals, such as only the C2 domain interacts directly with the membrane [Stoilova-McPhie 2002]. The light chain is oriented differently in the FVIII membrane-bound helical and 2D crystal structures based on electron microscopy data and the 3D structure solved by X-ray [Ngo 2008]. The flexibility of the FVIII-LC domain organization in different crystal packing (3D, 2D and helical) is essential to understand the FVIII membrane-bound organization and its significance for hemostasis. SSM acknowledge Professor Ed Egelman from the University of Virginia at Charlotseville for help and support with the IHRSR algorithm and helical analysis. This work is supported by a National Scientist Development grant from the American Heart Association: 10SDG3500034 and UTMB start up funds to SSM. The Cryo-EM and initial structure analysis was carried out at the NCMI facility, courtesy of Professor Wah Chiu, supported by a National Center for Research Resources grant P41RR02250.

Von Willebrand Factor-ADAMTS13 Interactions

Abstract SCI-18ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 repeats-13) is a plasma metalloenzyme that cleaves von Willebrand factor (vWF), thereby limiting excessive platelet adhesion and aggregation and attenuating the rate of thrombus formation at the site of injury. Severe deficiency of plasma ADAMTS13 activity results in thrombotic thrombocytopenic purpura (TTP), a potentially fatal syndrome, while mild to moderate deficiency of plasma ADAMTS13 activity is associated with an increased risk for myocardial infarction and stroke. Adamts13 null mice in the ApoE null background exhibit a dramatically increased systemic inflammation and early atherosclerosis. Thus, ADAMTS13 plays a crucial role in pathogenesis of arterial thrombotic disorders such as TTP and chronic inflammatory diseases such as atherosclerosis. vWF is the only known substrate for ADAMTS13 to date. Structure-function analysis demonstrates that the N-terminal portion of ADAMTS13 (MDTCS) is sufficient for proteolytic cleavage of vWF, while the C-terminal portion of ADAMTS13 (T2–8 and CUB) inhibits shear-dependent platelet aggregation via free thiols (-SH). The proteolytic cleavage of vWF by ADAMTS13 is dramatically accelerated by binding of Factor VIII (FVIII) and/or platelets under shear stress. A B-domainless FVIII (FVIII-SQ) or a light chain of FVIII (FVIII-LC) appears to be sufficient for the enhanced proteolysis of vWF by ADAMTS13 in vitro and in vivo using a murine model. In contrast, a heavy chain of FVIII (FVIII-HC) or a light chain lacking an acidic region (a3) (FVIII-LC#x2610;¢a3) exhibits no rate enhancing effect under the same conditions. These results suggest that the light chain, particularly the acidic (a3) region that contains a high-affinity vWF binding site is required for the cofactor activity in accelerating vWF proteolysis. Consistent with this hypothesis, mutations in the D'D3 domain of vWF naturally occurring in patients with type 2N von Willebrand disease that reduces FVIII binding also impair the FVIII-dependent proteolysis by ADAMTS13 under fluid shear stress. To develop novel therapeutics, we have constructed, expressed, and screened for a panel of ADAMTS13 variants with a modification of the exosites (motif A and motif B) in the spacer domain. We identified several ADAMTS13 variants that have enhanced specific activity and are also resistant to inhibition by autoantibodies against ADAMTS13 in patients with acquired idiopathic TTP. These gain-of-function and autoantibody-resistant ADAMTS13 variants may be further developed as potential therapeutics for acquired TTP with inhibitors. Disclosures:No relevant conflicts of interest to declare.

Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.

