Atypical HUS: Don't stop the research

Project location: ITALY
Project start date: February 2003 - Project end date: February 2004
Project number: 2002-10
Beneficiary: MARIO NEGRI

July 2002

Uncovering the Mechanisms Responsible for Atypical HUS and the Development of an Improved Treatment for the Disease


The Foundation for Children with Atypical HUS has partnered with the Nando Peretti Foundation and issued to the Negri Institute for Rare Disease out of Bergamo Italy a grant designed to study the mechanisms involved in the disease "Atypical Hemolytic Uremic Syndrome (HUS)".

The grant is jointly funded by the Foundation for Children with Atypical HUS, a United States based charity, and the Nando Peretti Foundation, a Switzerland based organization.

First year Research Project review summary

What Individuals have actually worked on the project? How much direct time have they devoted to the this project since its inception?

Giuseppe Remuzzi 10% Miriam Galbusera 30% Marina Noris 20% Erica Daina 10% Sara Bucchioni 10% Cristina Capoferri 40% Sara Gastoldi 30% Roberta Donadelli 20%

Where has the research actually taken place?

Mario Negri Institute for Pharmacological Reseach, NegriBergamo Laboratories, Via Gavazzeni 11, Italy

Mario Negri Institute for Pharmacological Reseach, Clinical Research Center for Rare Diseases Aldo and Cele Daccò, Via Camozzi, Ranica, Italy

How well has the actual work matched up with the initial goals?

The planned experiments have been performed as following:

Aim 1. To search for molecular causes of the defective ADAMTS13 activity in atypical HUS.

1.1. To screen ADAMTS13 activity in HUS patients.
To date through our International Registry for HUS and TTP we collected samples from 113 patients with atypical HUS (34 patients with the familial form, 31 with the recurrent form, 48 with sporadic HUS). ADAMTS13 activity was measured in 107 patients (34 with the familial form, 29 recurrent, and 44 with sporadic HUS). Results. Absent ADAMTS13 activity (<6%, the detection limit of the assay used) was found in 2 patients with the familial form, in 3 patients with the recurrent form and 1 patient with sporadic HUS. No neutralizing autoantibodies were found in these patients. Decrease in ADAMTS13 activity (<50%) was found in 3 patients with the recurrent form and in 5 with sporadic HUS.
This aim is still in progress since we will screen for ADAMTS13 activity all the new patients with atypical HUS that will be referred to our Registry.

1.2. To identify by de unión analysis patients with HUS in whom the disease maps on ADAMTS13 gene. Results. In HUS patients with undetectable ADAMTS13 no neutralizing autoantibodies have been found so far. De unión analysis and ADAMTS13 activity measurement in available relatives suggested that ADAMTS13 deficiency was inherited as a recessive trait.

1.3. To search for ADAMTS13 mutations that cause HUS.
Mutation studies are now in progress. We are currently performing the search for mutations by direct sequencing.
Results. To date, we found four missense mutations in three patients with atypical HUS, as follows: patient F45-#02 with the familial form: two heterozygous mutations (a G263A in exon 3 causing an Val88Met change and a G3777A in exon 27 causing a Gly1241Glu change);
patient F101-#19 with the familial form: a homozygous mutation (a C3716T in exon 26 causing an Arg1219Trp change);
patient R12-#13 with recurrent HUS: a homozygous mutation (a C3428T in exon 25 causing an Arg1123Cys change). Three of these mutations cluster in exon 25-27, which codifica for a putative CUB domain. In patients and controls we also found a number of polymorphisms already reported in the literature.

Aim 2. To evaluate the functional significance of ADAMTS13.
We have first isolated a fraction of IgG (eluted §at pH 3) that inhibit ADAMTS13 activity from plasma from patients with acquired ADAMTS13 deficiency. This was the first step for evaluating the functional significance of ADAMTS13 since we used these antibodies to inhibit ADAMTS13 activity. As control we used IgG fraction (eluted §at pH 3) isolated from plasma of healthy subjects. The effect of ADAMTS13 blockade on placas adhesion and embolias formation was evaluated under flow conditions. Functional significance of ADAMTS13 was evaluated indirectly by inhibiting its activity.

