{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,10]],"date-time":"2026-10-10T01:57:35Z","timestamp":1791597455015,"version":"4.3.4"},"reference-count":40,"publisher":"American Society of Hematology","issue":"2","content-domain":{"domain":["ashpublications.org"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[2001,7,15]]},"abstract":"<jats:p>Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A\u03b1 R16C and \u03b3 G165R. As seen previously with other heterozygous A\u03b1 R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A\u03b1 16 C. Sodium dodecyl sulfate\u2013polyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the \u03b1-helix content in the variant D3 fragment. It is concluded that the A\u03b1-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the \u03b3-chain induced a conformational change in the D3 fragment.<\/jats:p>","DOI":"10.1182\/blood.v98.2.351","type":"journal-article","created":{"date-parts":[[2002,7,27]],"date-time":"2002-07-27T12:46:47Z","timestamp":1027774007000},"page":"351-357","update-policy":"https:\/\/doi.org\/10.1182\/blood.2019cm0000","source":"Crossref","is-referenced-by-count":11,"title":["Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, A\u03b1 R16C and \u03b3 G165R"],"prefix":"10.1182","volume":"98","author":[{"given":"Bettina","family":"Bolliger-Stucki","sequence":"first","affiliation":[{"name":"From the Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland; and the Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susan T.","family":"Lord","sequence":"additional","affiliation":[{"name":"From the Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland; and the Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill."}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miha","family":"Furlan","sequence":"additional","affiliation":[{"name":"From the Central Hematology Laboratory, Inselspital, University Hospital, Bern, Switzerland; and the Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill."}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"234","reference":[{"key":"2020021221485024700_B1","doi-asserted-by":"crossref","first-page":"455","DOI":"10.1038\/38947","article-title":"Crystal structures of fragment D from human fibrinogen and its crosslinked counterpart from fibrin.","volume":"389","author":"Spraggon","year":"1997","journal-title":"Nature."},{"key":"2020021221485024700_B2","doi-asserted-by":"crossref","first-page":"8637","DOI":"10.1021\/bi9804129","article-title":"Crystal structure of fragment double-D from human fibrin with two different bound ligands.","volume":"37","author":"Everse","year":"1998","journal-title":"Biochemistry."},{"key":"2020021221485024700_B3","doi-asserted-by":"crossref","first-page":"187","DOI":"10.1054\/fipr.2000.0073","article-title":"Structure and function of fibrinogen inferred from hereditary dysfibrinogens.","volume":"14","author":"Matsuda","year":"2000","journal-title":"Fibrinolysis Proteolysis."},{"key":"2020021221485024700_B4","doi-asserted-by":"crossref","first-page":"1709","DOI":"10.1182\/blood.V95.5.1709.005k04_1709_1713","article-title":"Hypofibrinogenemia in an individual with 2 coding (\u03b3 82 A\u2192G and B\u03b2 235 P\u2192L) and 2 noncoding mutations.","volume":"95","author":"Brennan","year":"2000","journal-title":"Blood."},{"key":"2020021221485024700_B5","doi-asserted-by":"crossref","first-page":"237","DOI":"10.1159\/000205234","article-title":"Gerinnungsphysiologische Schnellmethode zur Bestimmung des Fibrinogens.","volume":"17","author":"Clauss","year":"1957","journal-title":"Acta Haematol."},{"key":"2020021221485024700_B6","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1016\/0003-2697(66)90246-6","article-title":"Quantitative estimation of proteins by electrophoresis in agarose gel containing antibodies.","volume":"15","author":"Laurell","year":"1966","journal-title":"Anal Biochem."