Management of bleeding disorders: basic science

Development of factor VIII (FVIII) inhibitors is the most severe and challenging complication of haemophilia A treatment and represents the highest economic burden for a chronic disease. Therefore, major research efforts are ongoing to optimize the therapeutic approaches able to minimize this complication. FVIII inhibitors have variable immuno-reactivity against different FVIII concentrates and generally have a lower reactivity against von Willebrand factor (VWF)-containing FVIII concentrates than plasma-derived FVIII (pdFVIII) or recombinant FVIII (rFVIII) that are devoid of VWF, in particular when the inhibitors are directed against the light chain of FVIII. This paper provides an overview of several in vitro and in vivo studies that compared three clinically available clinical FVIII products (Kogenate®, Bayer AG, Leverkusen, Germany; Advate®, Baxter Healthcare, Zurich, Switzerland; and Fanhdi®, Grifols S.A., Barcelona, Spain) in order to evaluate the functional actvity of the FVIII fractions in rFVIII that cannot bind VWF; explore the use of the thrombin generation assay (TGA) as a potential tool for optimizing the choice of FVIII concentrate for use in haemophilia A patients with inhibitors; compare the kinetics of the interactions between anti-FVIII antibodies and FVIII both in the presence/absence of VWF, using surface plasmon resonance.

P3–P3′ Residues Flanking Scissile Bonds in Factor VIII Modulate Rates of Substrate Cleavage and Procofactor Activation by Thrombin

Thrombin-catalyzed activation of factor VIII (FVIII) occurs through proteolysis at three P1 Arg residues: Arg(372) and Arg(740) in the FVIII heavy chain and Arg(1689) in the FVIII light chain. Cleavage at the latter two sites is relatively fast compared with cleavage at Arg(372), which appears to be rate-limiting. Examination of the P3-P3' residues flanking each P1 site revealed that those sequences at Arg(740) and Arg(1689) are more optimal for thrombin cleavage than at Arg(372), suggesting these sequences may impact reaction rates. Recombinant FVIII variants were prepared with mutations swapping scissile bond flanking sequences in the heavy chain individually and in combination with a second swap or with a P1 point mutation. Rates of generation of A1 and A3-C1-C2 subunits were determined by Western blotting and correlated with rates of cleavage at Arg(372) and Arg(1689), respectively. Rates of thrombin cleavage at Arg(372) were increased ~10- and ~3-fold compared with that of wild-type FVIII when it was replaced with P3-P3' residues flanking Arg(740) and Arg(1689), respectively, and these values paralleled increased rates of A2 subunit generation and procofactor activation. Positioning of more optimal residues flanking Arg(372) abrogated the need for initial cleavage at Arg(740) to facilitate this step. These results show marked changes in cleavage rates correlate with the extent of cleavage-optimal residues flanking the scissile bond and modulate the mechanism for procofactor activation.

Mass Spectrometry-assisted Study Reveals That Lysine Residues 1967 and 1968 Have Opposite Contribution to Stability of Activated Factor VIII

The A2 domain rapidly dissociates from activated factor VIII (FVIIIa) resulting in a dampening of the activity of the activated factor X-generating complex. The amino acid residues that affect A2 domain dissociation are therefore critical for FVIII cofactor function. We have now employed chemical footprinting in conjunction with mass spectrometry to identify lysine residues that contribute to the stability of activated FVIII. We hypothesized that lysine residues, which are buried in FVIII and surface-exposed in dissociated activated FVIII (dis-FVIIIa), may contribute to interdomain interactions. Mass spectrometry analysis revealed that residues Lys(1967) and Lys(1968) of region Thr(1964)-Tyr(1971) are buried in FVIII and exposed to the surface in dis-FVIIIa. This result, combined with the observation that the FVIII variant K1967I is associated with hemophilia A, suggests that these residues contribute to the stability of activated FVIII. Kinetic analysis revealed that the FVIII variants K1967A and K1967I exhibit an almost normal cofactor activity. However, these variants also showed an increased loss in cofactor activity over time compared with that of FVIII WT. Remarkably, the cofactor activity of a K1968A variant was enhanced and sustained for a prolonged time relative to that of FVIII WT. Surface plasmon resonance analysis demonstrated that A2 domain dissociation from activated FVIII was reduced for K1968A and enhanced for K1967A. In conclusion, mass spectrometry analysis combined with site-directed mutagenesis studies revealed that the lysine couple Lys(1967)-Lys(1968) within region Thr(1964)-Tyr(1971) has an opposite contribution to the stability of FVIIIa.

