Kathleen P. Pratt

Crystal structure of a 30 kDa C-terminal fragment from the γ chain of human fibrinogen

Abstract Background: Blood coagulation occurs by a cascade of zymogen activation resulting from minor proteolysis. The final stage of coagulation involves thrombin generation and limited proteolysis of fibrinogen to give spontaneously polymerizing fibrin. The resulting fibrin network is covalently crosslinked by factor XIIIa to yield a stable blood clot. Fibrinogen is a 340 kDa glycoprotein composed of six polypeptide chains, ( α β γ ) 2 , held together by 29 disulfide bonds. The globular C terminus of the γ chain contains a fibrin-polymerization surface, the principal factor XIIIa crosslinking site, the platelet receptor recognition site, and a calcium-binding site. Structural information on this domain should thus prove helpful in understanding clot formation. Results: The X-ray crystallographic structure of the 30 kDa globular C terminus of the γ chain of human fibrinogen has been determined in one crystal form using multiple isomorphous replacement methods. The refined coordinates were used to solve the structure in two more crystal forms by molecular replacement; the crystal structures have been refined against diffraction data to either 2.5 A or 2.1 A resolution. Three domains were identified in the structure, including a C-terminal fibrin-polymerization domain (P), which contains a single calcium-binding site and a deep binding pocket that provides the polymerization surface. The overall structure has a pronounced dipole moment, and the C-terminal residues appear highly flexible. Conclusions: The polymerization domain in the γ chain is the most variable among a family of fibrinogen-related proteins and contains many acidic residues. These residues contribute to the molecular dipole moment in the structure, which may allow electrostatic steering to guide the alignment of fibrin monomers during the polymerization process. The flexibility of the C-terminal residues, which contain one of the factor XIIIa crosslinking sites and the platelet receptor recognition site, may be important in the function of this domain.

T‐cell responses over time in a mild hemophilia A inhibitor subject: epitope identification and transient immunogenicity of the corresponding self‐peptide

Background: Antibodies that neutralize factor (F) VIII activity, clinically referred to as ‘inhibitors’, complicate the treatment of hemophilia A patients; current tolerance and bypass strategies are extremely costly and sometimes ineffective. The development of inhibitors requires T‐cell help. Objectives: We characterized T‐cell responses of a subject with mild hemophilia A with missense genotype A2201P for one year following his initial inhibitor response, with the goals of defining the primary epitope(s) and its (their) MHC Class II restriction. We investigated the possible involvement of regulatory T cells in modulating immune responses.Patients/methods: The subject developed high‐titer FVIII‐neutralizing antibodies (250 BU mL−1) that declined over time to 8 BU ml−1. His clotting activity was initially impaired (3%) but returned to baseline (8–10%) within four weeks. MHC Class II tetramers were used to analyze his CD4 T cells, which were stimulated with peptides spanning the C2 domain. Responses of total and CD25‐depleted CD4 cells to sequences containing A2201 (native), P2201 (hemophilic), and other predicted T‐cell epitopes were evaluated. Results and conclusions: An HLA‐DRA‐DRB1*0101 restricted T‐cell epitope containing the wild‐type A2201 sequence was identified. Interestingly, peptides containing A2201 were recognized by CD4 T cells at all time points, whereas a P2201 peptide was recognized only near the initial peak response. The responsiveness of CD25‐depleted CD4 cells to an A2201 peptide was enhanced 11 and 19 weeks following inhibitor detection, suggesting the possible involvement of CD4+CD25+ regulatory T cells in modulating immune responses. Patient‐derived T‐cell clones proliferated in response to C2 protein and to peptides containing A2201 but not P2201.

