ALBERT

Description: Platelet transfusions are widely used for patients with severe thrombocytopenia. There are, however, practical problems in the current donor-dependent platelet transfusions, such as the limited supply and risk of serious immune reactions. Thus, the development of new strategies for generating platelets for transfusion is crucial. Platelets have been differentiated from hematopoietic stem cells, fetal liver cells, embryonic stem cells, induced pluripotent stem cells, NF-E2-transduced fibroblasts, and preadipocytes. Here, among these cells preadipocytes, especially in the subcutaneous adipose tissue, could be ideal candidate cells for manufacturing megakaryocytes (MKs) and platelets, because (1) they are relatively easy to obtain large quantities and have ability to proliferate in vitro, (2) their differentiation does not require gene transfer, as they possess genes in relation to megakaryopoiesis and thrombopoiesis, such as p45NF-E2 and c-mpl, and (3) they differentiate into MKs and platelets using an endogenous thrombopoietin. Thus, to clarify the usefulness of preadipocytes as a donor-independent source for platelet transfusion, we compared both number and function between platelets derived from mouse subcutaneous preadipocytes and those from bone marrow mononuclear cells (BMMNCs), the established cell source for manufacturing platelets. First, BMMNCs were not feasible for their expansion in vitro and therefore the cells were directly seeded in MK lineage induction media. In contrast, preadipocytes were to be passaged 6 times without any morphological changes, and then cultured in MK lineage induction media for their differentiation into platelets. Thus, as assessed by CD41-positive platelet-sized cells, 106.2±5.0 ×105 or 3.9±1.0 ×105 platelets were obtained from 106 preadipocytes or 106 BMMNCs, respectively (p