Factor VIII Can Be Synthesized in Hemophilia A Mice Liver by Bone Marrow Progenitor Cell-Derived Hepatocytes and Sinusoidal Endothelial Cells

Hemophilia A (HA) is caused by mutation in factor VIII (FVIII) gene in humans; it leads to inadequate synthesis of active protein. Liver is the primary site of FVIII synthesis; however, the specific cell types responsible for its synthesis remain controversial. We propose that the severity of the bleeding disorder could be ameliorated by partial replacement of mutated liver cells by healthy cells in HA mice. The aim of this investigation was to study the cellular origin of FVIII by examining bone marrow cell therapy for treatment of HA in mice. Recipient liver was perturbed with either acetaminophen or monocrotaline to facilitate the engraftment and differentiation of lineage-depleted (Lin(-)) enhanced green fluorescent protein-expressing bone marrow cells. Immunohistochemical analysis of liver tissue was conducted to identify the donor-derived cells that expressed FVIII. This identification was confirmed by transmission electron microscopy and quantitative gene expression analysis. The phenotypic correction in HA mice was determined by tail-clip challenge and FVIII level in plasma by Chromogenix and activated partial thromboplastin time assays. Immunohistochemical analysis showed that von Willebrand factor and cytokeratin-18-expressing endothelial cells and hepatocytes, respectively, were obtained from BM-derived cells. Both cell types expressed FVIII light chain mRNA and protein, which was further confirmed by transmission electron microscopy. The transplanted HA mice showed FVIII activity in plasma (P<0.01) and survived tail-clip challenge (P<0.001). Thus, we conclude that BM-derived hepatocytes and endothelial cells can synthesize FVIII in liver and correct bleeding phenotype in HA mice.

Reconstitution of Recombinant Factor VIII In fVIII-/- mice Restores Von Willebrand Factor Homeostasis

