Research Article Antigenic Peptides Capable of Inducing ...downloads.hindawi.com/archive/2013/590329.pdf · Fundac ¸ ao Centro de Hematologia e Hemoterapia, Hemominas, Servic ¸o

Hindawi Publishing CorporationInternational Journal of PeptidesVolume 2013 Article ID 590329 6 pageshttpdxdoiorg1011552013590329

Research ArticleAntigenic Peptides Capable of Inducing SpecificAntibodies for Detection of the Major Alterations Found inType 2B Von Willebrand Disease

Marina de Oliveira Paro1 Cyntia Silva Ferreira1 Fernanda Silva Vieira1

Marcos Aureacutelio de Santana1 William Castro-Borges1

Maria Sueli Silva Namen-Lopes2 Sophie Yvette Leclercq3

Cibele Velloso-Rodrigues4 and Milton Heacutercules Guerra de Andrade1

1 Departamento de Ciencias BiologicasDECBI Instituto de Ciencias Exatas e BiologicasICEBUniversidade Federal de Ouro Preto Nucleo de Pesquisas em Ciencias BiologicasNUPEBCampus Universitario Morro do Cruzeiro 35400-000 Ouro Preto MG Brazil

2 Fundacao Centro de Hematologia e Hemoterapia Hemominas Servico de Pesquisa Alameda Ezequiel Dias 321Bairro Santa Efigenia 30130-110 Belo Horizonte MG Brazil

3 Fundacao Ezequiel Dias FUNED Rua Conde Pereira Carneiro 80 Bairro Gameleira 30510-010 Belo Horizonte MG Brazil4Departamento Basico Instituto de Ciencias Biologicas Universidade Federal de Juiz de Fora Campus de Governador ValadaresArea da Saude Avenida Doutor Raimundo Monteiro de Resende 330 Centro 35010-177 Governador Valadares MG Brazil

Correspondence should be addressed to Milton Hercules Guerra de Andrade miltonguerra00gmailcom

Received 11 March 2013 Revised 19 June 2013 Accepted 20 June 2013

Academic Editor Jean-Marie Zajac

Copyright copy 2013 Marina de Oliveira Paro et alThis is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in anymedium provided the originalwork is properly cited

Von Willebrand disease (VWD) is an inherited hemorrhagic disorder promoted by either quantitative or qualitative defects of thevon Willebrand factor (VWF) The disease represents the most common human coagulopathy afflicting 13 of the populationQualitative defects are subdivided into four subtypes and classified according to the molecular dysfunction of the VWF Thedifferential diagnosis of the VWD is a difficult task relying on a panel of tests aimed to assess the plasma levels and functionof the VWF Here we propose biochemical approaches for the identification of structural variants of the VWF A bioinformaticanalysis was conducted to design seven peptides amongwhich threewere representatives of specific amino acid sequences belongingto normal VWF and four encompassed sequences found in the most common VWD subtype 2B These peptides were used toimmunize mice after which peptide-specific immunoglobulins were purified This resulted in four Ig preparations capable ofdetecting alterations in the subtype 2B VWD plus additional three antibody fractions targeting the normal VWF The panel ofantibodies could serve many applications among them (1) assessment of VWF antigen interaction (2) VWF multimer analysisand (3) production of monoclonal antibodies against VWF for therapeutic purposes as in thrombotic thrombocytopenic purpura

1 Introduction

VonWillebrand disease (VWD) is an inherited hemorrhagicdisturbance related to quantitative andor qualitative defectsof the von Willebrand factor (VWF) [1 2] VWD prevalencevaries between 08 and 20 depending on the investigatedpopulation being considered the most common coagulopa-thy afflicting humans [3]

The VWF is a multimeric plasma protein composed ofa varying number of 250 kDa monomers which exhibits anessential role in primary haemostasis Some of its reportedfunctions are the attachment of platelets to subendothelialcollagen at injured sites (platelet plug formation) and protec-tion binding and transportation of coagulation factor VIII[4] Mechanisms leading to the disease present themselves ashighly diverse at the molecular level giving rise to a variety