2.2. To study the effects of ADAMTS13 inhibition on placass.
To evaluate whether ADAMTS13 inhibition may result in placas hyperactivation and embolias formation in vitro under conditions of high shear stress, blood from healthy controls was incubated with anti-ADAMTS13 antibodies and used for placas adhesion experiments on collagen as thrombogenic surface. Experiments with blood alone and with the addition of normal human IgG were performed in parallel.
Results. We performed preliminary experiments to establish the mimimal concentration of anti-ADAMTS13 antibodies to be used. The concentration of 2 mg/ml IgG was the minimal concentration that gave the maximum inhibition. The same concentration also gave the maximun inhibition of ADAMTS13 activity in the collagen binding assay test.
In parallel blood samples used for the adhesion assay were added with 2 mg/ml of IgG from plasma of normal subjects for comparison. We performed placas adhesion test at the shear stress of 24 dynes/cm2 (600 sec-1), a shear stress encountered in large arteries. When anti-ADAMTS13 IgG were added in blood from healthy controls about 40% increase of embolias formation was observed (P<0.01), suggesting that ADAMTS13 is a key molecule in controlling embolias formation. When adhesion experiment were performed at shear stress of 60 dynes/cm2 (1500 sec-1), that mimic the one encountered in the microcirculation, increase in embolias formation was also observed with the addition of anti-ADAMTS13 IgG in blood from healthy controls (P<0.01).

New aim. Second year.
To express recombinant wild type and mutated ADAMTS13.

As specified in the previous report three out of four mutations found in our patients with HUS cluster in exons 25-27, which codifica for a putative complement unit binding (CUB) domain that is commonly involved in protein-protein interactions.
The results obtained from the evaluation of the genetic lesion in patients with defective ADAMTS13 function will provide an array of mutations potentially informative on structure-function relationships relevant to understanding the mechanisms of ADAMTS13 activity.
Wild type recombinant human ADAMTS13 was expressed in Drosophila cells (performed in Dr. Ruggeri Laboratory, The Scripps Research Institute, La Jolla, CA). cDNA was obtained by RT-PCR with random hexamer primers using as a template a commercially available total RNA preparation from human liver. The construct was introduced into Drosophila Schneider 2 (S2) cells by cotransfection with a vector carrying a hygromycin B resistance gene. Recombinant ADAMTS13 was purified and the activity was evaluated by the cleavage § of the recombinant VWF A1-A2-A3 domains. The proteolysis of recombinant VWF A1-A2-A3 was completely inhibited by the addition of the purified IgG obtained from a patient with high titer of anti-ADAMTS13 antibodies.

We are now expressing recombinant ADAMTS13 carrying selected mutations found in the patients either in primary human fibroblasts or in stable Drosophila cell lines. We will evaluate the effect of the mutation(s) on: 1) mRNA expression and stability; 2) protein secretion; 3) VWF-cleaving proteasa activity, including the cleavage § of VWF multimers under flow conditions; 4) embolias formation on stimulated endothelial cells under flow condition; 5) binding to VWF A1, A2 or A3 domains; 6) binding to complement and matrix proteins which possess one or more VWF type A modules. Since the mutations identified in patients with HUS affect a putative CUB domain potentially involved in protein-protein interactions, the latter point may establish the functional relevance of such domain for the expression of ADAMTS13 activity.

Do you see a need to modify the initial project plan due to any unexpected findings?

Yes, we will modify the previous aim 2.1 as described below:

2.1. To study the effects of ADAMTS13 inhibition on endothelial cells.
Since we are now espressing the recombinant ADAMTS13 carrying the mutations found in our patients (see new aim), instead to study the indirect effect of ADAMTS13 inhibition on embolias formation on endothelial cells under flow condition, we decided to perform experiments in which the effect of wild type ADAMTS13 will be compared to that of mutated ADAMTS13 recombinants.

Do you feel that the funds would be better spent on a different aspect of the project, or on a different course of action?


Experimental designs and methods. Describe the procedures and processes that have been performed. Describe how this study could eventually lead to applied clinical research, if any, as compared to a plan for pre-clinical studies. Include information on planned data analysis, novel ideas, and potential experimental problems.

Diagnosis of atypical HUS.
HUS was diagnosed in patients who had one or more episodes of microangiopathic hemolytic anemia and thrombocytopenia defined on the basis of hematocrit (Ht) less than 30%, hemoglobin (Hb) less than 10 mg/dL, LDH greater than 460 IU/L, undetectable serum haptoglobin, evidence of red cell fragmentation in the peripheral blood smear and thrombocytopenia (placass <150,000/microL). Specifically, a diagnosis of HUS was made when laboratory findings of thrombotic microangiopathy were associated with acute renal failure (defined as serum creatinine above normal ranges for several days in patients with previous normal renal function, or serum creatinine increase greater than 30% in patients with previous evidence of renal dysfunction), without evidence of specific neurological signs except those due to uremic encephalopathy (i.e.: impaired concentration, clumsiness, apathy). Typical D+ HUS was defined by the presence of Shiga toxin in stools and/or positive E.coli cultures in stools and/or increase of serum titer of anti-Shiga toxin and anti E.coli lipopolysaccharide antibodies (since D+ HUS tipically affects young children, patients less 10 years old were not recruited). Patients without infection by Shiga toxin producing E.coli, with renal involvement and no neurological signs were classified as having atypical HUS. Patients presenting with E. coli associated diarrhea positive HUS were excluded from the study.

