Action of Thrombin in the Clotting of Fibrinogen

Bailey, Kenneth; Bettelheim, F. R.; Lóránd, L.; Middlebrook, W. R.

doi:10.1038/167233a0

Cited by 233 publications

(76 citation statements)

References 8 publications

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“…Cleavage of FPA occurs rapidly, exposes a polymerization site at the amino terminus of the a chain (52), and can result in fibrin polymerization without FPB cleavage as demonstrated by the action of reptilase (53). Cleavage of FPB by thrombin occurs more slowly (54,55), and exposes a secondary polymerization site that accelerates fibrin polymerization (56) and contributes to lateral organization of fibrin protofibrils (57). Additionally cleavage of FPB exposes a fibrin specific epitope, 115-21, for which monoclonal antibodies have been identified (58,59).…”

Section: Discussionmentioning

confidence: 99%

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Bunce

Sporn²,

Francis³

1992

J. Clin. Invest.

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Adhesion and spreading ofcultured human umbilical vein endothelial cells on fibrin surfaces of varying structure were characterized to understand better the interactions occurring between endothelium and fibrin at sites of vascular injury. Fibrin prepared with reptilase, which cleaves only fibrinopeptide A from fibrinogen, and fibrin prepared with thrombin, which cleaves both fibrinopeptide A and fibrinopeptide B, equally supported endothelial cell adhesion. In contrast, only fibrin made with thrombin mediated endothelial cell spreading, as assessed by fluorescence microscopy of cells stained with rhodamine phalloidin to identify actin stress fibers or by scanning electron microscopy. Fibrin prepared with reptilase failed to support cell spreading. To further investigate the role of the amino terminus of the fibrin 6 chain after fibrinopeptide B cleavage in promoting cell spreading, protease III from Crotalus atrox venom was used to specifically cleave the amino-terminal 42 residues of the fibrinogen B,) chain. After clotting with thrombin, this fibrin derivative lacking B#1-42 failed to support significant cell spreading. Spreading on fibrin was unaffected by depletion of Weibel-Palade bodies from endothelial cells, indicating that the spreading was independent of stimulated von Willebrand factor release. We conclude that endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and involves residues 1542 of the fibrin 8 chain. (J. Clin. Invest.

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Section: Discussionmentioning

confidence: 99%

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Bunce

Sporn²,

Francis³

1992

J. Clin. Invest.

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“…The ratio is usually expressed as the percentage of the total nitrogen which appears in the clot (% nitrogen clottable). It should be noted that there is evidence that a peptide is split from fibrinogen during the clotting process (Bailey, Bettelheim, Lorand & Middlebrook, 1951;Lorand, 1952). For bovine fibrinogen the peptide nitrogen split off is close to 3*0-3.5% of the fibrinogen nitrogen.…”

Section: Purification Of Human Fibrinogenmentioning

confidence: 99%

The purification of human fibrinogen

Kekwick

Mackay

Nance

et al. 1955

Biochemical Journal

162

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For investigations concerning the mode of action of the clearing factor, estimations of free fatty acids in mixtures of normal rat plasma and lipaemic dog plasma were performed using a modification of van de Kamer's (1948) method as described for faeces. Because the method developed may be of value for similar or other uses, it is given here. In a 100 ml. flask 2 ml. of the plasma-mixture, containing either heparin or citrate as an anticoagulant, are run into 6 ml. of phosphoric acid solution (metaphosphoric acid, 50 g., NaCl, 250 g. water to 1 1.). After a few minutes 16 ml. of ethanol, containing 0-4 % amyl alcohol, are added, followed by 20 ml. of distilled light petroleum (b.p. 40-60°). The flask is stoppered and the mixture shaken by hand for 1 min. After separation, the upper layer is filtered through dry paper and the funnel covered in order to minimize evaporation. Care should be taken to avoid contamination with traces of the other phase. Ten ml. of the light petroleum extract is evaporated on a boiling-water bath and the residue dried in vacuo. Neutralized ethanol (5 ml.) containing 0-6 mg. of thymol blue per 100 ml. are added and the residue dissolved by boiling gently under reflux for 3 min. Finally, the fatty acids are titrated under a stream of N2 with O-O0N-NaOH.The results are corrected for a blank (usually about 0060 ml. of 001N-NaOH), obtained by running 2 ml. of water through the same procedure.The reproducibility of the method suffices for many purposes. In twenty-four estimations giving results ranging from 021 to 210 0,-equiv. of fatty acids per 2 ml. of plasma tested, the differences between duplicate determinations were found to have a standard deviation of + 0-14 ,u-equiv.