2 International Journal of Peptides

of clinical outcomes Through different laboratory criteria itis possible to identify three primary types of the disease [5]Alterations on the plasma levels of VWF are associated withVWD types 1 and 3 whereas structural and functional defectsof VWF result in VWD type 2 [3 6ndash11] VWD types 1 and3 reflect respectively partial and complete deficiency of theVWF VWD type 2 is also classified into four subtypes Insubtype 2A there is enhanced platelet adhesion caused bythe selective deficiency of high molecular mass multimers ofVWF (HMW) In subtype 2B VWD it is observed increasedaffinity of VWF by platelet glycoprotein Ib (GpIb) associatedwith loss of HMW-VWF and mild thrombocytopenia Incontrast in subtype 2M a pronounced reduction in plateletadhesion is described even considering the relatively normalsize of the VWF multimers Variants of the VWF in thesubtype 2N display reduced affinity by coagulation factorVIII (FVIII) Altogether these six VWD types correlate withdiverse clinical outcomes each requiring adequate therapeu-tic interventions [4 12] Whilst some patients with type 2BVWD can be treated with desmopressin patients who donot show a satisfactory response to this drug should receiveVWFFVIII-containing products [13 14]

Considering the current difficulties associated with diag-nosis of the qualitative defects of the VWF the developmentof novel biochemical approaches that would allow detectionof such molecular alterations is of great biotechnologicalinterest The possibility of a precise and direct diagnosis ofqualitative defects in the VWF would certainly permit appli-cation of a better oriented medical approach to afflictedpatients In this context it is worth emphasizing that anti-bodies capable of detecting structural variants of the VWFare not commercially available The design of peptides forsynthesis and further generation of antibodies to be employedin the identification of structural alterations in the VWFmight therefore represent a convenient way forward

It has previously been shown that amino acid substitu-tions R1306W R1308C V1316M and R1341Q account for 90prevalence of the subtype 2B VWD [6 8] These mutationsin the A1 domain of VWF are responsible for a ldquogain-of-functionrdquo defect allowing for an increased affinity of largemultimers to platelets in the circulation [15] In the presentinvestigation we have generated a panel of antipeptide-specific antibodies useful at detecting the aforementionedstructural variants of the VWF

2 Materials and Methods

21 Ethics Statement All experiments involving mice wereconducted according to approved guidelines for animal useand care defined by the Local Ethics Committee on AnimalExperimentation (CEUAUFOP) Healthy individuals wereinformed previously of the investigatory nature of this studyand after giving their consent plasma samples were obtainedunder the procedures approved by the Local Ethics Commit-tee from Hemominas Foundation MG Brazil

22 Selection of VWF Peptides for Synthesis As stated previ-ously the VWF amino acid substitutions R1306W R1308C

V1316M and R1341Q are collectively found in the majority ofsubtype 2B VWD patients [6] In order to design signaturepeptides that would represent such mutations Homo sapi-ens VWF sequences were retrieved from the InternationalSociety on Thrombosis and Haemostasis database avail-able at httpwwwvwfgroupshefacuk Predicted peptidesequences for synthesis obeyed the following criteria (1)peptide size should be within the 8 to 10 mer range (2) pep-tides should contain one aromatic residue for estimationof peptide yield after synthesis and HPLC purification (inthe absence of an aromatic residue this was added atthe peptide C-terminal) (3) the location of the peptidesequences in the crystal structure of the VWF (available athttpwwwpdbjorg) revealed by the Swiss-Pdb Viewer 37should demonstrate their exposure to the solvent meaningthat highly hydrophobic peptide sequences were not consid-ered in this study

The selected peptide sequences were then aligned toMusmusculus VWF sequence using ClustalW2 (httpwwwebiacukToolsmsaclustalw2) to reveal species specific aminoacid differences as predictors for successful production ofanti-VWF peptide antibodies in mice