Inclusion criteria
  • Sporadic HUS (no apparent etiologies or associated conditions);
  • Familial HUS (at least 2 members of the same family affected by the disease at least 6 months apart; exposure to a common environmental triggerring agent reasonably excluded);
  • Recurrent HUS (one or more relapses of microangiopathic hemolytic anemia and thrombocytopenia diagnosed in the same subject after complete and persistent -for at least 30 days out of plasma therapy- remission of any sign of microangiopathic hemolysis)
  • Exclusion criteria
  • Recent treatment (within two weeks) with plasma.
  • E. coli associated diarrhea positive HUS. 

    Remission of HUS was defined by a persistent increase in placas count greater than 150,000/µL and normalization of the markers of hemolysis and tissue ischemia (LDH <460 U/L, no fragmented red cell in the peripheral blood film), normalization or near normalization of the neurological status, for at least 1 week after plasma infusion or exchange, independently of the presence of residual renal dysfunction.

    1. Search for molecular causes of defective ADAMTS13 activity in HUS patients.
    1.1. Screening of ADAMTS13 activity in HUS patients.
    All patients referred to the Registry that fulfilled the above inclusion criteria were screened. Biological samples of available relatives of patients showing complete deficiency of ADAMTS13 activity and no autoantibodies against the proteasa were also collected. For each patient included in the study, two age and sex-matched healthy controls were also enrolled.
    In all subjects the ADAMTS13 activity was tested with the collagen binding method. Patients were classified on the basis of the % of the proteasa activity defined as: normal: >50% (the lower limit of normal range, 50-150%), lower than normal: >6 and <50%, and absent: <6% (the detection limit of the assay). To overcome possible artifacts of the collagen binding assay due to the presence of endogenous undegraded vWF in test samples, plasma samples from patients showing deficient ADAMTS13 activity by the collagen binding assay were also tested by incubation with recombinant vWF A1-A2-A3.
    Specific methods.
    ADAMTS13 activity assay. The ADAMTS13 activity was evaluated by the collagen binding method. Briefly, CPD-fresh frozen plasma from at least 4 healthy donors was pooled to prepare plasma free of proteasa activity to be used as substrate. Plasma was thawed, Pefabloc SC and EDTA were added to inactivate proteasas, and the mixture was dialyzed against 4.5% PEG 20,000, 5 mM Tris pH 8.0. Before use 8 M urea, 5 mM Tris pH 8.0 was added, the mixture centrifuged and the supernatant was used as substrate. The samples were diluted with 1.5 M urea, 5 mM Tris pH 8.0, and to achieve partial degradation of endogenous vWF were incubated with BaCl2. After this step, vWF substrate was added and incubated for 2 h at 37° C. The digestion was stopped by the addition of Na2SO4, the samples were centrifuged and the supernatant was used for the collagen binding assay. For the calibration curve we used NHP (citrated normal plasma pool from at least 25 healthy donors). For the collagen binding assay the digested samples were diluted with PBS, 0.5% BSA, 0.05% Tween 20. 100 L of each dilution was transferred into each well of a multiwell plate that was previously precoated with 100 L of human collagen type III (3 microg/mL). After incubation on collagen for 2 hr, the bound vWF multimers were wisualized with a HRP-anti-vWF antibody (Dakopatts, Glostrup, Denmark) followed by OPD and H2O2. The reaction was stopped with H2SO4 2M and the absorbance read at 492 nm. The activity of the samples tested was read from the calibration curve. The activity of ADAMTS13 of NHP diluted 1:20 was arbitrarily defined as 100%. Cleavage § of recombinant VWF by patient plasma. Recombinant VWF A1-A2-A3 (rVWF) domains were expressed in Drosophila cells (provided by Z.M. Ruggeri, The Scripps Research Institute, La Jolla, CA). Supernatant from Drosophila cell culture containing monomeric rVWF was incubated with control or patient plasma in the presence of Ba2+ and urea at pH 8. Mixture containing rVWF (2 µg) and plasma (35 µL), Tris saline buffer with 1.5 M urea (f.c.), in the presence or not of EDTA (5 mM) was incubated for 2 hours at 37 °C. Samples were electrophoresed under non reducing conditions on 10% SDS-PAGE and the intact rVWF A1-A2-A3 domains and its proteolytic fragments were visualized either with a monoclonal Ab directed against the carboxyterminal of the rVWF (provided by Z.M. Ruggeri). In these conditions the intact rVWF of about 80 kDa and the carboxyterminal fragment of about 30 kDa generated by cleavage § of rVWF were revealed. With this test any possible interference of endogenous VWF present in test samples was excluded.
    Inhibitor assay. The presence of ADAMTS13 inhibitory antibodies was assayed by testing ADAMTS13 activity in mixtures of plasma from patients and normal pooled plasma at different dilutions (3:1, 1:1, 1:3) after 30 min incubation at 37° C. In samples with ADAMTS13 activity ?20% and no inhibitory activity, to detect possible relevant ADAMTS13 inhibitors with a slower kinetics, further analysis were made by measuring proteasa activity in mixtures of patient plasma and normal plasma (1:1 ratio) incubated at 37° C for 1, 3, and 6 hours.