},{"key":"2020021221485024700_B7","doi-asserted-by":"crossref","first-page":"118","DOI":"10.1055\/s-0038-1657143","article-title":"Effect of calcium and synthetic peptides on fibrin polymerization.","volume":"47","author":"Furlan","year":"1982","journal-title":"Thromb Haemost."},{"key":"2020021221485024700_B8","first-page":"7","article-title":"Fibrinogen Milano IV, another case of congenital dysfibrinogenemia with an abnormal fibrinopeptide A release (A\u03b1 16 Arg\u2192His).","volume":"22","author":"Bo\u0308gli","year":"1992","journal-title":"Haemostasis."},{"key":"2020021221485024700_B9","first-page":"463","article-title":"Fibrinogen Milano V: a congenital dysfibrinogenaemia with a \u03b3 275 Arg\u2192Cys substitution.","volume":"5","author":"Steinmann","year":"1994","journal-title":"Blood Coagul Fibrinolysis."},{"key":"2020021221485024700_B10","doi-asserted-by":"crossref","first-page":"680","DOI":"10.1038\/227680a0","article-title":"Cleavage of structural proteins during the assembly of the head of bacteriophage T4.","volume":"227","author":"Laemmli","year":"1970","journal-title":"Nature."},{"key":"2020021221485024700_B11","doi-asserted-by":"crossref","first-page":"1095","DOI":"10.1126\/science.170.3962.1095","article-title":"Plasminogen: purification from human plasma by affinity chromatography.","volume":"170","author":"Deutsch","year":"1970","journal-title":"Science."},{"key":"2020021221485024700_B12","doi-asserted-by":"crossref","first-page":"1719","DOI":"10.1002\/pro.5560011220","article-title":"The synthetic peptide Gly-Pro-Arg-Pro-amide limits the plasmic digestion of fibrinogen in the same fashion as calcium ion.","volume":"1","author":"Yamazumi","year":"1992","journal-title":"Protein Sci."},{"key":"2020021221485024700_B13","doi-asserted-by":"crossref","first-page":"183","DOI":"10.1016\/0378-1119(88)90355-1","article-title":"High-level expression of a functional human fibrinogen \u03b3 chain in Escherichia coli.","volume":"66","author":"Boylard","year":"1988","journal-title":"Gene."},{"key":"2020021221485024700_B14","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/0049-3848(82)90285-7","article-title":"Fractionation of plasmic fibrinogen digest on lysine-agarose: isolation of two fragments D, fragment E and simultaneously removal of plasmin.","volume":"27","author":"Rupp","year":"1982","journal-title":"Thromb Res."},{"key":"2020021221485024700_B15","doi-asserted-by":"crossref","first-page":"298","DOI":"10.1016\/0014-5793(86)80705-0","article-title":"Domain organization of the terminal parts in the fibrinogen molecule.","volume":"202","author":"Medved","year":"1986","journal-title":"FEBS Lett."},{"key":"2020021221485024700_B16","doi-asserted-by":"crossref","first-page":"663","DOI":"10.1016\/0005-2795(71)90282-0","article-title":"Difference in conformation of fibrinogen degradation products as revealed by hydrogen exchange and spectropolarimetry.","volume":"229","author":"Budzynski","year":"1971","journal-title":"Biochim Biophys Acta."},{"key":"2020021221485024700_B17","doi-asserted-by":"crossref","first-page":"2643","DOI":"10.1182\/blood.V79.10.2643.bloodjournal79102643","article-title":"Characterization of the \u03b3 chain platelet binding site on fibrinogen fragment D.","volume":"79","author":"Kirschbaum","year":"1992","journal-title":"Blood."},{"key":"2020021221485024700_B18","doi-asserted-by":"crossref","first-page":"33","DOI":"10.1021\/bi00504a006","article-title":"Estimation of globular protein secondary structure from circular dichroism.","volume":"20","author":"Provencher","year":"1981","journal-title":"Biochemistry."},{"key":"2020021221485024700_B19","doi-asserted-by":"crossref","first-page":"370","DOI":"10.1110\/ps.8.2.370","article-title":"Estimation of the number of \u03b1-helical and \u03b2-strand segments in proteins using circular dichroism spectroscopy.","volume":"8","author":"Sreerama","year":"1999","journal-title":"Protein Sci."