ALBERT

Description: Abstract 2200 ADAMTS13 specifically cleaves multimeric von Willebrand factor (VWF) into smaller molecules to reduce its high reactivity with platelets. The disintegrin-like (D) domain, adjacent to the catalytic domain of ADAMTS13, plays an important role in the process of VWF cleavage. In this study, we aimed to elucidate critical peptide sequences in D-domain involved in the interaction with VWF. A series of partially overlapping peptide sequences, approximately 20 amino acids in length, covering the D-domain, were synthesized and the inhibitory effects on the catalytic activity of plasma ADAMTS13 was examined using FRETS-VWF73 assay. Consequently, some synthetic peptides were selected and the minimal length necessary for the inhibitory effect was determined as TFAREHLDMCQALSC (peptide323-337). Removal of the amino-terminal threonine diminished the inhibitory effect moderately, although deletion of the carboxyl-terminal cysteine abolished it completely. According to the amino acids alignment of ADAMTS family, this peptide sequence is not conserved, highlighting the specific role in the interaction with its substrate. From the recent analysis of crystal structure, amino-terminal half of the peptide323-337, TFAREHL (323-329), was disordered and designated as the variable (V) loop, which creates one of VWF-binding exosites (Akiyama, et al. Proc Natl Acad Sci USA. 2009; 106:19274-9). We hypothesized that the amino-terminal amino acids of the peptide323-337 contribute to VWF binding, whereas the carboxyl-terminal amino acids allow the structural stability of the peptide conformation. To evaluate the effect of carboxyl-terminal cysteine at 337, other synthetic peptides with alanine, serine, glycine or phenylalanine instead of the cysteine (C337A, C337S, C337G, or C337F) were tested about their inhibitory effects on the catalytic activity. Interestingly, C337A, C337S, C337G peptides exhibited slightly weaker inhibitory effects on VWF73 catalysis, although C337F peptide showed stronger inhibition than wild-type sequence, suggesting that the residue 337 regulates the characteristics of the peptide323-337. From the results of peptide screening, the amino- and carboxyl-terminal amino acids of the peptide323-337, TFAREHLDMCQALSC, likely play key roles in the inhibitory effects; therefore, the middle part of the sequence, HLDMC, was replaced by 5 alanines (AAAAA) or reversed sequence CMDLH. Surprisingly, the converted peptides still retained the equivalent level of inhibitory effects, indicating both sides of the amino- and carboxyl-terminal amino acids were especially significant in the interaction with VWF. In conclusion, we characterized the peptide sequence, TFAREHLDMCQALSC (323-337), in D-domain. The peptide clearly inhibited the cleavage of VWF73 and the both sides of amino- and carboxyl-terminal amino acids seemed especially important. The peptide sequence is supposed to bind to VWF for the precise cleavage in the process of proteolysis. By modifying this peptide sequence, such variant ADAMTS13 as gain-of-function recombinants might be developed, leading to an alternative anti-thrombotic drug. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1167 Background: Cleavage of ultra-large von Willebrand factor (UL-VWF) multimer by ADAMTS13 is an essential process to maintain the appropriate VWF response to platelet in blood flow. The production of autoantibodies to ADAMTS13 causes the accumulation of UL-VWF multimer, resulting in the formation of platelet-rich thrombi in microcirculation, which is considered as a pivotal pathophysiology of acquired thrombotic thrombocytopenic purpura (TTP). Previous studies showed that the major recognition sites of the autoantibodies resided in the cysteine-rich and spacer domains, which include substrate binding exosites. To date, an ELISA kit to measure the total IgG autoantibodies to whole ADAMTS13 molecule is available, and the domain-specific autoantibodies were detected with recombinant truncated proteins using western blot or immunoprecipitation methods. Objective: The aim of this study was to perform a radioimmunoprecipitation assay to quantify the domain-specific autoantibodies to ADAMTS13 in acquired TTP. This approach was expected to allow us the sensitive detection and the precise quantification of these autoantibodies, leading to the understanding of pathophysiology and clinical course of TTP. Materials and methods: Two kinds of 35S-methionine labeled antigens, MDTCS and T2-8/CUB, were prepared using in vitro transcription/translation kit (TNT Quick Coupled Transcription/Translation system, Promega). MDTCS was a peptide from amino acid residue-1 to 687, including metalloprotease, disintegrin-like, TSP1-1, cysteine-rich and spacer domains, containing the substrate binding exosites and the catalytic site. T2-8/CUB was a peptide from residue-686 to 1427, including seven TSP1 repeats and two CUB domains, which are associated with the shear-related function in blood flow. The correct synthesis and molecular size of the two radiolabeled antigens were confirmed with SDS-PAGE. First, to establish the quantitative assay, each of the antigens, MDTCS or T2-8/CUB, was mixed with mouse anti-ADAMTS13 monoclonal antibodies, whose epitopes were already well examined in our previous study (ASH Annual Meeting 2009 114:3182). The immune complex was precipitated with protein G beads and the beads were washed, and then applied in a liquid scintillation counter. Second, each of the antigens was similarly immunoprecipitated and quantified with IgG samples purified from 12 acquired TTP patients. As a control, IgG samples from healthy subjects with no histories of autoimmune disease were used. Results: Each of the radiolabeled antigens was expressed as a single band with expected molecular size with SDS-PAGE and successfully immunoprecipitated with monoclonal anti-ADAMTS13 antibodies corresponding to each epitopes, indicating the intact conformation of the antigens. Furthermore, this assay system showed the appropriate dose-dependent escalation curve according to the addition of the monoclonal antibodies, verifying its quantitative analysis. The titration of IgG sample from TTP patients using each antigen, MDTCS or T2-8/CUB, revealed that all of the samples exhibited significantly higher titers to both of the antigens than the control IgG samples, indicating the reliable sensitivity of this assay. Conclusion: We performed a quantitative analysis of the domain-specific autoantibodies to ADAMTS13 in TTP using the radioimmunoprecipitation assay. This sensitive approach may enable us to clarify the relationship between the epitopes of autoantibodies to ADAMTS13 and the clinical characteristics of TTP. Disclosures: Matsumoto: Alexion Pharma: Membership on an entity's Board of Directors or advisory committees. Fujimura:Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Alexion Pharma: Membership on an entity's Board of Directors or advisory committees.