Lineages of human T-cell clones, including T helper 17/T helper 1 cells, isolated at different stages of anti-factor VIII immune responses

The development of neutralizing antibodies (inhibitors) after factor VIII (FVIII) infusions is a serious complication that affects approximately one-quarter of hemophilia A patients who have access to replacement therapy. To investigate the differentiation of naive T cells into FVIII-specific helper T cells that promote B-cell activation and antibody secretion, HLA-DRA-DRB1*0101-restricted T-cell clones that respond to a specific epitope in FVIII were isolated from a mild hemophilia A subject (the proband) 19 weeks and 21 months after his development of a high-titer inhibitor. Clones responding to the same epitope were also isolated from his multiply infused brother, who has not developed a clinically significant inhibitor. The 19-week proband clones were T helper (T(H))17/T(H)1- or T(H)1/T(H)2-polarized, whereas all 8 clones isolated 21 months postinhibitor development were T(H)2-polarized cells. In contrast, all 6 clones from the brother who did not develop an inhibitor were T(H)1-polarized, indicating that tolerance to FVIII can be maintained even with circulating T(H)1-polarized cells that respond vigorously to in vitro FVIII stimulation. This is the first evidence that T(H)17/T(H)1-polarized cells play a role in hemophilic immune responses to FVIII. Furthermore, this is the first report of successful isolation and expansion of antigen-specific human T(H)17/T(H)1 clones using standard culture conditions.

Induction of partial immune tolerance to factor VIII through prior mucosal exposure to the factor VIII C2 domain

Summary.  Background: The development of anti‐factor VIII (FVIII) neutralizing antibodies (inhibitors) is a significant obstacle to FVIII replacement therapy. Objective: As mucosal administration of an antigen may induce immune tolerance we have evaluated the efficacy of mucosal antigen exposure to achieve tolerance to FVIII. Methods: We investigated the effects of oral and nasal administration of the purified FVIII C2 domain (FVIII‐C2) to FVIII‐deficient BALB/c mice prior to FVIII protein challenge. Mice received oral or nasal doses of FVIII‐C2, followed by a subcutaneous challenge of either FVIII‐C2 or FVIII. The development of anti‐FVIII inhibitors, cytokine production by splenocytes in vitro, and adoptive transfer assays were analyzed. Results and Conclusions: Mucosal administration of FVIII‐C2 decreases the titer of anti‐FVIII‐C2 inhibitors after FVIII‐C2 challenge, and decreases the percentage of FVIII‐C2 specific antibodies after challenge with full‐length FVIII. Tolerance induction to FVIII‐C2 is associated with increased IL‐10 production by splenocytes in vitro, and can be adoptively transferred to naïve mice. This study is the first to demonstrate that tolerance to the FVIII‐C2 domain can be induced via the mucosal route. Based on these results, the potential use of FVIII‐specific mucosal tolerance induction as an immunotherapy treatment for anti‐FVIII inhibitor development warrants further investigation.

ELISA system for detection of immune responses to FVIII: a study of 246 samples and correlation with the Bethesda assay.

Summary.  Inhibitors of FVIII are usually IgG polyclonal antibodies that develop as alloimmune responses in patients with congenital haemophilia A or as autoimmune responses resulting in acquired haemophilia. Their recognition can be difficult, especially when the titre is low. Furthermore, results from a Bethesda assay often require several days as samples are referred to a specialty laboratory. The aim of this study is to assess the utility of an ELISA system for detecting immune responses to FVIII. A total of 246 plasma samples submitted from 176 individuals with immune responses to FVIII, as verified with the Bethesda assay, and samples from 50 control subjects were tested for the presence of FVIII‐specific IgG using an ELISA‐based assay. Paired sera from 18 of the patients were also tested by the ELISA. Of the 246 samples that were positive for a FVIII inhibitor by the Bethesda assay, 235 (95.5%) were also positive by ELISA. The regression coefficient, using Log BU was r = 0.82. The correlation data were strengthened when 27 inhibitor samples were diluted further. There was a strong correlation between ELISA results for the 18‐paired serum and plasma samples (r = 0.99). There is a strong correlation between the ELISA and Bethesda methods in detecting immune responses to FVIII. The ELISA provides rapid screening that could be available well in advance of confirmation by the Bethesda assay.