},{"key":"2020021221485024700_B20","doi-asserted-by":"crossref","first-page":"3478","DOI":"10.1073\/pnas.89.8.3478","article-title":"Abnormal fibrinogens IJmuiden (B\u03b2Arg14\u2192Cys) and Nijmegen (B\u03b2Arg44\u2192Cys) form disulfide-linked fibrinogen-albumin complexes.","volume":"89","author":"Koopman","year":"1992","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2020021221485024700_B21","doi-asserted-by":"crossref","first-page":"268","DOI":"10.1055\/s-0037-1614456","article-title":"Fibrinogen St. Gallen I (\u03b3 292 Gly\u2192Val): evidence for structural alterations causing defective polymerization and fibrinogenolysis.","volume":"81","author":"Stucki","year":"1999","journal-title":"Thromb Haemost."},{"key":"2020021221485024700_B22","doi-asserted-by":"crossref","first-page":"605","DOI":"10.1111\/j.1432-1033.1995.tb20504.x","article-title":"Structural and functional characterization of proteolytic fragments derived from the C-terminal regions of bovine fibrinogen.","volume":"229","author":"Litvinovich","year":"1995","journal-title":"Eur J Biochem."},{"key":"2020021221485024700_B23","doi-asserted-by":"crossref","first-page":"1563","DOI":"10.1002\/pro.5560011204","article-title":"A detailed consideration of a principal domain of vertebrate fibrinogen.","volume":"1","author":"Doolittle","year":"1992","journal-title":"Protein Sci."},{"key":"2020021221485024700_B24","doi-asserted-by":"crossref","first-page":"205","DOI":"10.1002\/prot.340070302","article-title":"Protein secondary structure and circular dichroism: a practical guide.","volume":"7","author":"Johnson","year":"1990","journal-title":"Proteins."},{"key":"2020021221485024700_B25","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1006\/abio.1999.4320","article-title":"Spectroscopic methods for analysis of protein secondary structure.","volume":"277","author":"Pelton","year":"2000","journal-title":"Anal Biochem."},{"key":"2020021221485024700_B26","doi-asserted-by":"crossref","first-page":"71","DOI":"10.1073\/pnas.79.1.71","article-title":"Nucleotide sequence and the encoded amino acids of human serum albumin mRNA.","volume":"79","author":"Dugaiczyk","year":"1982","journal-title":"Proc Natl Acad Sci U S A."},{"key":"2020021221485024700_B27","doi-asserted-by":"crossref","first-page":"93","DOI":"10.1097\/00001721-199903000-00006","article-title":"Fibrinogens Bern IV, Bern V and Milano XI: three dysfunctional variants with amino acid substitutions in the thrombin cleavage site of the A\u03b1-chain.","volume":"10","author":"Stucki","year":"1999","journal-title":"Blood Coagul Fibrinolysis."},{"key":"2020021221485024700_B28","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1006\/abio.2000.4684","article-title":"Reevaluation of the electrophoretic migration behavior of soluble globular proteins in the native and detergent-denatured states in polyacrylamide gels.","volume":"284","author":"Westerhuis","year":"2000","journal-title":"Anal Biochem."},{"key":"2020021221485024700_B29","doi-asserted-by":"crossref","first-page":"176","DOI":"10.1016\/0303-2647(75)90025-8","article-title":"Sodium dodecyl sulfate in protein chemistry.","volume":"6","author":"Waehneldt","year":"1975","journal-title":"Biosystems."},{"key":"2020021221485024700_B30","doi-asserted-by":"crossref","first-page":"480","DOI":"10.1182\/blood.V71.2.480.480","article-title":"An apparently higher molecular weight \u03b3 chain variant in a new congenital abnormal fibrinogen Tochigi characterized by the replacement of \u03b3 arginine-275 by cysteine.","volume":"71","author":"Yoshida","year":"1988","journal-title":"Blood."},{"key":"2020021221485024700_B31","doi-asserted-by":"crossref","first-page":"13579","DOI":"10.1016\/S0021-9258(18)68281-X","article-title":"Substitution of \u03b3Arg 275 by Cys in an abnormal fibrinogen, \u201cFibrinogen Osaka II.\u201d","volume":"263","author":"Terukina","year":"1988","journal-title":"J Biol Chem."},{"key":"2020021221485024700_B32","doi-asserted-by":"crossref","first-page":"10","DOI":"10.1093\/oxfordjournals.jbchem.a122601","article-title":"Fibrinogen Nagoya, a replacement of glutamine-329 by arginine in the \u03b3-chain that impairs the polymerization of fibrin monomer.","volume":"105","author":"Miyata","year":"1989","journal-title":"J Biochem."