23 Synthesis Characterization and Production of Poly-clonal Antibodies against VWF Peptides Peptides selectedfor synthesis were representatives of the normal and alteredversions of the VWF found in the major subtypes of type 2VWD (Table 1) Peptides were synthesized using the solid-phase protocol essentially as described by Merrifield [16]Briefly the support matrix consisted of Rink Amide ResinHL (Merck Germany) at 078mmolg for an expectedmaximum yield of 40 120583M of peptides per synthesis usingFmoc-derivatized amino acids Synthetic peptides were thenpurified via reversed-phase chromatography using a C18column (Shim-pack CLD-ODS Shimadzu Japan) on a Shi-madzu HPLC system Selected peptide peaks were recoveredfor analysis through direct injection onto an electrospray-operating mass spectrometer (LCMS-IT-ToF ShimadzuJapan) in positive ionization mode and capillary voltage setto 4500VMass spectrometric data were acquired over 10msafter which mz values obtained were compared with theexpected molecular masses for the synthesized peptides

Synthetic peptides representatives of both normal andaltered VWF were individually coupled to the highly immu-nogen carrier protein keyhole limpet hemocyanin (KLHSigma) at 1 1 ratio (1mg peptide 1mg KLH) using glutaral-dehyde as the crosslinking agent [17] Polyclonal anti-KLHpeptide antibodies were raised in male Swiss mice aged tenweeks Immunization regimen consisted of three intraperi-toneal administrations of 50120583g of theKLH-peptide conjugateprepared in 10 aluminium hydroxide as adjuvant each ata 15 days interval Blood was withdrawn after 45 days post-immunization

24 Immobilization of Synthetic Peptides on Sepharose-4Band Purification of Anti-VWF Specific ImmunoglobulinsAffinity columns (02mL) containing immobilized syntheticpeptides at approximately 10mgmL were produced as

International Journal of Peptides 3

Table 1 Proposed synthetic peptides for detection of the major qualitative alterations found in VWD type 2B

Major qualitativealterations found inVWD type 2B

Designed 8 mer peptides for synthesis Theoretical[M + H]+

Observed[M + H]+

Respective peptide fromM musculus VWF

R1306W MEWLRISY 109754 111356lowast

MERLHISR1308C MERLCISY 101447 101450Respective peptide found in H sapiens VWF MERLRISY 106756 108257lowast

V1316M SQKWVRMA 100552 102054lowastSQKRIRVA

Respective peptide found in H sapiens VWF SQKWVRVA 97355 97257[minus1H+]

R1341Q RPSELQRY 104855 104857RPSELRRRespective peptide found in H sapiens VWF RPSELRRY 107659 107662

lowastPeptide mass containing an oxidized methionine

described previously [18]The antisera frommice immunizedwith KLH coupled to altered VWF peptides were loadedindividually in affinity columns bearing the correspondingnormal versions of the VWF peptide This procedure aimedthe subtraction of antibodies targeting the normal VWFApproximately 100 120583L of the unbound fraction was collectedfor analysis during the first chromatographic step whilstthe remaining were immediately submitted to further sixrounds of chromatography to guarantee complete removalof antinormal VWF antibodies For each chromatographicstep bound fractions were eluted in 5mL 01M glycine pH26 and collected in tubes containing 5mL of 04M Tris-HClpH 80 for pH neutralization Alkaline phosphatase labeledanti-mouse IgG at 1 2000 dilution was used to detect thepresence of IgGs found in both nonretained and retainedfractions from each chromatographic step using the westernblotting technique [19]

The next approach involved confirmation that antibodiestargeting the normal VWF have been thoroughly subtractedafter the six passages in affinity columns containing immo-bilized altered VWF peptides For this purpose 1mL ofpooled plasma from human healthy donors (119899 = 5) was firstprecipitated in 20 ethanol to enrich for normal VWFApproximately 10 120583g aliquots of the precipitated plasma wereresuspended in protein loading buffer and loaded in threedifferent lanes for separation on a 7 SDS-PAGEThe gel wastransferred to a PVDF (polyvinylidene fluoride) membraneand each lane western blotted with either a 100 120583L fractionobtained from the crude antisera or the two 100120583L non-retained fractions recovered from the first and sixth chro-matographic steps

