The specificity of the fibrinogen-thrombin reaction

Hogg, Desmond H.; Blombäck, Birger

doi:10.1016/0049-3848(74)90057-7

Cited by 36 publications

(5 citation statements)

References 15 publications

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“…Despite the diminution of o2-antiplasmin its inhibitor capacity still prevents a relevant fibrinogenolysis by free plasmin whose catalytic efficiency on fibrinogen as calculated from published data (60, 6t) is about 50-200 fold lower than that of thrombin (31, 32,54,55). The practically constant heparin cofactor II activity in our experiments is explained by the small amounts of thrombin generated.…”

Section: Discussionmentioning

confidence: 93%

Tissue Thromboplastin Induced Reversible DIC and Heparin-Enhanced Inhibitors in Dogs

Marbet

Griffith

1986

Thromb Haemost

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SummaryReversible acute disseminated intravascular coagulation (DIC) has been induced in dogs by intravenous injection of homologous tissue thromboplastin. There was no measurable consumption of antithrombin III and heparin cofactor II even if fibrinogen was reduced during DIC by more than 80% of its baseline. The prothrombin level remained practically constant. These data correspond to the generation of a few nanomoles of thrombin in vivo with subsequent pseudo-first order inactivation by the major thrombin inhibitors. An ex vivo measure of the pseudo-first order rate constant (dynamic thrombin inhibitory capacity, DTIC) was a sensitive probe of circulating heparin. There was no change of DTIC during DIC in the absence of exogenous heparin suggesting that heparin-like endogenous glycosaminoglycans were not released in substantial amounts. Pretreatment with heparin efficiently inhibited the development of tissue thromboplastin induced DIC. This animal model may serve as a tool for the study of glycosaminoglycan anticoagulants in vivo.

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Section: Discussionmentioning

confidence: 93%

Tissue Thromboplastin Induced Reversible DIC and Heparin-Enhanced Inhibitors in Dogs

Marbet

Griffith

1986

Thromb Haemost

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“…3 Residues of fibrinogen contributing to this excep tional specificity have been localized to the first 51 amino acids of the Aα-chain. 4,5 This region has been further dissected to explore subsites, assigning roles (in order of importance) to fragments F1-F23, F33-F44, and F45-F51 6 (fibrinogen residues are prefixed by the letter "F", D-phenylalanyl-L-prolyl-L-arginine chloromethyl-ketone (PPACK) residues by "I", and hirudin residues by "H"). Two separate sites in thrombin outside S1 have been proposed as conferring this additional specificityone apolar, in the unprimed region, 7 ' 8 and one basic postcleavage site (the fibrinogen recognition exosite).…”