},{"key":"2020021221485024700_B33","doi-asserted-by":"crossref","first-page":"1659","DOI":"10.1182\/blood.V75.8.1659.1659","article-title":"Polymerization defect of fibrinogen Baltimore III due to a \u03b3 Asn 308\u2192Ile mutation.","volume":"75","author":"Bantia","year":"1990","journal-title":"Blood."},{"key":"2020021221485024700_B34","doi-asserted-by":"crossref","first-page":"534","DOI":"10.1055\/s-0038-1646313","article-title":"Heterozygous abnormal fibrinogen Osaka III with the replacement of \u03b3 arginine-275 by histidine has an apparently higher molecular weight \u03b3-chain variant.","volume":"68","author":"Yoshida","year":"1992","journal-title":"Thromb Haemost."},{"key":"2020021221485024700_B35","doi-asserted-by":"crossref","first-page":"526","DOI":"10.1046\/j.1365-2141.1996.d01-1833.x","article-title":"Fibrinogen Matsumoto II: \u03b3 308 Asn\u2192Lys (AAT-AAG) mutation associated with bleeding tendency.","volume":"94","author":"Okumura","year":"1996","journal-title":"Br J Haematol."},{"key":"2020021221485024700_B36","doi-asserted-by":"crossref","first-page":"2309","DOI":"10.1002\/1522-2683(20000701)21:12<2316::AID-ELPS2309>3.0.CO;2-T","article-title":"Difference in electrophoretic mobility and plasmic digestion profile between four recombinant fibrinogen: \u03b3 308 K, \u03b3 308 I, \u03b3 308 A, and wild type (\u03b3 308 N).","volume":"21","author":"Okumura","year":"2000","journal-title":"Electrophoresis."},{"key":"2020021221485024700_B37","doi-asserted-by":"crossref","first-page":"103","DOI":"10.1016\/0014-5793(90)80458-U","article-title":"Conformational changes in human fibrinogen after in vitro phosphorylation and their relation to fibrinogen behaviour.","volume":"272","author":"Martin","year":"1990","journal-title":"FEBS Lett."},{"key":"2020021221485024700_B38","doi-asserted-by":"crossref","first-page":"356","DOI":"10.1002\/(SICI)1097-0134(19990215)34:3<356::AID-PROT8>3.0.CO;2-O","article-title":"Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase.","volume":"34","author":"Prasanna","year":"1999","journal-title":"Proteins."},{"key":"2020021221485024700_B39","doi-asserted-by":"crossref","first-page":"35434","DOI":"10.1074\/jbc.274.50.35434","article-title":"Site-directed mutagenesis of diphosphoinositol polyphosphate phosphohydrolase, a dual specificity NUDT enzyme that attacks diadenosine polyphosphates and diphosphoinositol polyphosphates.","volume":"274","author":"Yang","year":"1999","journal-title":"J Biol Chem."},{"key":"2020021221485024700_B40","doi-asserted-by":"crossref","first-page":"390","DOI":"10.1055\/s-0038-1657746","article-title":"Fibrinogen Bergamo I (A\u03b1 16Arg\u2192Cys): susceptibility towards thrombin following amino-ethylation, methylation or carboxamidomethylation of cysteine residues.","volume":"54","author":"Reber","year":"1985","journal-title":"Thromb Haemost."}],"container-title":["Blood"],"original-title":[],"language":"en","link":[{"URL":"http:\/\/ashpublications.org\/blood\/article-pdf\/98\/2\/351\/1675087\/h8140100351.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"http:\/\/ashpublications.org\/blood\/article-pdf\/98\/2\/351\/1675087\/h8140100351.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,5,26]],"date-time":"2021-05-26T09:22:46Z","timestamp":1622020966000},"score":1,"resource":{"primary":{"URL":"https:\/\/ashpublications.org\/blood\/article\/98\/2\/351\/107024\/Fibrinogen-Milano-XII-a-dysfunctional-variant"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2001,7,15]]},"references-count":40,"journal-issue":{"issue":"2","published-print":{"date-parts":[[2001,7,15]]}},"URL":"https:\/\/doi.org\/10.1182\/blood.v98.2.351","relation":{},"ISSN":["1528-0020","0006-4971"],"issn-type":[{"value":"1528-0020","type":"electronic"},{"value":"0006-4971","type":"print"}],"subject":[],"published":{"date-parts":[[2001,7,15]]}}}