The combined eluates from each chromatographic stepcontaining immunoglobulins targeting a given altered VWFpeptide were lyophilized resuspended in 1mL of phosphatebuffer pH 74 and individually loaded onto the Sepharose-4Baffinity column bearing the respective altered VWF peptideBound IgGs targeting the altered VWF peptide were elutedexactly as described above

25 Production of Carrier Albumins Bearing Altered VWFSynthetic Peptides andTheir Recognition by Anti-VWF SpecificAntibodies Aiming to produce a model sample for detection

of alteredVWF through the western blotting technique puri-fied albumin was conjugated to two representative syntheticpeptides (SQKWVRMA and RPSELQRY Table 1) [20] at a1 1 ratio respectively Briefly to each 1mg of the respectivepeptides it was added 17120583L of N-N1015840diisopropylcarbodiimidediluted 1 25 in dimethylformamide (DMF) and 20120583L of1M N-hydroxysuccinimide (also diluted in DMF) Reactionproceeded during 5min at room temperature after which50120583L of 04M sodium acetate (inDMF)was added After fur-ther 2min incubation at room temperature 1mL of bovineserum albumin (BSA Sigma) at 1mgmL prepared in 005Mammonium bicarbonate pH 75 was combined Couplingreactions occurred during 1 h followed by dialysis of eachpreparation in 01M ammonium acetate over 24 h Peptide-derivatized albumins were lyophilized and resuspended in100 120583L of saline

Five 120583g aliquots of peptide-conjugated albumin wereloaded into three lanes and separated through 12 SDS-PAGE followed byCoomassie staining A replica gel was pro-duced and transferred to a PVDF membrane Antipeptide-specific IgG purified through affinity chromatography atapproximately 015 120583g120583L was used as the primary anti-body at a final dilution of 1 500The reaction was allowed toproceed for 3 h at room temperature Development of thereactive band was achieved using alkaline phosphataselabeled anti-mouse IgG (Sigma) at 1 2000 dilution for2 h followed by addition of the alkaline phosphatase sub-strates nitroblue tetrazolium5-bromo-4-chloro-3-indolyl-phosphate (NBTBCIP Sigma) as per the manufacturerrsquosinstructions

3 Results and Discussion

In this study four amino acid sequences here termed alteredVWF peptides (MEWLRISY MERLCISY SQKWVRMAand RPSELQRY) were synthesized for generation of anti-bodies in mice These were intended for recognition of themost common qualitative defects of VWF found in subtype2B VWD Three additional peptide sequences MERLRISYSQKWVRVA and RPSELRRY representatives of the respec-tive normal versions of the VWF peptides were also pro-duced and immobilized onto Sepharose 4B for depleting


250

1 2 3

(kDa)Approx Mr

(a)

250

4 5 6

(kDa)Approx Mr

(b)

50

25

66

66

87 9 10

8998400

9998400

10998400

(kDa)Approx Mr

(kDa)Approx Mr

(c)

Figure 1 SDS-PAGE and western blotting approaches featuring the major steps involved in the production of a panel of anti-(KLH-peptide)antibodies for detection of VWD subtype 2B (a) Lane 1 10 SDS-PAGE profile of pooled human plasma after enrichment for normalVWF using ethanol precipitation Lane 2 western blotting for detection of normal VWF at approximately 250 kDa using the commerciallyavailable antibody against VWFFVIIImdashnote the presence of additional protein bands being recognized particularly at lower mass Lane 3a representative Western blotting reaction obtained with the generated panel of anti-(KLH-peptide) antibodies targeting both normal andaltered VWF (b) Detection of normal VWf by western blotting following the subtractive affinity chromatography Lane 4 detection of VWFprior to depletion of antibodies against the normal factor Lanes 5 and 6 detection of VWF using the nonretained fractions from the firstand sixth chromatographic steps respectivelymdashnote that 6 column passages proved sufficient for complete removal of anti-(KLH-peptide)antibodies (c)Lane 7 10SDS-PAGEprofile representative of the eluates obtained after affinity purification of antibodies targeting specificallyaltered VWf peptidesmdashnote the presence of IgG heavy and light chains at approximately 50 and 25 kDa respectively Lanes 8 9 and 10 10SDS-PAGE profile of nonderivatized BSA BSA-(RPSELRR) and BSA-(SQKRIRVA) respectively Lanes 81015840 91015840 and 101015840 corresponding westernblotting reactions obtained using control sera anti-(KLH-RPSELRR) and anti-(KLH-SQKRIRVA) respectively

out immunoglobulins targeting the normal VWF from theantisera raised with the altered VWF peptides Given that thecorresponding Mus musculus VWF peptides are MERLHISSQKRIRVA and RPSELRR we anticipated that the observedamino acid differences between human and mice VWFwould justify the peptides capabilities for generating specificantibodies in mice (Table 1)