AbstractAbstract 2213Coagulation factor VIII (FVIII) binds von Willebrand factor (VWF) at the D′D3 domain with high affinity. FVIII almost always circulates as a FVIII-VWF complex in blood. Our recent study has demonstrated that FVIII accelerates proteolytic cleavage of VWF by ADAMTS13 under mechanically induced fluid shear stress (Cao et al, PNAS, 2008). In this study, we sought to determine: 1) the domain of FVIII required for the enhancing effect on VWF proteolysis by ADAMTS13 under these conditions; 2) the physiological relevance of this enhancing effect on plasma VWF homeostasis in a murine model. First, we employed the novel vortex-based shear assay developed in the laboratory to assess the rate enhancing effect of various recombinant FVIII variants on VWF proteolysis by ADAMTS13. In these experiments, purified plasma VWF (150 nM) was incubated for 10 min with recombinant ADAMTS13 (50 nM) in the presence of various concentrations of recombinant FVIII variants (0-20 nM) in a total volume of 20 μl under constant fluid shear generated with a bench top mini-vortexer. This maneuver appears to produce ∼75 dyn/cm2 of fluid shear stress. Proteolytic cleavage product (∼350 kDa, the dimer of two C-terminal fragments linked by a disulfide bond) was then determined by 5% SDS-polyacrylamide gel electrophoresis and Western blotting. We showed that addition of a recombinant light chain of FVIII (FVIII-LC) increased the formation of proteolytic cleavage product as a function of increasing FVIII-LC concentrations. This rate enhancing effect was similar to that of full-length recombinant FVIII and B-domainless FVIII (FVIII-SQ). However, addition of a heavy chain of FVIII (FVIII-HC) or a light chain variant lacking the acidic (a3) region (FVIII-Δa3LC) did not increase the formation of cleavage product under the same conditions. These results suggest that FVIII-light chain is sufficient for accelerating VWF proteolysis by ADAMTS13 under physiological conditions. The rate enhancing effect of FVIII-LC depends on its high affinity interaction with VWF. Second, we determined plasma VWF antigen and multimer distribution in fVIII-/- and fVIII+/+ (WT) mice with same genetic background (C57BL/6) prior to and after reconstitution with recombinant FVIII-SQ and variants via a hydrodynamic approach. Plasma VWF antigen was determined by a sandwich ELISA assay and plasma VWF multimers were determined by 1% agarose gel electrophoresis and Western blotting. We showed that plasma VWF antigen levels in the fVIII-/- mice (n=18) were increased by ∼2.0 fold as compared with those in the WT mice (n=14). No difference in the ratio of ultra large (UL)-VWF to dimer was observed between the fVIII-/- mice and the WT mice (p>0.05). These data suggest that lack of FVIII may impair plasma VWF homeostasis. To assess whether plasma FVIII affects VWF proteolysis in vivo, plasma VWF multimer distribution was determined by agarose (1%) gel electrophoresis and Western blotting in the fVIII-/- mice 48 hours after injection of a series of endotoxin-free expression plasmids encoding various FVIII variants/fragments (i.e. plasmids diluted in 2 ml normal saline and injected into tail vein within 5 seconds). We showed that the ratio of the UL-VWF multimers to the dimer in plasma of fVIII-/- mice receiving normal saline alone was 1.70 ± 0.59 (means ± SD) (n=10). However, the ratios in plasma of fVIII-/- mice receiving plasmids encoding canine FVIII-SQ, human FVIII-SQ, human FVIII-HC+LC, and FVIII-LC were 0.44 ± 0.37 (n=20), 0.88 ± 0.18 (n=9), 0.40 ± 0.20 (n=5), and 0.97 ± 0.29 (n=9), respectively. These ratios were dramatically reduced compared with that in fVIII-/- mice receiving normal saline alone (p values<0.05∼0.001). Our hydrodynamic injection approach resulted in plasma levels of FVIII-SQ, FVIII-LC+HC and FVIII-LC between 150% and 200% of the WT. In contrast, a hydrodynamic injection of plasmid encoding human FVIII-HC or FVIII-Δa3LC did not prevent the accumulation of plasma UL-VWF multimers in fVIII-/- mice. We therefore conclude that FVIII, through the high affinity binding interaction between its light chain and D′D3 domain of VWF, may play a critical role in maintaining VWF homeostasis under (patho) physiological conditions. Disclosures:No relevant conflicts of interest to declare.

Domain specificity of factor VIII inhibitors during immune tolerance induction in patients with haemophilia A

Frequent administration of high dosages factor VIII (FVIII), so-called immune tolerance induction (ITI), provides an efficient strategy to eradicate inhibitory antibodies in patients with haemophilia A. At present, our knowledge on the characteristics of inhibitory antibodies in patients undergoing ITI is limited. In this study we characterized the domain specificity of FVIII inhibitors in 11 haemophilia A patients during ITI. In three of six patients who were successfully tolerized, inhibitory antibodies were directed predominantly against the FVIII light chain. In two other patients within this group, a significant contribution of A2 antibodies was observed which did not change during treatment. In the sixth patient the relative contribution of A2 inhibitors declined which coincided with an increase in antilight chain antibodies. In four of five patients who failed ITI, A2 inhibitors were observed. In two patients the contribution of A2 inhibitors increased during treatment, while in two other patients the contribution of A2 inhibitor remained constant. The fifth patient had inhibitory antibodies predominantly directed against the FVIII light chain. Overall, our findings revealed changes in domain specificity of FVIII antibodies in five of 11 patients analysed. Remarkably, antibodies exclusively directed towards the light chain of FVIII were predominantly observed in patients who were successfully tolerized.