Structural Studies of Blood Coagulation Factor VIII in Protein Complexes

A deficiency in blood coagulation factor VIII (fVIII) is responsible for the inherited bleeding disorder hemophilia A, which affects approximately 1 in 5000 males. The development of inhibitory antibodies is a significant issue faced by hemophilia A patients receiving therapeutic infusions of fVIII. The C-terminal C2 domain of fVIII has been shown to be highly immunogenic and the site of binding for numerous antibodies of both the classical and non-classical classifications. A detailed understanding of the structural components involved in C2-antibody binding interactions is vital for the development of improved therapeutics for hemophilia patients. Here we present the structure of the classical antibody 3E6 bound to human C2 at 2.6 Å resolution. Previous studies have suggested that pairs of classical and non-classical antibodies may exhibit binding cooperativity. Comparison of the C2:3E6 structure with a previously determined ternary structure of C2 bound simultaneously to both 3E6 and the non-classical antibody G99 reveals that cooperativity is not the result of dramatic conformational changes at the binding interface. Rather, changes in B-factor ratios between the C2 complexes and the isolated C2 domain suggest that dynamic changes upon 3E6 binding may lead to more favorable binding of a second antibody on the protein’s opposite face. Studies of the fVIII C2 domain were then extended to attempts to understand the structural basis for the interactions between fVIII and its circulatory partner von Willebrand factor (vWF), which is crucial for maintaining fVIII levels in plasma. The inability for vWF to bind fVIII is the basis for another blood coagulation disorder, von Willebrand’s disease type 2N, which can lead to hemophilia-type levels of fVIII in the blood and subsequent bleeding episodes. Previous publications have suggested a role for C2 in the binding of vWF. Attempts were made to formulate protein complexes between two vWF domain mutants, TIL\u27E\u27 and D\u27D3, and the human C2 domain, but were ultimately unsuccessful. This outcome corroborates recent publications suggesting that the fVIII C1 domain may play a more active role in binding vWF than the C2 domain. In order to further study this possibility, complexes were formed between recombinant B-domain deleted fVIII and TIL’E’ or D’D3 and X-ray crystal trials initiated. Hopefully, greater understanding of the structural interactions between fVIII and other proteins will lead to improved therapies for patients suffering from fVIII-related diseases

A Novel Depletion Technique for Studying the Role of Protein L12 in the Activation of Ribosome-Dependent GTPases

The ribosome is a complex macromolecular machine that is responsible for the synthesis of proteins from a nucleic acid template. This process is largely regulated by various protein translation factors, many of which are GTPases. Ribosome-dependent GTPase activity has been observed to be coincident with the presence of ribosomal protein L12. Of current interest is to understand how L12 interacts with the GTPase factors on the 705 ribosome. A key to the investigation of these interactions is to produce ribosomes fully depleted of L12 for comparisons of factor activity and binding in the presence and absence of this protein. Here, we present a novel two-step depletion protocol that takes advantage of the JE28 ribosomes\u27 engineered C-terminal (His)G-tag chromosomally encoded on protein L12. Fully depleted ribosomes were shown to be absent of L12 in Western blotting studies. Furthermore, these 705 ribosomes were shown not to stimulate ribosome-dependent GTP hydrolysis by translation factor EF-G in malachite green GTP hydrolysis assays. This population of ribosomes purified in the complete absence of protein L12 will make possible investigations of factor binding and ribosome-dependent GTP hydrolysis to further elucidate the role of L12 in translation

Structure of the Human Factor VIII C2 Domain in Complex with the 3E6 Inhibitory Antibody.

Blood coagulation factor VIII is a glycoprotein cofactor that is essential for the intrinsic pathway of the blood coagulation cascade. Inhibitory antibodies arise either spontaneously or in response to therapeutic infusion of functional factor VIII into hemophilia A patients, many of which are specific to the factor VIII C2 domain. The immune response is largely parsed into “classical” and “non-classical” inhibitory antibodies, which bind to opposing faces cooperatively. In this study, the 2.61Å resolution structure of the C2 domain in complex with the antigen-binding fragment of the 3E6 classical inhibitory antibody is reported. The binding interface is largely conserved when aligned with the previously determined structure of the C2 domain in complex with two antibodies simultaneously. Further inspection of the B factors for the C2 domain in various X-ray crystal structures indicates that 3E6 antibody binding decreases the thermal motion behavior of surface loops in the C2 domain on the opposing face, thereby suggesting that cooperative antibody binding is a dynamic effect. Understanding the structural nature of the immune response to factor VIII following hemophilia A treatment will help lead to the development of better therapeutic reagents

The 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain and binding analysis to anti-human C2 domain antibodies and phospholipid surfaces.