After immunization the obtained antisera were testedfor their ability to specifically recognize the VWF enriched

from plasma samples of human healthy donors Figure 1(a)lane 3 is a representative reactivity obtained for all antiseraraised with either the altered or normal versions of VWFpeptides A unique band at approximately 250 kDa wasobserved coinciding exactly with a major band at the samemass observed in Figure 1(a) lane 2 This was probed witha commercially available antiserum against VWF-FVIII Incontrast the latter antibody preparation do recognize otherprotein bands (particularly of lower masses) in the sample


(Figure 1(a) lane 2) revealing that the antisera generatedherein aremore suitable for specific recognition of the humanVWF

The result obtained in Figure 1(a) lane 3 is a proof thatthe antisera produced using altered VWF peptides also con-tain IgGs targeting the normal VWF Aiming to removethose immunoglobulins from the preparations a subtractionexperimentwas conducted Firstly each individual antiserumobtained by immunization with a given altered VWF peptidewas submitted to affinity chromatography on a Sepharose4B column containing the respective immobilized normalVWF peptide Through collection of both non-retainedand retained fractions during 6 chromatographic rounds itwas observed complete removal of IgGs targeting normalVWF This can be visualized in Figure 1(b) (lanes 4 5and 6) which represents the enriched plasma probed withthe antiserum prior to affinity chromatography (lane 4) orprobed with the retained fractions from the first (lane 5) andsixth (lane 6) column passages From this result we expectedthe immunoglobulins targeting specifically the altered VWFpeptides to be found in the combined non-retained fractions

Isolation of anti-altered VWF specific immunoglobulinswas achieved through new rounds of affinity chromatogra-phies employing Sepharose 4B bearing immobilized alteredVWF peptides Bound immunoglobulins were eluted fromeach individual column and the presence of specific IgGs(heavy and light chains at approx 50 and 25 kDa resp) con-firmed by western blotting using alkaline phosphataselabeled anti-mouse IgG (Figure 1(c) lane 7)

Given the experienced difficulty in obtaining a plasmasample from a VWD patient for whom a qualitative alter-ation of the subtype 2B has been undoubtedly confirmedwe have engineered a model protein as a manner to testthe diagnostic usefulness of our produced antisera In thisapproach nonconjugated BSA BSA-SQKWVRMA plusBSA-RPSELQRY were separated using 1D 12 SDS-PAGEand the gel lanes western blotted with their respective pep-tide-specific antisera As shown in Figure 1(c) nonconjugatedalbumin which served as a control was not detected by any ofthe anti-altered VWF peptide specific antiserum (Figure 1(c)represented by lane 881015840) In contrast the two derivatizedalbumins were recognized by the respective antisera gener-ated against the two aforementioned altered VWF peptides(Figure 1(c) lanes 991015840 and 10101015840)

Finally it is worth mentioning that the protocol used toproduce the chimera albumins guarantees minimal couplingof the peptides This should have resulted in a more realisticmodel samples being produced Such information is of rel-evance considering that high sensitivity for detecting quali-tative alterations in the VWF in human plasma is obviouslydesirable

4 Conclusions

In this study a combination of bioinformatic analysis peptidesynthesis and affinity chromatography allowed the genera-tion of antipeptide specific antibodies capable of recognizingthe most common qualitative defects associated with type

2B VWD These should provide speed and innovation whenpotentially applied to the diagnosis of human VWD