HLA-DR-restricted T-cell responses to Factor VIII epitopes in a mild haemophilia A family with missense substitution A2201P

Summary.  An HLA‐DRA‐DRB1*0101‐restricted T‐cell epitope in the factor VIII (FVIII) C2 domain occurred in a mild haemophilia A patient with missense substitution FVIII‐A2201P. His T cells responded to synthetic peptides FVIII2186–2205 and FVIII2194–2213 (J Thromb Haemost 2007; 5: 2399). T cells from family members with genotype FVIII‐A2201P were analysed to determine if FVIII‐specific T cells occur in individuals with a haemophilic mutation but no clinically significant inhibitor response. Fluorescent MHC class II tetramers corresponding to subjects’HLA‐DRB1 types were loaded with 20‐mer peptides and utilized to label antigen‐specific CD4+ T cells. T‐cell responses to peptides spanning the FVIII‐C2 sequence were evaluated. T cells recognizing specific peptides were cloned, and antigen specificity was verified by proliferation assays. Plasma and/or purified IgG samples were tested for FVIII inhibitory activity. CD4+ T cells and T‐cell clones from two brothers who shared the DRB1*0101 allele responded to FVIII2194–2213. A haemophilic cousin’s HLA‐DRA‐DRB1*1104‐restricted response to FVIII2202–2221 was detected only when CD4+CD25+ cells were depleted. A great uncle and two obligate carriers had no detectable FVIII‐C2‐specific T cells. Concentrated IgG from the brother without a clinical inhibitor response showed a low‐titre FVIII inhibitor. FVIII‐specific T cells and inhibitory IgG were found in a previously infused, haemophilic subject who had a sub‐clinical FVIII inhibitor. CD4+CD25+ depleted T cells from a non‐infused haemophilic cousin recognized an overlapping FVIII epitope, indicating a latent HLA‐DRA‐DRB1*1104‐restricted T‐cell response to FVIII. Specific T‐cell responses to FVIII can occur without clinically significant inhibitors.

Dangerous liaisons: how the immune system deals with factor VIII

Summary.  Only a fraction of patients with hemophilia A develop a neutralizing antibody (inhibitor) response to therapeutic infusions of factor VIII. Our present understanding of the underlying causes of the immunogenicity of this protein is limited. In the past few years, insights into the uptake and processing of FVIII by antigen‐presenting cells (APCs) have expanded significantly. Although the mechanism of endocytosis remains unclear, current data indicate that FVIII enters APCs via its C1 domain. Its subsequent processing within endolysosomes allows for presentation of a heterogeneous collection of FVIII‐derived peptides on major histocompatibility complex (MHC) class II, and this peptide–MHC class II complex may then be recognized by cognate effector CD4+ T cells, leading to anti‐FVIII antibody production. Here we aim to summarize recent knowledge gained about FVIII processing and presentation by APCs, as well as the diversity of the FVIII‐specific T‐cell repertoire in mice and humans. Moreover, we discuss possible factors that can drive FVIII immunogenicity. We believe that increasing understanding of the immune recognition of FVIII and the cellular mechanisms of anti‐FVIII antibody production will lead to novel therapeutic approaches to prevent inhibitor formation in patients with hemophilia A.

T-cell responses in two unrelated hemophilia A inhibitor subjects include an epitope at the factor VIII R593C missense site