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the "non-classical" antibody epitopes. Furthermore, antibody-binding results illustrate that the "classical" 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed

The 1.7 Å X-Ray Crystal Structure of the Porcine Factor VIII C2 Domain and Binding Analysis to Anti-Human C2 Domain Antibodies and Phospholipid Surfaces

The factor VIII C2 domain is essential for binding to activated platelet surfaces as well as the cofactor activity of factor VIII in blood coagulation. Inhibitory antibodies against the C2 domain commonly develop following factor VIII replacement therapy for hemophilia A patients, or they may spontaneously arise in cases of acquired hemophilia. Porcine factor VIII is an effective therapeutic for hemophilia patients with inhibitor due to its low cross-reactivity; however, the molecular basis for this behavior is poorly understood. In this study, the X-ray crystal structure of the porcine factor VIII C2 domain was determined, and superposition of the human and porcine C2 domains demonstrates that most surface-exposed differences cluster on the face harboring the “non-classical” antibody epitopes. Furthermore, antibody-binding results illustrate that the “classical” 3E6 antibody can bind both the human and porcine C2 domains, although the inhibitory titer to human factor VIII is 41 Bethesda Units (BU)/mg IgG versus 0.8 BU/mg IgG to porcine factor VIII, while the non-classical G99 antibody does not bind to the porcine C2 domain nor inhibit porcine factor VIII activity. Further structural analysis of differences between the electrostatic surface potentials suggest that the C2 domain binds to the negatively charged phospholipid surfaces of activated platelets primarily through the 3E6 epitope region. In contrast, the G99 face, which contains residue 2227, should be distal to the membrane surface. Phospholipid binding assays indicate that both porcine and human factor VIII C2 domains bind with comparable affinities, and the human K2227A and K2227E mutants bind to phospholipid surfaces with similar affinities as well. Lastly, the G99 IgG bound to PS-immobilized factor VIII C2 domain with an apparent dissociation constant of 15.5 nM, whereas 3E6 antibody binding to PS-bound C2 domain was not observed

A New Era in the Ethics of Human Embryonic Stem Cell Research

Scientific progress in human embryonic stem cell (hESC) research and increased funding make it imperative to look ahead to the ethical issues generated by the expected use of hESCs for transplantation. Several issues should be addressed now, even though phase I clinical trials of hESC transplantation are still in the future. To minimize the risk of hESC transplantation, donors of materials used to derive hESC lines will need to be recontacted to update their medical history and screening. Because of privacy concerns, such recontact needs to be discussed and agreed to at the time of donation, before new hESC lines are derived. Informed consent for phase I clinical trials of hESC transplantation also raises ethical concerns. In previous phase I trials of highly innovative interventions, allegations that trial participants had not really understood the risk and benefits caused delays in subsequent trials. Thus, researchers should consider what information needs to be discussed during the consent process for hESC clinical trials and how to verify that participants have a realistic understanding of the study. Lack of attention to the special ethical concerns raised by clinical trials of hESC transplantation and their implications for the derivation of new hESC lines may undermine or delay progress toward stem cell therapies

ELISA of human and porcine C2/mAb interactions.

<p>Binding of human (open circles) and porcine (closed squares) His₆-C2 domain to immobilized (A) 3E6 mAb and (B) G99 mAb. Bound His₆-C2 was detected with Ni-NTA-alkaline phosphatase. (C) Bethesda assay for 3E6 and G99 with human or porcine BDD fVIII.</p

PS membrane and PS-bound fVIII C2 ELISA results for factor VIII C2 domain variants and inhibitory antibodies.

<p>(A) Comparison of human (open circles) and porcine (closed squares) factor VIII C2 domains binding to PS membrane surfaces. (B) Comparison of human factor VIII C2 domain (open circles) with the human K2227A C2 domain mutant (closed squares). (C) Comparison of human factor VIII C2 domain (open circles) with the human K2227E C2 domain mutant (closed squares). (D) Binding of G99 (open circles) and 3E6 (closed circles) to PS-bound fVIII C2 domain. Bound His₆-C2 was detected with Ni-NTA-alkaline phosphatase, and bound IgG was detected with a AP-conjugated goat anti-mouse mAb.</p

X-ray crystal structure of the porcine factor VIII C2 domain.

<p>(A) Ribbon diagram presentation of the 1.7 Å X-ray crystal structure. Displayed residues are solvent-exposed hydrophobic and basic residues proposed to interact with platelet surfaces. (B) Superposition of the human (pdb#: 1D7P, magenta) and porcine (pdb#: 4MO3, green) factor VIII C2 domain X-ray crystal structures. (3) Sequence alignment of human and porcine factor VIII C2 domains. Highlighted residues represent sequence differences (orange: 3E6 mAb binding region, blue: G99 mAb binding region, red: BO2C11 mAb binding region, cyan: G99 and BO2C11 binding region).</p

Structural representation of sequence differences proximal to inhibitory antibody epitopes.

<p>(A) The 3E6 mAb epitope face. (B) The G99 mAb epitope face. Proximal residues are defined as <5 Å C2 domain/mAb intermolecular distances (orange: 3E6 mAb binding region, blue: G99 mAb binding region, red: BO2C11 mAb binding region, cyan: G99 and BO2C11 binding region, green: >5 Å from all mAbs)</p