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: Background ADAMTS13-binding immunoglobulin G (IgG)-type autoantibodies are present in patients with acquired thrombotic thrombocytopenic purpura (TTP), thereby causing severe deficiency of plasma ADAMTS13 activity. Some specific autoantibodies directly inhibit the enzymatic activity by interfering with the access of its substrate, von Willebrand factor (VWF), particularly in the spacer domain, although others bind to almost all of the domains in the molecule, possibly leading to the acceleration of the clearance from blood stream. Not only the inhibitor titer, but the importance of ADAMTS13 autoantibody titer was highlighted by many previous clinical studies, associating with the prognosis such as recurrence rate. Objective We targeted to establish a novel high-sensitive assay to measure ADAMTS13-binding IgG autoantibody titer using three kinds of radioisotope-labeled antigens, ADAMTS13 whole molecule, MDTCS and T2-8/CUB, and aimed to analyze the association between the autoantibody titer and the clinical characteristics of TTP patients. Materials and Methods Human Cell-Free Protein Expression System (Takara #3281, Shiga, Japan) was used to synthesize radioisotope-labeled antigens. ADAMTS13 cDNA corresponding to whole molecule (A13), metalloprotease to spacer domains (MDTCS) and TSP1-2 to CUB2 domains (T2-8/CUB) were cloned into an expression vector pT7-IRES and mixed with Cell Lysate containing T7 RNA polymerase, methionine-free amino acids, ATP, translation enhancement factor and 35S-methionine. The correct synthesis and molecular size of the radiolabeled antigens were checked with SDS-PAGE. To assess the utility as a quantitative assay, each of the antigens was mixed with mouse anti-ADAMTS13 monoclonal antibodies, whose epitopes were determined in our previous study (Thromb Res. 2012; 130(3):e79-83), and the immune complex was precipitated with protein G beads, washed and measured in a liquid scintillation counter. Plasma samples from acquired TTP patients were tested to quantify the autoantibody titers using radiolabeled A13, MDTCS and T2-8/CUB antigens, respectively. As a control, plasma samples from healthy subjects with no histories of autoimmune disease were also tested. Results Each of the radiolabeled antigens was detected as a single band at the correct molecular weight size and successfully immnoprecipitated with several mouse anti-ADAMTS13 monoclonal antibodies, indicating the intact molecular conformation of the synthesized proteins using the cell-free protein synthesis system. Moreover, the appropriate dose-dependent escalation curves in accordance with the addition of the monoclonal antibodies were observed, thereby confirming the utility of the assay as a quantitative analysis. We tested TTP patient plasma at onset (n=5) and were able to detect ADAMTS13 autoantibody titers with each of the radiolabeled A13, MDTCS and T2-8/CUB antigens. We next applied this assay for monitoring ADAMTS13 autoantibody titer in a clinical course of TTP patient. The patient developed the first episode of TTP at the age of 2 month and treated with steroid pulse and plasma exchange therapy for six consecutive days. Remission was once achieved but 6 months later from the onset, the second episode of TTP occurred and the patient was treated with 11 plasma exchange and rituximab at the dose of 375 mg/m2 once a week for 4 weeks. Plasma samples at onset, after the first 6 consecutive plasma exchange and after rituximab administration were examined about autoantibody titer using this assay. Interestingly, the titers remained high even after the plasma exchange but declined clearly after the rituximab treatment, whereas no reduction of total IgG level was observed. These findings suggest that the autoantibody titration using this assay might be useful to assess the effect of treatment and associate with the prognosis related to the recurrence. Conclusion We developed a novel quantitative radioimmunoprecipitation assay to measure ADAMTS13 autoantibody titer related to three antigens, ADAMTS13 whole molecule, MDTCS and TSP2-8/CUB. This assay may serve not only as a diagnostic test but as a monitoring index to evaluate the prognosis of TTP. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3182 Poster Board III-119 Fourteen mouse anti-ADAMTS13 monoclonal antibodies (MoAb#1∼#13, A10) were individually analyzed for their precise epitope peptide sequences in each domain using lambda phage surface display system. A phage library expressing random peptide fragments of ADAMTS13 on its surface was constructed, thereby selecting phage clones bound to each MoAbs immobilized on microtiter plates. Binding epitope sequences for eleven MoAbs were defined, although MoAb#3, #4 and #5 were not clarified. Among 11 epitope-determined MoAbs, epitopes were relatively short (6 to 23 amino acids) in MoAb#1, #2, #8, #11, #12 and #13, recognizing metallopretease, disintegrin-like, TSP1-4, TSP1-8, CUB1 and C-terminus domains, respectively. On the other hand, epitopes were relatively long (49 to 72 amino acids) in A10, MoAb#6, #7, #9 and #10, recognizing disintegrin-like, TSP1-2, TSP1-3, TSP1-5 and TSP1-7 domains, respectively. MoAb#1, #2 and A10 demonstrated inhibitory effects on the cleavage activity of ADAMTS13 evaluated by FRETS-VWF73 assay. MoAb#1 recognized Gln159 to Asp166 in the metalloprotease domain, and MoAb#2 and A10 recognized Asn308 to Glu327, Tyr305 to Glu376 in the disintegrin-like domain, respectively. From findings using C-terminal truncated mutants of ADAMTS13, MoAb#3 and #5 were supposed to recognize TSP1-1 and spacer domain, respectively, although only C-terminal tail peptide sequences were selected from both of the screening, suggesting the possibility of intramolecular association between the C-terminal region and TSP1-1/spacer domains. MoAb#4 was supposed to recognize disintegrin-like domain, although we could not obtain any significant ADAMTS13 peptide sequence from the screening. We speculate that these 3 epitope-undetermined MoAbs may recognize complex conformational structure of ADAMTS13. Alternatively, intact peptide structure of ADAMTS13 might not be expressed properly on the phage surface. In conclusion, we defined precise epitope sequences of 11 monoclonal anti-ADAMTS13 antibodies. Three of them, recognizing metalloprotease or disintegrin-like domains inhibited the cleavage activity of ADAMTS13. Analysis of the epitope sequences may elucidate the correlation between the molecular conformation and the catalytic activity. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1144 Introduction: A flow-chamber system was developed to evaluate the growth of platelet thrombus formation (PTF) quantitatively using whole blood under various shear stress conditions. This device, T-TAS (Total Thrombus-formation Analysis System, Fujimori Kogyo Co., Yokohama, Kanagawa), analyzes the process of PTF by monitoring the continuous pressure increase in the capillary of microchip where whole blood flows, using two kinds of thrombogenic surfaces (PL chip: coated with collagen, AR chip: coated with collagen plus tissue factor). In the current study, we characterized this system using whole blood samples from healthy subjects by comparing the measurements with those of other standard platelet function tests. Materials and Methods: Whole blood samples were collected from 32 healthy volunteers with hirudin (PL chip) or 3.2% sodium citrate (AR chip) as anticoagulants. For AR chip, CaCl2 with corn trypsin inhibitor was mixed immediately before the testing. The samples were individually applied on the system to measure the PTF starting time (T10: time to reach 10 kPa), occlusion time (OT: T60, time to reach 60 kPa for PL chip; T80, 80 kPa for AR chip), and AUC (area under the flow pressure curve: AUC10, until 10 min for PL chip; AUC30, until 30 min for AR chip) under various shear rates (1000, 1500, 2000 s−1 for PL chip; 300 s−1 for AR chip). Platelet function of the blood sample was also tested using platelet aggregometry (collagen, ADP, ristocetin, and epinephrine as agonists), PFA-100 (C/EPI-, C/ADP-CT: closure time) and VerifyNow P2Y12 assay (PRU). Results: In PL chip, T10 was correlated with C/EPI- and C/ADP-CT, and AUC10 was correlated with C/EPI-CT under all of the shear conditions. The correlation was enhanced in accordance with the increase of the shear rates. In addition, T60 and AUC10 were correlated with AUC of collagen-induced aggregation curve of platelet aggregometry. In AR chip, T10–80, reflecting the rate of thrombus growth, was likely correlated with C/ADP-CT. Measured values from VerifyNow P2Y12 assay was not significantly associated with those from this system. Interestingly, platelet numbers were significantly correlated with all of the measurements with AR chip, and partially with those with PL chip. Conclusion: In healthy subjects, PTF starting time and AUC with PL chip, and the growth rate of PTF with AR chip, seemed associated with PFA-100 measurements, indicating its characteristics related to shear induced PTF. However, the values from this system showed a rare correlation with those from platelet aggregometry and VerifyNow P2Y12 assay. This system may allow us to identify the parameters of individuals' thrombogenicity independent of those related to other platelet function tests, under whole blood flow conditions. Disclosures: Matsubara: Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees. Ohnishi:Fujimori Kogyo Co.: Employment. Hosokawa:Fujimori Kogyo Co.: Employment. Murata:Medico's Hirata: Honoraria; Advisory Committees on VerifyNow: Membership on an entity's Board of Directors or advisory committees.

