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Analysis on the molecular species and concentration of circulating ADAMTS13 in Blood
Kenji Soejima1, Hitomi Nakamura, Masaki Hirashima
1Research Department 1, The Chemo-Sero-Therapeutic Research Institute, Kyokushikawabe, Kikuchi, Kumamoto 869-1298. soejima@kaketsuken.or.jp
Abstract:
ADAMTS13 is the metalloprotease responsible for the proteolytic degradation of von Willebrand factor (VWF). A severe deficiency of this VWF-cleaving protease activity causes thrombotic thrombocytopenic purpura. This protease, comprising 1,427 amino acid residues, is composed of multiple domains, i.e., a preproregion, a metalloprotease domain, a disintegrin-like domain, a thrombospondin type-1 motif (Tsp1), a cysteine-rich domain, a spacer domain, seven Tsp1 repeats, and two CUB domains. We prepared one polyclonal and seven monoclonal antibodies recognizing distinct epitopes spanning the entire ADAMTS13 molecule. Of these antibodies, two of the monoclonal ones, which recognize the disintegrin-like and cysteine-rich/spacer domains, respectively, abolished the hydrolytic activity of ADAMTS13 toward both a synthetic substrate, FRETS-VWF73, and the natural substrate, VWF. In addition, these antibodies blocked the binding of ADAMTS13 to VWF. These results revealed that the region between the disintegrin-like and cysteine-rich/spacer domains interacts with VWF. Employing these established polyclonal and monoclonal antibodies, we examined the molecular species of ADAMTS13 circulating in the blood by immunoprecipitation followed by Western blot analysis, and estimated the plasma concentration of ADAMTS13 by enzyme-linked immunosorbent assay. These studies indicated that the major fraction of ADAMTS13 in blood plasma consisted of the full-length form. The concentration of ADAMTS13 in normal plasma was approximately 0.5-1 microg/ml.
Insights
Researchers developed antibodies to study ADAMTS13, a protease deficient in thrombotic thrombocytopenic purpura. These antibodies identified key interaction sites and confirmed full-length ADAMTS13 is dominant in blood plasma.
Area of Science:
- Biochemistry
- Immunology
- Hematology
Background:
- ADAMTS13 is a metalloprotease crucial for degrading von Willebrand factor (VWF).
- Deficiency in ADAMTS13 activity leads to thrombotic thrombocytopenic purpura (TTP).
- ADAMTS13 possesses a complex structure with multiple functional domains.
Purpose of the Study:
- To develop antibodies for characterizing ADAMTS13 structure and function.
- To identify the ADAMTS13 domains involved in VWF binding and activity.
- To determine the molecular forms and plasma concentration of ADAMTS13 in circulation.
Main Methods:
- Generation of polyclonal and monoclonal antibodies against ADAMTS13.
- Assessment of antibody effects on ADAMTS13 proteolytic activity and VWF binding.
- Immunoprecipitation, Western blot, and ELISA for analyzing plasma ADAMTS13.
Main Results:
- Two monoclonal antibodies targeting the disintegrin-like and cysteine-rich/spacer domains inhibited ADAMTS13 activity and VWF binding.
- These findings pinpoint the region between the disintegrin-like and cysteine-rich/spacer domains as critical for VWF interaction.
- The predominant form of ADAMTS13 in normal plasma is full-length, with concentrations around 0.5-1 microg/ml.
Conclusions:
- Novel antibodies provide tools to probe ADAMTS13 function and VWF interaction.
- The disintegrin-like and cysteine-rich/spacer domains are essential for ADAMTS13's interaction with VWF.
- Full-length ADAMTS13 is the major circulating form, with established normal plasma concentration ranges.
