Localization of a cross-link donor site in the α-chain of human fibrin

Corcoran, Doris; Ferguson, Earl W.; Fretto, L J; McKee, Patrick A.

doi:10.1016/0049-3848(80)90016-x

Cited by 25 publications

(16 citation statements)

References 6 publications

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“…Although the present data may therefore represent an overestimate of cross-linking information, several lines of evidence indirectly support the specific ␣ chain cross-linking findings obtained. Identification of Lys 508 as a donor site by lysine labeling is consistent with the previously reported involvement of the region A␣ 477-517 (CNBr IX), with its single lysine residue, in ␣ polymer formation (21). Identification of Lys 539 cross-linked to the acceptor probe is in keeping with the recent observation that a mono- Table IV) Cross-linked peptide 5 (1.04 nmol) was subjected to NH 2 -terminal sequencing for 12 steps, through the COOH-terminal residues of its donor and acceptor components.…”

Section: Discussionsupporting

confidence: 75%

Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Sobel

Gawinowicz

1996

Journal of Biological Chemistry

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Biochemical studies of fibrin cross-linking were conducted to identify the specific A␣ chain lysine residues that potentially serve as Factor XIII a amine donor substrates during ␣ polymer formation. A previously characterized Factor XIII a fibrin lysine labeling system was employed to localize sites of donor activity based on their covalent incorporation of a synthetic peptide acceptor substrate analog modelled after the NH 2 -terminal cross-linking domain of ␣ 2 antiplasmin. Peptide-decorated fibrin was prepared using purified fibrinogen as the starting material. Cyanogen bromide digestion, immunoaffinity chromatography, high pressure liquid chromatography (HPLC), and enzyme-linked immunosorbent assay (anti-peptide) methodologies were employed to isolate purified CNBr fibrin fragments whose structures included the acceptor probe in cross-linked form and, therefore, represented regions of (amine) donor activity. Five ␣ chain CNBr fragments (within A␣ 208 -610) and one ␥ chain CNBr fragment (␥ 385-411) were the only portions of fibrin found associated with the acceptor peptide, based on collective sequencing, mass, and compositional data. Trypsin digestion, HPLC, and enzyme-linked immunosorbent assay (anti-peptide) methodologies were used to isolate smaller derivatives whose structures included an ␣ chain tryptic cleavage product (the donor arm) cross-linked to the trypsin-resistant synthetic peptide (the acceptor arm). Biochemical characterization and quantitative peptide recovery data revealed that 12 of the 23 potential lysine donor residues within ␣ 208 -610 had incorporated the peptide probe, whereas ␥ chain donor activity was due solely to peptide cross-linking at (␥) Lys , and/or Lys 219 responsible for the remaining proportion (2-5%, each). The collective findings extend current models proposed for the mechanism of ␣ polymer formation, raise questions concerning the physiological role of multiple ␣ chain donor sites, and, most importantly, provide specific information that should facilitate future efforts to identify the respective lysine and glutamine partners involved in native fibrin ␣ chain cross-linking.The structure-function relationships involved in fibrinogen's transition to the cross-linked fibrin gel that forms the hemostatically active portion of a thrombus has been the subject of intense investigation for more than two decades. During this time, the complete primary structure of this large molecule has been elucidated (1-6), its domainal architecture has been characterized (7-9), and the mechanisms involved in the initial events of the transition, i.e. thrombin cleavage and fibrin polymerization, have been defined (as reviewed in Ref. 10). Although these aspects of fibrinogen biochemistry are well understood, less is known about the final stages of fibrin formation in which Factor XIII a cross-links are introduced between neighboring fibrin molecules to stabilize the alignments created during polymerization.Factor XIII a , or plasma transglutaminase, catalyzes the introduction of ⑀-(␥-glutamy...

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Section: Discussionsupporting

confidence: 75%

Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Sobel

Gawinowicz

1996

Journal of Biological Chemistry

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“…In a slower reaction, crosslinking can also occur between α chains of the adjacent molecules. Re spective acceptor sites are located in the middle sec tion of each α chain at positions 328 and 366, while donator sites are located at positions 508 and prob ably 556, and 562 close to the carboxy end (position 610) [20]. The crosslinkages between α chains, however, are split off in a very early stage of fibri nolysis inbound in small peptides and are no consti tuents of the crosslinked fibrin derivatives dealt with in this report.…”

Section: Structures Of Crosslinked Fibrin Derivativesmentioning

confidence: 76%

Detection and Relevance of Crosslinked Fibrin Derivatives in Blood

Graeff¹,

Hafter²

1982

Semin Thromb Hemost

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“…Intermolecular cross linking between the chains of fibrin involves a number of acceptor and donor sites which are likely to interact on a restricted, but somewhat random, basis (Chen & Doolittle, 1971). Corcoran & Ferguson (1980) have suggested that the cross link acceptor sites are in the chain at glutamine residues 328 and/or 366. These form " -(glutamyl) lysine cross links with donor residues 508 and possibly also lysine residues 556 or 562.…”

Section: Discussionmentioning

confidence: 99%

Fibrinogen Lincoln: a new truncated α chain variant with delayed clotting

et al. 1996

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A patient referred for preoperative investigation of prolonged bleeding and easy bruising was found to have increased thrombin and reptilase times; however, the thrombin catalysed release of fibrinopeptides A and B was normal. Analysis of five other family members, spanning three generations, indicated that three had a similar defect and suggested autosomal dominant inheritance. Non-reducing SDS-PAGE of purified fibrinogen from affected individuals showed that the 340 kD form of their fibrinogen ran as a doublet. SSCP (single-stranded conformational polymorphism) analysis of exon 5 of the A alpha gene, which encodes the C-terminal half of the chain, confirmed the presence of a mutation. Cycle sequencing of PCR amplified DNA revealed a 13 base pair deletion (nt 4758-4770), resulting in a frame-shift at Ala 475, which translates as four new amino acids before terminating at a new stop codon (-476His-Cys-Leu-Ala-Stop). The presence of a circulating truncated A alpha chain was confirmed when SDS-PAGE gels were probed with an alpha chain specific antisera; which showed that the variant A alpha chain comigrated with gamma chains. The truncation results in a variant A alpha chain with a deletion of 131 amino acids (480-610), and four new amino acids at the C-terminal.

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Localization of a cross-link donor site in the α-chain of human fibrin

Cited by 25 publications

References 6 publications

Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Identification of the α Chain Lysine Donor Sites Involved in Factor XIIIa Fibrin Cross-linking

Detection and Relevance of Crosslinked Fibrin Derivatives in Blood

Fibrinogen Lincoln: a new truncated α chain variant with delayed clotting

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