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.

Description: The metalloprotease ADAMTS13 cleaves multimeric von Willebrand factor (VWF) to regulate VWF-mediated thrombus formation. We planned to search core epitopes of ADAMTS13 that is required for its binding to VWF. We constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using immobilized VWF as a probe. After the first screening, the C-terminus of the spacer domain from Arg670 to Glu684 (termed as epitope-1) and the middle of the cysteine-rich domain from Arg484 to Arg507 (epitope-2) were determined as epitopes. When we added the synthetic epitope-1 peptide to the second screening, a new site, from Pro618 to Glu641 (epitope-3), was found in the middle of spacer domain. While the presence of synthetic epitope-2 peptide did not affect the subsequent screening, the presence of epitope-3 peptide enhanced the isolation of clones encoding epitope-1. These results suggest that ADAMTS13 epitopes-1, -2 and -3 may interact with each other for their binding to VWF. From screening in the presence of any combination or all of the three synthetic peptides, however, no new VWF binding site was uncovered. To examine the effect of divalent metal cations on the binding of ADAMTS13 epitopes to immobilized VWF, screening was carried out in the presence or absence of 5 mM of EDTA. No new epitope site was found. We next explored inhibitory effect of the synthetic epitope peptides on ADAMTS13 protease activity using recombinant ADAMTS13 and FRETS-VWF73 as a substrate. Synthetic epitopes-2 and -3 peptides markedly inhibited the cleavage of VWF by ADAMTS13, while the synthetic epitope-1 peptide did not as efficiently as epitopes-2 and -3. The stronger inhibitory effect of epitope-3 peptide than that of epitope-1 peptide was confirmed by SDS-agarose gel electrophoresis analysis of cleavage products of denatured multimeric VWF molecules by recombinant ADAMTS13. This was consistent with the dissociation constants for the three synthetic peptides with immobilized VWF determined by surface plasmon resonance, in which epitopes-2 and -3 have higher affinities for VWF than that of epitope-1. The results described above suggest that ADAMTS13 may initially bind to immobilized VWF through the sites of epitope-1 and epitope-2 with relatively weak affinity. The binding of epitope-1 to VWF may subsequently induce the conformational change of VWF, thereby exposing a binding site for epitope-3 for the efficient catalytic cleavage of VWF by ADAMTS13.

Description: Anti-ADAMTS13 autoantibodies are considered to play pivotal roles in the pathophysiology of acquired thrombotic thrombocytopenic purpura (TTP). They inhibit the ADAMTS13 function resulting in the appearance of ultra-large von Willebrand factor (VWF) multimers. Major binding sites of the autoantibodies were reported to be in the cysteine-rich/spacer domains. To clarify the precise peptide sequences recognized by anti-ADAMTS13 IgG autoantibodies, we constructed a random cDNA fragment library expressing various peptides of ADAMTS13 on the surface of lambda phage and screened the library using purified IgG from 13 TTP patients. Diverse peptide sequences were obtained from almost entire ADAMTS13 domains such as metalloprotease, disintegrin, TSP1-1, cysteine-rich, spacer, TSP1- 2, 3, 4, 5, 7, 8 and CUB1. In particular, we detected an identical 26 amino-acid epitope sequence in the C-terminus of spacer domain from Gly662 to Val687 (sp662–687) shared by 5 TTP patients. Moreover, the peptide sequence was exactly included in one of the VWF binding epitope sites that we previously determined (Blood110 (11), 795a, 2007). We then assessed the impact of specific autoantibody to ADAMTS13 activity measured by FRETS-VWF73 or EIA and ADAMTS13 inhibitor titer in each of TTP patient plasma. However, both of the ADAMTS13 activity and inhibitor titer seemed not correlated with the existence of specific sp662–687 IgG autoantibody. These observations suggest that the autoantibody to sp662–687 may be one specific feature of TTP, although other epitopes are also involved in the pathogenesis of the disorder.