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confidence: 99%

A Model for the Specificity of Fibrinogen Cleavage by Thrombin

Stubbs¹,

Bode²

1993

Semin Thromb Hemost

112

168

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The specific cleavage of fibrinogen by the serine proteinase thrombin (EC 3.4.21.5) initiates the polymer ization of fibrin monomers, a primary event in blood clot formation. 1 Fibrinogen (M r 340,000) is a covalently linked dimer of three peptide chains, with stoichiometry (Aα,Bβ,γ) 2 . 2 The cleavage releases two peptides, fibrinopeptides A (FPA) and B (FPB) from the amino-termini of chains Act and Bβ, respectively, thereby revealing recognition sites for aggregation with the 7 chain.Thrombin exhibits primarily a trypsin-like specific ity, that is, a preference for arginyl P1 residues 3 (see Bode and Stubbs in this issue of Seminars for nomencla ture). The cleavage of fibrinogen by thrombin represents a very specific reaction, however. Of the 376 Arg/LysXaa bonds in the fibrinogen molecule, thrombin cleaves in the native state only four, releasing the fibrinopeptides. 3 Residues of fibrinogen contributing to this excep tional specificity have been localized to the first 51 amino acids of the Aα-chain. 4,5 This region has been further dissected to explore subsites, assigning roles (in order of importance) to fragments F1-F23, F33-F44, and F45-F51 6 (fibrinogen residues are prefixed by the letter "F", D-phenylalanyl-L-prolyl-L-arginine chloromethyl-ketone (PPACK) residues by "I", and hirudin residues by "H"). Two separate sites in thrombin outside S1 have been proposed as conferring this additional specificityone apolar, in the unprimed region, 7 ' 8 and one basic postcleavage site (the fibrinogen recognition exosite). [9][10][11] This recognition exosite appears to be in volved in the binding of other proteins, including fibrin itself, thrombomodulin (a cell surface protein that allows thrombin activation of protein C, which in turn shuts down thrombin production) and hirudin (the potent anti coagulant from the saliva of the leech Hirudo medicinalis). 12-14 Hirugen, a sulfated dodecapeptide correspond ing to the hirudin C-terminus, and the nonsulfated C-terminal peptide hirudin (45-65) are competitive inhib itors of fibrinogen cleavage by thrombin, while allowing peptidase activity on small synthetic substrates. 15,16 These sites have recently been identified in a series of crystallographic studies 17-23 (see Bode and Stubbs in this issue). The ternary complex of human α-thrombin with peptides derived from FPA and hirudin 22 (FPA-Hthrombin) allows a structural explanation for the extreme specificity of the fibrinogen-thrombin interaction; a spec ificity whose disruption can lead to severe clotting disor ders. 24 The covalently bound (N-acetylated) decapeptide chloromethylketone (Ac-AspF7-PheF8-LeuF9-AlaF10-GluFll-GlyF12-GlyF13-GlyF14-ValF15-ArgF16-CMK) exhibits little in the way of regular secondary structure (Fig. 1). The acetylated amino-terminal points away from the enzyme, whereby the preceding six residues could extend freely into solution. Asp F7 begins a short stretch (F8-F10) of α-helical conformation with a hydro gen bond from its carboxylate O δ1 to the amide nitrogen of Ala F10. This irregularly hydr...

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“…The release of FPA and FPB was described by Blomback & Vestermark (1957), who estimated a Km for thrombin release of FPA to be approx. 5 pM (Hogg & Blomback, 1974); Nossel et al (1976) found a roughly similar value, namely 2.99,UM. Others have found higher values (Hermans & McDonagh, 1982).…”

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confidence: 65%

Formation of soluble fibrin oligomers in purified systems and in plasma

Alkjærsig¹,

Fletcher²

1983

Biochemical Journal

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The kinetic parameters for release of fibrinopeptide A (FPA) from human fibrinogen by thrombin are: Km = 2.3 X 10(-6)M and Vmax. = 1.1 X 10(-10)mol of FPA/s per unit of thrombin; for fibrin formation, Km is similar to that for FPA release, but, the conditions of the present study, Vmax. was approximately half of that for FPA release. The formation of fibrin polymer before the sol-gel transition was studied by gel-permeation chromatography combined with effluent analysis for fibrinogen antigen and residual FPA. Polymer formation in purified fibrinogen incubated with thrombin proceeded as a bimolecular association of exposed sites in a manner predicted by probability calculations and assuming random FPA cleavage. Each oligomer consisted of n molecules of fibrin monomer and two fibrinogen molecules, each of the latter lacking one FPA molecule, i.e. each oligomer, regardless of molecular size, retains two FPA molecules. The addition of 5 mM-CaCl2 to the reaction mixture changed the rate of polymer formation, so that dimer was no longer the prevalent oligomer; in the presence of Ca2+, the trimer was the oligomer in highest concentration. The polymers formed in the presence of calcium were similar in composition to those without, i.e. 2 mol of FPA/mol of oligomer. EDTA-treated plasma samples incubated for short periods of time, 30s or less, with thrombin ranging in concentration up to 1 N.I.H. unit/ml did not form clots during the 10-15 min period of observation until they were applied to the column, though a large proportion of the available FPA was cleaved (maximum 45%). The soluble polymers in plasma were mostly of the high-Mr variety (tetramer and greater); these high-Mr polymers contained less than 2 mol of FPA/mol of polymer, whereas dimer and trimer in plasma were similar to those in the purified systems, i.e. 2 mol of FPA/mol.

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The specificity of the fibrinogen-thrombin reaction

Cited by 36 publications

References 15 publications

Tissue Thromboplastin Induced Reversible DIC and Heparin-Enhanced Inhibitors in Dogs

Tissue Thromboplastin Induced Reversible DIC and Heparin-Enhanced Inhibitors in Dogs

A Model for the Specificity of Fibrinogen Cleavage by Thrombin

Formation of soluble fibrin oligomers in purified systems and in plasma

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