Abbreviations

VWF VonWillebrand factorVWD VonWillebrand disease

Conflict of Interests

Authors do not declare conflict of interests

Acknowledgments

Thiswork was supported in part by the ConselhoNacional deDesenvolvimento Cientıfico e Tecnologico (MCTCNPqCT-SAUDE no 572010ndashClinical Genetics) and Fundacao deAmparo a Pesquisa do Estado de Minas Gerais (FAPEMIG-CDS-APQ-03515-10 and CBB-APQ-03096-09)

References

[1] U Budde and R Schneppenheim ldquovon Willebrand factor andvon Willebrand diseaserdquo Reviews in Clinical and ExperimentalHematology vol 5 no 4 pp 335ndash368 2001

[2] A H James ldquoVonWillebrand disease in women awareness anddiagnosisrdquoThrombosis Research vol 124 no 1 pp S7ndashS10 2009

[3] E J Werner E H Broxson E L Tucker D S Giroux J Shultsand T C Abshire ldquoPrevalence of von Willebrand disease inchildren a multiethnic studyrdquo Journal of Pediatrics vol 123 no6 pp 893ndash898 1993

[4] D Lillicrap ldquoVon Willebrand disease-Phenotype versus geno-type deficiency versus diseaserdquo Thrombosis Research vol 120no 1 pp S11ndashS16 2007

[5] S Keeney and A M Cumming ldquoThe molecular biology of vonwillebrand diseaserdquo Clinical and Laboratory Haematology vol23 no 4 pp 209ndash230 2001

[6] G Castaman A B Federici F Rodeghiero and P M Man-nucci ldquoVon Willebrandrsquos disease in the year 2003 towards thecomplete identification of gene defects for correct diagnosis andtreatmentrdquo Haematologica vol 88 no 1 pp 94ndash108 2003

[7] G Castaman J C J Eikenboom R M Bertina and F Rodegh-iero ldquoInconsistency of association between type 1 von Wille-brand disease phenotype and genotype in families identified inan epidemiological investigationrdquoThrombosis and Haemostasisvol 82 no 3 pp 1065ndash1070 1999

[8] DGinsburg J E Sadler TAbe et al ldquoVonWillebranddisease adatabase of point mutations insertions and deletionsrdquoThrom-bosis and Haemostasis vol 69 no 2 pp 177ndash184 1993

[9] F Rodeghiero G Castaman and A Tosetto ldquoVon Willebrandfactor antigen is less sensitive than ristocetin cofactor for thediagnosis of type I von Willebrand diseasemdashresults based onan epidemiological investigationrdquoThrombosis and Haemostasisvol 64 no 3 pp 349ndash352 1990

[10] J E Sadler ldquoA revised classification of von Willebrand dis-ease For the Subcommittee on von Willebrand Factor of theScientific and Standardization Committee of the InternationalSociety on Thrombosis and Haemostasisrdquo Thrombosis andHaemostasis vol 71 no 4 pp 520ndash525 1994


[11] J E Sadler T Matsushita Z Y Dong E A Tuley and L AWestfield ldquoMolecular mechanism and classification of vonWil-lebrand diseaserdquoThrombosis and Haemostasis vol 74 no 1 pp161ndash166 1995

[12] J E Sadler U Budde J C J Eikenboom et al ldquoUpdate on thepathophysiology and classification of vonWillebrand disease areport of the Subcommittee on von Willebrand factorrdquo Journalof Thrombosis and Haemostasis vol 4 no 10 pp 2103ndash21142006

[13] M S Enayat A M Guilliatt W Lester J T Wilde M D Will-iams and F G H Hill ldquoDistinguishing between type 2B andpseudo-von Willebrand disease and its clinical importancerdquoBritish Journal of Haematology vol 133 no 6 pp 664ndash6662006

[14] F Rodeghiero G Castaman and A Tosetto ldquoOptimizing treat-ment of von Willebrand disease by using phenotypic andmolecular datardquo Hematology vol 2009 pp 113ndash123 2009

[15] DMeyer E Fressinaud L Hilbert A-S Ribba J-M Lavergneand CMazurier ldquoType 2 vonWillebrand disease causing defec-tive von Willebrand factor-dependent platelet functionrdquo BestPractice and Research vol 14 no 2 pp 349ndash364 2001