Summary.  Background: Development of neutralizing anti‐factor (F)VIII antibodies (‘inhibitors’) is a serious clinical problem in hemophilia A. Increased inhibitor risk has been associated with certain FVIII missense substitutions, including R593C in the A2 domain. Objectives: The aim of the present study was to identify T‐cell epitopes in FVIII and characterize T‐cell responses in two unrelated hemophilia A subjects sharing F8‐R593C and HLA‐DRB1*1101 genotypes. We hypothesized that the hemophilic substitution site coincides with an important T‐cell epitope. Patients/methods: The binding affinities of peptides for recombinant HLA‐DR proteins were measured and compared with epitope prediction results. CD4+ T cells were stimulated using peptides and stained with fluorescent, peptide‐loaded tetramers. Results: The inhibitor subjects, but not HLA‐matched controls, had high‐avidity HLA‐DRB1*1101‐restricted T‐cell responses against FVIII589–608, which contains the hemophilic missense site. Antigen‐specific T cells secreted Th1 and Th2 cytokines and proliferated in response to FVIII and FVIII592–603. FVIII589–608 bound with physiologically relevant (micromolar) IC50 values to recombinant DR0101, DR1101 and DR1501 proteins. Conclusions: Hemophilia A patients with R593C missense substitutions and these HLA haplotypes had an increased incidence of inhibitors in our cohorts, supporting a paradigm in which presentation of FVIII epitopes containing the wild‐type R593 influences inhibitor risk in this hemophilia A sub‐population.

B-Cell and T-Cell Epitopes in Anti-factor VIII Immune Responses

Adequate hemostasis is achieved for many hemophilia A patients by infusion of plasma-derived or recombinant factor VIII (FVIII), but unfortunately, a significant subset of patients develop an immune response in which anti-FVIII antibodies, referred to clinically as “inhibitors,” interfere with its procoagulant activity. Inhibitors are the subset of anti-FVIII antibodies that bind to surfaces on FVIII (B-cell epitopes) that are important for its proper functioning in coagulation. Less antigenic FVIII molecules may be designed by identifying and then modifying the amino acid sequences of inhibitor B-cell epitopes. Conversely, characterization of these epitopes can yield important information regarding functionally important surfaces on FVIII. The production of inhibitor antibodies is driven by T cells. T cells recognize FVIII as foreign when FVIII-derived peptides bind to major histocompatibility complex (MHC) class II molecules on the surface of antigen-presenting cells. The class II–peptide complexes must then be recognized by T-cell receptors (TCRs). T-cell stimulation requires sustained association of antigen-presenting cells and T cells through formation of a class II–peptide–TCR complex, and peptide sequences that mediate this association are termed “T-cell epitopes.” MHC class II tetramers that bind FVIII-derived peptides and recognize antigen-specific TCRs are proving useful in the characterization of human leukocyte antigen-restricted T-cell responses to FVIII.

Six Amino Acid Residues in a 1200 Å2 Interface Mediate Binding of Factor VIII to an IgG4κ Inhibitory Antibody

The development of neutralizing anti-factor VIII (FVIII) antibodies complicates the treatment of many hemophilia A patients. The C-terminal C2 domain is a particularly antigenic FVIII region. A crystal structure of recombinant FVIII-C2 bound to an Fab fragment of the patient-derived monoclonal antibody BO2C11, which recognizes an immunodominant inhibitor epitope on FVIII and blocks its ability to bind von Willebrand factor (VWF) and phospholipids, revealed that 15 amino acids in FVIII contact this antibody. Forty-three recombinant FVIII-C2 proteins, each with a surface-exposed side chain mutated to alanine or another residue, were generated, and surface plasmon resonance studies were carried out to evaluate effects of these substitutions on BO2C11/FVIII-C2 binding affinity. Thermodynamic analysis of experiments carried out at three temperatures indicated that one beta hairpin turn at the antigen-antibody interface (FVIII-F2196, N2198, M2199 and F2200) plus two non-contiguous arginines (FVIII-R2215 and R2220), contributed appreciably to the affinity. B-domain-deleted (BDD) FVIII-F2196A, FVIII-F2196K and FVIII-M2199A were generated and characterized. Their pro-coagulant activities and binding to VWF were similar to those of WT-BDD-FVIII, and FVIII-F2196K avoided neutralization by BO2C11 and murine inhibitory mAb 1B5. This study suggests specific sites for amino acid substitutions to rationally design FVIII variants capable of evading immunodominant neutralizing anti-FVIII antibodies.