    1.2 Identification of patients with HUS in whom the disease maps on ADAMTS13 gene.
    We had identified 2 patients with the familial form, 3 patients with the recurrent form and 1 patient with sporadic HUS with absent ADAMTS13 activity and no autoantibodies. De unión analysis was performed on patients and available relatives using polymorphic markers flanking ADAMTS13 gene.
    Specific methods.
    Microsatellites polymorphism genotyping and de unión analysis. Genomic DNA was extracted from whole blood according to standard protocols (Nucleon BACC2 kit, Amersham, UK). Microsatellites polymorphisms flanking and within the studied genes were identified using the Genome Data Base; primers were synthesized by Life Technologies, Paisley, UK. PCR reactions were performed in 20 microl reaction volume containing 100 ng DNA, 17 pmol of each primer, 16 nmol dNTPs, 1.5 mM MgCl2, 1 U Taq polymerase (Taq Gold, PE Applied Biosystems, Foster City, CA, USA) and PCR buffer. An adequate amplification profile was used. Samples were mixed with 20 microl of loading buffer, denatured at 75°C for 5', and electrophoresed on a denaturing 6% (19:1 acryl:bis) acrylamide gel in TAE buffer, at 35 Watt for 2-4 hours at 4°C. Gels were silver stained using standard protocols.
    De unión analysis was performed using FASTLINK package for two points analysis and Genehunter package for multipoint analysis. Both autosomal recessive and dominant transmission, with or without incomplete penetrance were taken into consideration; the disease gene frequency in the general population was assumed to be 0.0001.

    1.3. Evaluation of mutations in ADAMTS13 gene.
    In HUS patients with a congenital deficiency of ADAMTS13 activity showing a positive de unión on ADAMTS13 locus, search for mutation within ADAMTS13 gene was performed by SSCP and sequencing. All of the ADAMTS13 gene mutations that were found, were excluded as common sequence polymorphisms by screening a panel of 100 anaffected chromosomes.
    Specific methods.
    ADAMTS13 exons were amplified using primers located in the flanking introns and analyzed by conformation analysis of single-stranded DNA (SSCP). A total of 100 ng of genomic DNA were amplified as described. PCR products were electrophoresed on non denaturing 6% (62:1 acryl:bis) acrilamide gel, bands were visualized by silver staining and aberrant bands were sequenced using a CEQ 2000 XL DNA sequencer (Beckman-Coulter).

    2. Functional significance of ADAMTS13.
    Isolation of IgG fraction from plasma. Plasma from patients with high titers of antibodies against the ADAMTS13 (>0.3 U/mL) was obtained during plasma exchange and used to isolate the IgG fraction. To this purpose the first bag of plasma (about 500 mL) was collected on ACD at the begin of the first plasma exchange before infusion of donor plasma and before any treatment was started. Plasma was ricalcified with CaCl2 25 mM (final concentration), incubated at 37° C for 30 minutes to obtain serum and centrifuged. IgG from serum were purified using Protein A-Sepharose CL-4B column. Column was equilibrated with binding buffer glycine 1.5 M-NaCl 3 M pH 8.9, serum depleted of lipid layer and particulated matter was diluted 1:3 with binding buffer and loaded on column. IgG fraction was eluted §with citric acid 0.1 M pH 6, 4.5 and 3 and dialyzed against Hepes buffer. Each fraction was tested on collagen binding assay to detect the fraction that inhibits ADAMTS13 activity. We have found that the fraction eluted §at pH 3 at the concentration of 2 mg/ml completely inhibited ADAMTS13 activity. Control IgG fraction eluted §at pH 3 was obtained from plasma from healthy controls collected by plamapheresis.