[16] R B Merrifield ldquoSolid-phase peptide synthesisrdquo Advances inEnzymology and Related Areas of Molecular Biology vol 32 pp221ndash296 1969

[17] D Drenckhahn T Jons and F Schmitz ldquoChapter 2 productionof polyclonal antibodies against proteins and peptidesrdquoMethodsin Cell Biology vol 37 pp 7ndash56 1993

[18] I Matsumoto Y Mizuno and N Seno ldquoActivation of sepharosewith epichiorohydrin and subsequent immobilization of ligandfor affinity adsorbentrdquo Journal of Biochemistry vol 85 no 4 pp1091ndash1098 1979

[19] H Towbin T Staehelin and J Gordon ldquoElectrophoretic trans-fer of proteins frompolyacrylamide gels to nitrocellulose sheetsprocedure and some applicationsrdquo Proceedings of the NationalAcademy of Sciences of the United States of America vol 76 no9 pp 4350ndash4354 1979

[20] Bangs Laboratories Covalent Coupling Protocols Bangs Labo-ratories 2008

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International Journal of

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Zoology


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Signal TransductionJournal of


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Advances in

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Volume 2014

Stem CellsInternational



Enzyme Research



Microbiology


of clinical outcomes Through different laboratory criteria itis possible to identify three primary types of the disease [5]Alterations on the plasma levels of VWF are associated withVWD types 1 and 3 whereas structural and functional defectsof VWF result in VWD type 2 [3 6ndash11] VWD types 1 and3 reflect respectively partial and complete deficiency of theVWF VWD type 2 is also classified into four subtypes Insubtype 2A there is enhanced platelet adhesion caused bythe selective deficiency of high molecular mass multimers ofVWF (HMW) In subtype 2B VWD it is observed increasedaffinity of VWF by platelet glycoprotein Ib (GpIb) associatedwith loss of HMW-VWF and mild thrombocytopenia Incontrast in subtype 2M a pronounced reduction in plateletadhesion is described even considering the relatively normalsize of the VWF multimers Variants of the VWF in thesubtype 2N display reduced affinity by coagulation factorVIII (FVIII) Altogether these six VWD types correlate withdiverse clinical outcomes each requiring adequate therapeu-tic interventions [4 12] Whilst some patients with type 2BVWD can be treated with desmopressin patients who donot show a satisfactory response to this drug should receiveVWFFVIII-containing products [13 14]

Considering the current difficulties associated with diag-nosis of the qualitative defects of the VWF the developmentof novel biochemical approaches that would allow detectionof such molecular alterations is of great biotechnologicalinterest The possibility of a precise and direct diagnosis ofqualitative defects in the VWF would certainly permit appli-cation of a better oriented medical approach to afflictedpatients In this context it is worth emphasizing that anti-bodies capable of detecting structural variants of the VWFare not commercially available The design of peptides forsynthesis and further generation of antibodies to be employedin the identification of structural alterations in the VWFmight therefore represent a convenient way forward

It has previously been shown that amino acid substitu-tions R1306W R1308C V1316M and R1341Q account for 90prevalence of the subtype 2B VWD [6 8] These mutationsin the A1 domain of VWF are responsible for a ldquogain-of-functionrdquo defect allowing for an increased affinity of largemultimers to platelets in the circulation [15] In the presentinvestigation we have generated a panel of antipeptide-specific antibodies useful at detecting the aforementionedstructural variants of the VWF

2 Materials and Methods

21 Ethics Statement All experiments involving mice wereconducted according to approved guidelines for animal useand care defined by the Local Ethics Committee on AnimalExperimentation (CEUAUFOP) Healthy individuals wereinformed previously of the investigatory nature of this studyand after giving their consent plasma samples were obtainedunder the procedures approved by the Local Ethics Commit-tee from Hemominas Foundation MG Brazil

