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Title: Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools

Authors:
; ; ; ; ; ; ; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1434379
Resource Type:
Journal Article: Published Article
Journal Name:
Science Translational Medicine
Additional Journal Information:
Journal Volume: 9; Journal Issue: 372; Related Information: CHORUS Timestamp: 2018-01-12 15:49:37; Journal ID: ISSN 1946-6234
Publisher:
American Association for the Advancement of Science (AAAS)
Country of Publication:
United States
Language:
English

Citation Formats

Lamberth, Kasper, Reedtz-Runge, Stine Louise, Simon, Jonathan, Klementyeva, Ksenia, Pandey, Gouri Shankar, Padkjær, Søren Berg, Pascal, Véronique, León, Ileana R., Gudme, Charlotte Nini, Buus, Søren, and Sauna, Zuben E. Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools. United States: N. p., 2017. Web. doi:10.1126/scitranslmed.aag1286.
Lamberth, Kasper, Reedtz-Runge, Stine Louise, Simon, Jonathan, Klementyeva, Ksenia, Pandey, Gouri Shankar, Padkjær, Søren Berg, Pascal, Véronique, León, Ileana R., Gudme, Charlotte Nini, Buus, Søren, & Sauna, Zuben E. Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools. United States. doi:10.1126/scitranslmed.aag1286.
Lamberth, Kasper, Reedtz-Runge, Stine Louise, Simon, Jonathan, Klementyeva, Ksenia, Pandey, Gouri Shankar, Padkjær, Søren Berg, Pascal, Véronique, León, Ileana R., Gudme, Charlotte Nini, Buus, Søren, and Sauna, Zuben E. Wed . "Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools". United States. doi:10.1126/scitranslmed.aag1286.
@article{osti_1434379,
title = {Post hoc assessment of the immunogenicity of bioengineered factor VIIa demonstrates the use of preclinical tools},
author = {Lamberth, Kasper and Reedtz-Runge, Stine Louise and Simon, Jonathan and Klementyeva, Ksenia and Pandey, Gouri Shankar and Padkjær, Søren Berg and Pascal, Véronique and León, Ileana R. and Gudme, Charlotte Nini and Buus, Søren and Sauna, Zuben E.},
abstractNote = {},
doi = {10.1126/scitranslmed.aag1286},
journal = {Science Translational Medicine},
number = 372,
volume = 9,
place = {United States},
year = {Wed Jan 11 00:00:00 EST 2017},
month = {Wed Jan 11 00:00:00 EST 2017}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1126/scitranslmed.aag1286