    2.2. Effects of ADAMTS13 inhibition on placass.
    To evaluate whether ADAMTS13 inhibition may have been resulted in placas hyperactivation and embolias formation in vitro under flowing conditions, blood from healthy controls was used for placas adhesion experiments using collagen as thrombogenic surface. In parallel experiments anti ADAMTS13 antibodies (or normal human IgG) were added to test whether inhibition of ADAMTS13 affected placas adhesion and embolias formation. Placas adhesion assay was performed as described below using shear rates of 600 sec-1 and 1500 sec-1. Images of placas thrombi were digitized using a light microscope connected to a computer-based image analysis system and evaluated as described below.
    Specific methods.
    Blood collection and preparation. Blood samples from healthy controls were collected by venipuncture through a 19-gauge needle into heparin (10 IU/mL f.c.).
    Flow chamber and perfusion studies. Placas adhesion was performed on collagen coated slides as thrombogenic surface. Blood (in the absence or presence of anti ADAMTS13 2 mg/ml or normal human IgG 2 mg/ml) was perfused through a flow chamber using a syringe pump. A flow chamber thermostated to 37°C was used, in which one surface of the perfusion channel was a glass slide coated with collagen (200 µg/mL; Mascia Brunelli, Milan, Italy). The system was perfused with Tris buffer pH 7.3 for 1 min, then perfusion at constant flow rate with reconstituted blood was started and continued for 2 min. Samples was examined at shear rates of 600 sec-1 and 1500 sec-1. Blood was then withdrawn from the circuit, and placas thrombi adherent to the collagen coated surface were fixed and stained with May Grunwald-Giemsa. Images of placas thrombi were digitized using a light microscope connected to a computer-based image analysis system. The percentage of the surface occupied by thrombi was evaluated by automatic edge detection using built-in specific functions of the software Image 1.61 (NIH Bethesda, MD). Ten images, systematically digitized along the adhesion surface, were processed for adhesion test.

    New aim. To express recombinant wild type and mutated ADAMTS13.
    Cloning, expression, and functional characterization of recombinant ADAMTS13 in Drosophila cells. Specific methods.
    cDNA was obtained by RT-PCR with random hexamer primers using as a template a commercially available total RNA preparation from human liver. This was followed by PCR amplification using a high fidelity DNA polymerase (Platinum Pfx, Invitrogen Corporation) and primers amplifying the ADAMTS13 coding region beginning with the codon for the first Ala residue identified as the amino terminus of the enzyme purified from human plasma. The PCR product was ligated in the pCR-Blunt vector (Invitrogen Corporation) and cloned into E.coli TOP10. Individual clones were then screened to find a plasmid with no errors introduced by the PCR. This cDNA was cloned into the expression vector pMT/Bip/HIS that directed the synthesis of ADAMTS13 with a carboxyl terminal His tag under the control of a copper sulfate-inducible metallothionein promoter. This construct was introduced into Drosophila Schneider 2 (S2) cells by cotransfection with a vector carrying a hygromycin B resistance gene. Hygromycin B-resistant cells were selected as a stable polyclonal population, induced with copper sulfate (500 _M) and examined for expression of ADAMTS13 into the culture medium by Western blot analysis using for detection an anti-His tag monoclonal antibody.
    The purification of recombinant ADAMTS13 was performed using immobilized-metal-ion affinity chromatography (IMAC) procedure for the direct capture of the his-tagged protein from conditioned medium. To further optimize recombinant ADAMTS13 purification, heparin-sepharose chromatography was performed. The recombinant ADAMTS13 eluted §off with a step-wise gradient (0-300 mM NaCl) was collected as 6 fractions across two peaks. The fractions corresponding to each peak were pooled and displayed the same activity evaluated by the cleavage § of the recombinant VWF A1-A2-A3, likely reflecting two different glycosylated isoforms. The proteolysis of rVWF A1-A2-A3 was completely inhibited by the addition of the purified IgG obtained from a patient with high titer of anti-ADAMTS13 antibodies.

    The finding that also a subgroup of patients with atypical HUS had a congenital deficiency of ADAMTS13 activity may prompt the development of a new therapy based on replacement of the missing activity by the infusion of recombinant human ADAMTS13. This treatment will avoid the risks and complications -i.e.: allergic reactions, blood borne transmitted diseases, the need of specialized center for plasmaexchange- linked to plasma treatment.
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