22 Selection of VWF Peptides for Synthesis As stated previ-ously the VWF amino acid substitutions R1306W R1308C

V1316M and R1341Q are collectively found in the majority ofsubtype 2B VWD patients [6] In order to design signaturepeptides that would represent such mutations Homo sapi-ens VWF sequences were retrieved from the InternationalSociety on Thrombosis and Haemostasis database avail-able at httpwwwvwfgroupshefacuk Predicted peptidesequences for synthesis obeyed the following criteria (1)peptide size should be within the 8 to 10 mer range (2) pep-tides should contain one aromatic residue for estimationof peptide yield after synthesis and HPLC purification (inthe absence of an aromatic residue this was added atthe peptide C-terminal) (3) the location of the peptidesequences in the crystal structure of the VWF (available athttpwwwpdbjorg) revealed by the Swiss-Pdb Viewer 37should demonstrate their exposure to the solvent meaningthat highly hydrophobic peptide sequences were not consid-ered in this study

The selected peptide sequences were then aligned toMusmusculus VWF sequence using ClustalW2 (httpwwwebiacukToolsmsaclustalw2) to reveal species specific aminoacid differences as predictors for successful production ofanti-VWF peptide antibodies in mice

23 Synthesis Characterization and Production of Poly-clonal Antibodies against VWF Peptides Peptides selectedfor synthesis were representatives of the normal and alteredversions of the VWF found in the major subtypes of type 2VWD (Table 1) Peptides were synthesized using the solid-phase protocol essentially as described by Merrifield [16]Briefly the support matrix consisted of Rink Amide ResinHL (Merck Germany) at 078mmolg for an expectedmaximum yield of 40 120583M of peptides per synthesis usingFmoc-derivatized amino acids Synthetic peptides were thenpurified via reversed-phase chromatography using a C18column (Shim-pack CLD-ODS Shimadzu Japan) on a Shi-madzu HPLC system Selected peptide peaks were recoveredfor analysis through direct injection onto an electrospray-operating mass spectrometer (LCMS-IT-ToF ShimadzuJapan) in positive ionization mode and capillary voltage setto 4500VMass spectrometric data were acquired over 10msafter which mz values obtained were compared with theexpected molecular masses for the synthesized peptides

Synthetic peptides representatives of both normal andaltered VWF were individually coupled to the highly immu-nogen carrier protein keyhole limpet hemocyanin (KLHSigma) at 1 1 ratio (1mg peptide 1mg KLH) using glutaral-dehyde as the crosslinking agent [17] Polyclonal anti-KLHpeptide antibodies were raised in male Swiss mice aged tenweeks Immunization regimen consisted of three intraperi-toneal administrations of 50120583g of theKLH-peptide conjugateprepared in 10 aluminium hydroxide as adjuvant each ata 15 days interval Blood was withdrawn after 45 days post-immunization

24 Immobilization of Synthetic Peptides on Sepharose-4Band Purification of Anti-VWF Specific ImmunoglobulinsAffinity columns (02mL) containing immobilized syntheticpeptides at approximately 10mgmL were produced as





Observed[M + H]+

















250

1 2 3

(kDa)Approx Mr

(a)

250

4 5 6

(kDa)Approx Mr

(b)

50

25

66

66

87 9 10

8998400

9998400

10998400

(kDa)Approx Mr

(kDa)Approx Mr

(c)











4 Conclusions



Abbreviations




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





Observed[M + H]+

















250

1 2 3

(kDa)Approx Mr

(a)

250

4 5 6

(kDa)Approx Mr

(b)

50

25

66

66

87 9 10

8998400

9998400

10998400

(kDa)Approx Mr

(kDa)Approx Mr

(c)











4 Conclusions



Abbreviations




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


250

1 2 3

(kDa)Approx Mr

(a)

250

4 5 6

(kDa)Approx Mr

(b)

50

25

66

66

87 9 10

8998400

9998400

10998400

(kDa)Approx Mr

(kDa)Approx Mr

(c)











4 Conclusions



Abbreviations




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology







4 Conclusions



Abbreviations




Acknowledgments


References





























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology








Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

Documents

Research Article Antigenic Peptides Capable of Inducing ...downloads.hindawi.com/archive/2013/590329.pdf · Fundac ¸ ao Centro de Hematologia e Hemoterapia, Hemominas, Servic ¸o