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“…The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.The specific cleavage of fibrinogen by the serine proteinase thrombin initiates the polymerisation of fibrin monomers, a primary event in blood clot formation [4]. Fibrinogen (340 kDa) is a covalently linked dimer of three peptide chains, with stoichiometry (Aa, BP,y), [5].…”

mentioning

confidence: 99%

“…The specific cleavage of fibrinogen by the serine proteinase thrombin initiates the polymerisation of fibrin monomers, a primary event in blood clot formation [4]. Fibrinogen (340 kDa) is a covalently linked dimer of three peptide chains, with stoichiometry (Aa, BP,y), [5].…”

mentioning

confidence: 99%

The interaction of thrombin with fibrinogen

Stubbs

Oschkinat

Mayr

et al. 1992

European Journal of Biochemistry

217

303

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The structure of the ternary complex of human a-thrombin with a covalently bound analogue of fibrinopeptide A and a C-terminal hirudin peptide has been determined by X-ray diffraction methods at 0.25 nm resolution. Fibrinopeptide A folds in a compact manner, bringing together hydrophobic residues that slot into the apolar binding site of human a-thrombin. Fibrinogen residue Phe8 occupies the aryl-binding site of thrombin, adjacent to fibrinogen residues Leu9 and Val15 in the S2 subsite. The species diversity of fibrinopeptide A is analysed with respect to its conformation and its interaction with thrombin. The non-covalently attached peptide fragment hirudin(54 -65) exhibits an identical conformation to that observed in the hirudin-thrombin complex. The occupancy of the secondary fibrinogen-recognition exosite by this peptide imposes restrictions on the manner of fibrinogen binding. The surface topology of the thrombin molecule indicates positions PI' -P3', differ from those of the canonical serine-proteinase inhibitors, suggesting a mechanical model for the switching of thrombin activity from fibrinogen cleavage to protein-C activation on thrombomodulin complex formation. The multiple interactions between thrombin and fibrinogen provide an explanation for the narrow specificity of thrombin. Structural grounds can be put forward for certain congenital clotting disorders.The specific cleavage of fibrinogen by the serine proteinase thrombin initiates the polymerisation of fibrin monomers, a primary event in blood clot formation [4]. Fibrinogen (340 kDa) is a covalently linked dimer of three peptide chains, with stoichiometry (Aa, BP,y), [5]. The cleavage releases two peptides, fibrinopeptides A and B, from the N-termini of chains Aa and Bfl respectively, thereby revealing recognition sites for aggregation with the y chain.Thrombin exhibits primarily a trypsin-like specificity, i.e. a preference for P1 arginine residues [6]. The cleavage of fibrinogen by thrombin represents a very specific reaction however; of the 376 Arg/Lys-Xaa bonds in the fibrinogen molecule, thrombin cleaves only four, releasing the fibrinopeptides [6]. Residues of fibrinogen contributing to this exceptional specificity have been localised to the first 51 amino acids of the Acc chain [7, 81. This region has been further dissected to explore subsites, assigning roles (in order of im-Enzyrnr. Thrombin (EC 3.4 .21 .S).Nomencluture. The peptide and subsite nomenclature is that suggested by Schechter and Berger 111: amino acid residues of substrates are numbered P1, P2, P3 etc. towards the amino terminus, and P1 ', P2', P3' etc. towards the carboxy terminus from the reactive-sitc bond; the complementary subsites of the enzyme are numbered S1, S2, S3 etc. and SI', S2', S3' etc., respectively. Thrombin residues are numbered according to the chymotrypsinogen system [2], with inserted residues marked by a lower-case suffix [ 3 ] . Fibrinogen residues are prefixed by the letter F, hirudin residues by the letter H and uPhe-Pro-Arg-MeC1 residues by the le...

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Action of Thrombin in the Clotting of Fibrinogen

Cited by 233 publications

References 8 publications

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

Endothelial cell spreading on fibrin requires fibrinopeptide B cleavage and amino acid residues 15-42 of the beta chain.

The purification of human fibrinogen

The interaction of thrombin with fibrinogen

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