Image by Erhabor Osaro
Researchers say they have uncovered a new avenue for therapeutic intervention in thrombotic thrombocytopenic purpura (TTP).
The team discovered how autoimmune antibodies in a TTP patient recognize and bind to ADAMTS13.
They believe this knowledge could help them alter ADAMTS13 to produce a therapeutic enzyme that can elude recognition by autoimmune antibodies yet still retain its ability to cleave von Willebrand factor.
Such an enzyme could be given to TTP patients in the hospital to speed recovery and cut the cost of treatment.
Long Zheng, MD, PhD, of the University of Alabama at Birmingham, and his colleagues described this work in PNAS.
The researchers found that 5 small loops in ADAMTS13’s amino acid sequence are necessary for autoantibodies to bind to ADAMTS13.
Cutting or substituting several amino acids out of any of the 5 loops prevented binding. Small deletions in a loop also left the enzyme unable to cleave von Willebrand factor.
“This was really surprising,” Dr Zheng said. “It’s like a table with 5 legs. If you take 1 away, it should still stand, but, somehow, it collapsed. This suggests that you need the coordinated activity of all 5.”
Thus, it appears that the autoimmune antibodies in TTP patients inhibit the enzyme by physically blocking the recognition site of ADAMTS13 for von Willebrand factor.
Analyses of autoantibodies from 23 more TTP patients revealed that most use the same binding site. This suggests modifying the ADAMTS13 enzyme by protein engineering may be able to help a wide range of TTP patients.
Details of the research
Dr Zheng and his colleagues first isolated messenger RNAs that code single chains of variable regions of monoclonal antibodies from B cells collected from patients with acquired TTP.
The team used phage display to select the messenger RNAs that code specific antibodies that bind and inhibit ADAMTS13. These monoclonal antibodies were then expressed in E coli cells, purified, and biochemically characterized.
Three inhibitory monoclonal antibodies were selected for further study by hydrogen-deuterium exchange coupled with mass spectrometry. This technology uses amine hydrogen exchange with deuterium on each amino acid residue except proline.
After the reaction was stopped, the protein was cut into small pieces (or peptide fragments) and run through high-performance liquid chromatography for separation and mass spectrometry for identification.
Antibody binding sites were detected by their ability to block the hydrogen and deuterium exchange, as compared with ADAMTS13 that was unbound.
One of the 3 high-affinity probes selected by phage display was used for the competition experiments against polyclonal autoimmune antibodies from 23 TTP patients.
The results show that this particular binding epitope is common among patients with acquired autoimmune TTP.
