Andrew Yee1,2, Manhong Dai3, Stacy E Croteau4, Jordan A Shavit5, Steven W Pipe5,6, David Siemieniak2,7, Fan Meng3,8, David Ginsburg2,5,7,9. 1. Department of Pediatrics, Baylor College of Medicine, Houston, TX, USA. 2. Life Sciences Institute, University of Michigan, University of Michigan Medical School, Ann Arbor, MI, USA. 3. Michigan Neuroscience Institute, University of Michigan Medical School, Ann Arbor, MI, USA. 4. Department of Pediatrics, Boston Children's Hospital, Harvard Medical School, Boston, MA, USA. 5. Department of Pediatrics, University of Michigan Medical School, Ann Arbor, MI, USA. 6. Department of Pathology, University of Michigan Medical School, Ann Arbor, MI, USA. 7. Howard Hughes Medical Institute, University of Michigan Medical School, Ann Arbor, MI, USA. 8. Department of Psychiatry, University of Michigan Medical School, Ann Arbor, MI, USA. 9. Departments of Internal Medicine and of Human Genetics, University of Michigan Medical School, Ann Arbor, MI, USA.
Abstract
BACKGROUND: Correction of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti-VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD). OBJECTIVE: Locate inhibitor-reactive regions within VWF using phage display. METHODS: We screened a phage library displaying random, overlapping fragments covering the full-length VWF protein sequence for binding to a commercial anti-VWF antibody or to immunoglobulins from three type 3 VWD patients who developed VWF inhibitors in response to treatment with plasma-derived VWF. Immunoreactive phage clones were identified and quantified by next-generation DNA sequencing (NGS). RESULTS: Next-generation DNA sequencing markedly increased the number of phages analyzed for locating immunoreactive regions within VWF following a single round of selection and identified regions not recognized in previous reports using standard phage display methods. Extending this approach to characterize VWF inhibitors from three type 3 VWD patients (including two siblings homozygous for the same VWF gene deletion) revealed patterns of immunoreactivity distinct from the commercial antibody and between unrelated patients, though with notable areas of overlap. Alloantibody reactivity against the VWF propeptide is consistent with incomplete removal of the propeptide from plasma-derived VWF replacement products. CONCLUSION: These results demonstrate the utility of phage display and NGS to characterize diverse anti-VWF antibody reactivities.
BACKGROUND: Correction of von Willebrand factor (VWF) deficiency with replacement products containing VWF can lead to the development of anti-VWF alloantibodies (i.e., VWF inhibitors) in patients with severe von Willebrand disease (VWD). OBJECTIVE: Locate inhibitor-reactive regions within VWF using phage display. METHODS: We screened a phage library displaying random, overlapping fragments covering the full-length VWF protein sequence for binding to a commercial anti-VWF antibody or to immunoglobulins from three type 3 VWD patients who developed VWF inhibitors in response to treatment with plasma-derived VWF. Immunoreactive phage clones were identified and quantified by next-generation DNA sequencing (NGS). RESULTS: Next-generation DNA sequencing markedly increased the number of phages analyzed for locating immunoreactive regions within VWF following a single round of selection and identified regions not recognized in previous reports using standard phage display methods. Extending this approach to characterize VWF inhibitors from three type 3 VWD patients (including two siblings homozygous for the same VWF gene deletion) revealed patterns of immunoreactivity distinct from the commercial antibody and between unrelated patients, though with notable areas of overlap. Alloantibody reactivity against the VWF propeptide is consistent with incomplete removal of the propeptide from plasma-derived VWF replacement products. CONCLUSION: These results demonstrate the utility of phage display and NGS to characterize diverse anti-VWF antibody reactivities.
Authors: Andrew Yee; Robert D Gildersleeve; Shufang Gu; Colin A Kretz; Beth M McGee; Keisha M Carr; Steven W Pipe; David Ginsburg Journal: Blood Date: 2014-05-21 Impact factor: 22.113
Authors: Marije Baaij; Karin P M van Galen; Rolf T Urbanus; Jeannet Nigten; Jeroen H C Eikenboom; Roger E G Schutgens Journal: Br J Haematol Date: 2015-04-07 Impact factor: 6.998
Authors: Zachary M Huttinger; Laura M Haynes; Andrew Yee; Colin A Kretz; Matthew L Holding; David R Siemieniak; Daniel A Lawrence; David Ginsburg Journal: Sci Rep Date: 2021-09-22 Impact factor: 4.379