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  • Factor VIIa (FVIIa) consists of a {gamma}-carboxyglutamic acid (Gla) domain, two epidermal growth factor-like domains, and a protease domain. FVIIa binds seven Ca{sup 2+} ions in the Gla, one in the EGF1, and one in the protease domain. However, blood contains both Ca{sup 2+} and Mg{sup 2+}, and the Ca{sup 2+} sites in FVIIa that could be specifically occupied by Mg{sup 2+} are unknown. Furthermore, FVIIa contains a Na{sup +} and two Zn{sup 2+} sites, but ligands for these cations are undefined. We obtained p-aminobenzamidine-VIIa/soluble tissue factor (sTF) crystals under conditions containing Ca{sup 2+}, Mg{sup 2+}, Na{sup +}, and Zn{supmore » 2+}. The crystal diffracted to 1.8{angstrom} resolution, and the final structure has an R-factor of 19.8%. In this structure, the Gla domain has four Ca{sup 2+} and three bound Mg{sup 2+}. The EGF1 domain contains one Ca{sup 2+} site, and the protease domain contains one Ca{sup 2+}, one Na{sup +}, and two Zn{sup 2+} sites. {sup 45}Ca{sup 2+} binding in the presence/absence of Mg{sup 2+} to FVIIa, Gla-domainless FVIIa, and prothrombin fragment 1 supports the crystal data. Furthermore, unlike in other serine proteases, the amide N of Gly{sup 193} in FVIIa points away from the oxyanion hole in this structure. Importantly, the oxyanion hole is also absent in the benzamidine-FVIIa/sTF structure at 1.87{angstrom} resolution. However, soaking benzamidine-FVIIa/sTF crystals with D-Phe-Pro-Arg-chloromethyl ketone results in benzamidine displacement, D-Phe-Pro-Arg incorporation, and oxyanion hole formation by a flip of the 192-193 peptide bond in FVIIa. Thus, it is the substrate and not the TF binding that induces oxyanion hole formation and functional active site geometry in FVIIa. Absence of oxyanion hole is unusual and has biologic implications for FVIIa macromolecular substrate specificity and catalysis.« less
  • An umbilical vein model was designed in which washed vein segments are filled with a reaction mixture containing factor VIIa, Ca(+)+, and a substrate, either 3H-factor IX or 3H-factor X. The vein wall provides the tissue factor (TF) for factor VIIa/TF complexes that activate the substrates as measured by activation peptide release. The model was developed to study TF induced on venous endothelium in situ. However, unlike previous studies with TF expressed on cultured umbilical vein endothelial cells, factors IX and X were activated without first having to expose the vein wall to a perturbing stimulus. Histologic studies revealed thatmore » washing the vein and mixing the reaction mixture before subsampling had disrupted the endothelium. Immunostaining with anti-TF antibodies revealed no staining of endothelium but intense staining in extensions of Wharton's jelly penetrating fenestrations of the muscularis media of the vein. Thus, the model provided data on factor VIIa/TF formed, not on endothelium, but within the mucoid connective tissue of Wharton's jelly. It is known that factor VIIa/TF formed with TF in suspension or with TF expressed on the surface of cultured cells activates factor X more rapidly than factor IX. In contrast, in the umbilical vein model, when each substrate was present in an 88 nmol/L concentration, factors IX and X were activated at equivalent rates (mean activation rate for factor IX, 18.8 +/- 3.6 nmol/L/h; for factor X, 17.8 +/- 2.9 nmol/L/h; n = 9 paired vein segments). These data strengthen the evidence that factor VIIa/TF activation of factor IX represents a key initial reaction of coagulation in tissues. These results also show that data obtained with factor VIIa/TF complexes formed on the surface of cultured cells need not hold for factor VIIa/TF complexes formed in extracellular matrix.« less
  • Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.
  • Aminoisoquinoline and isoquinoline groups have successfully replaced the more basic P1 benzamidine group of an acylsulfonamide factor VIIa inhibitor. Inhibitory activity was optimized by the identification of additional hydrophobic and hydrophilic P' binding interactions. The molecular details of these interactions were elucidated by X-ray crystallography and molecular modeling. We also show that decreasing the basicity of the P1 group results in improved oral bioavailability in this chemotype.
  • Vessel wall tissue factor (TF) is exposed to blood upon vascular damage which enables association with factor VIIa (FVIIa). This leads to initiation of the blood coagulation cascade through localization and allosteric induction of FVIIa procoagulant activity. To examine the docking pathway of the FVIIa-TF complex, various residues in the extracellular part of TF (sTF) that are known to interact with FVIIa were replaced with cysteines labelled with a fluorescent probe. By using stopped-flow fluorescence kinetic measurements in combination with surface plasmon resonance analysis, we studied the association of the resulting sTF variants with FVIIa. We found the docking trajectorymore » to be a sequence of events in which the protease domain of FVIIa initiates contact with sTF. Thereafter, the two proteins are tethered via the first epidermal growth factor-like and finally the {gamma}-carboxyglutamic acid (Gla) domain. The two labelled sTF residues interacting with the protease domain of FVIIa bind or become eventually ordered at different rates, revealing kinetic details pertinent to the allosteric activation of FVIIa by sTF. Moreover, when the Gla domain of FVIIa is removed the difference in the rate of association for the remaining domains is much more pronounced.« less