Transpeptidation Mechanism in Blood Clotting

Lóránd, L.; Konishi, Kazuhiko; Jacobsen, Ann-Christin

doi:10.1038/1941148a0

Cited by 118 publications

(78 citation statements)

References 7 publications

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“…The activity of a protease previously identified in cement of the barnacle C. fragilis was shown to be enhanced in the presence of Ca 2+ ions (Dougherty, 1996;Dougherty, 1997). Transglutaminases in both vertebrates and invertebrates are Ca 2+ dependent (Lorand et al, 1962;Fuller and Doolittle, 1971;Folk, 1980). The results of our cement polymerization assays with EDTA and EGTA, however, were inconclusive.…”

Section: + Dependencementioning

confidence: 58%

“…Proteolytic activity results in enzyme and structural protein activation, enabling bonding interactions of fibrin monomers. Integrity of assembled fibrin monomers is brought about through transglutaminase-mediated cross-linking (Lorand et al, 1962;Shen and Lorand, 1983;Lorand, 2000;Collet et al, 2005). Invertebrate blood coagulation involves similar enzymatic mechanisms: converging cascades of trypsin-like serine proteases in horseshoe crabs (Muta and Iwanaga, 1996;Iwanaga et al, 1998;Iwanaga, 2002); a transglutaminase (Fuller and Doolittle, 1971;Kopacek et al, 1993;Chen et al, 2005) showing homology to vertebrate transglutaminase (Wang et al, 2001); and proteolytic activation (Durliat and Vranckx, 1981;Madaras et al, 1981;Theopold et al, 2004) in crustaceans.…”

Section: Introductionmentioning

confidence: 99%

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Barnacle cement: a polymerization model based on evolutionary concepts

Dickinson

Vega

Wahl

et al. 2009

Journal of Experimental Biology

121

134

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SUMMARYEnzymes and biochemical mechanisms essential to survival are under extreme selective pressure and are highly conserved through evolutionary time. We applied this evolutionary concept to barnacle cement polymerization, a process critical to barnacle fitness that involves aggregation and cross-linking of proteins. The biochemical mechanisms of cement polymerization remain largely unknown. We hypothesized that this process is biochemically similar to blood clotting, a critical physiological response that is also based on aggregation and cross-linking of proteins. Like key elements of vertebrate and invertebrate blood clotting, barnacle cement polymerization was shown to involve proteolytic activation of enzymes and structural precursors, transglutaminase cross-linking and assembly of fibrous proteins. Proteolytic activation of structural proteins maximizes the potential for bonding interactions with other proteins and with the surface. Transglutaminase cross-linking reinforces cement integrity. Remarkably, epitopes and sequences homologous to bovine trypsin and human transglutaminase were identified in barnacle cement with tandem mass spectrometry and/or western blotting. Akin to blood clotting, the peptides generated during proteolytic activation functioned as signal molecules, linking a molecular level event (protein aggregation) to a behavioral response (barnacle larval settlement). Our results draw attention to a highly conserved protein polymerization mechanism and shed light on a long-standing biochemical puzzle. We suggest that barnacle cement polymerization is a specialized form of wound healing. The polymerization mechanism common between barnacle cement and blood may be a theme for many marine animal glues. Supplementary material available online at

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Section: + Dependencementioning

confidence: 58%

Section: Introductionmentioning

confidence: 99%

Barnacle cement: a polymerization model based on evolutionary concepts

Dickinson

Vega

Wahl

et al. 2009

Journal of Experimental Biology

121

134

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“…Factor XIII (fXIII) 1 is a member of the class of enzymes known as transglutaminases, which catalyze displacement of amide ammonia at the ␥-position in glutamine residues by replacing it with another amine, usually an ⑀-amino group from a suitable lysine residue (1)(2)(3)(4). Formation of ⑀-(␥-glutamyl)-lysine isopeptide bonds functions in both intra-and intermolecular cross-linking of proteins.…”

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confidence: 99%

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

Mitkevich

Shainoff

DiBello

et al. 1998

Journal of Biological Chemistry

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Coagulation factor XIIIa, plasma transglutaminase (endo-␥-glutamine:⑀-lysine transferase EC 2.3.2.13) catalyzes isopeptide bond formation between glutamine and lysine residues and rapidly cross-links fibrin clots. A monoclonal antibody (5A2) directed to a fibrinogen A␣-chain segment 529 -539 was previously observed from analysis of end-stage plasma clots to block fibrin ␣-chain cross-linking. This prompted the study of its effect on nonfibrinogen substrates, with the prospect that 5A2 was inhibiting XIIIa directly. It inhibited XIIIa-catalyzed incorporation of the amine donor substrate dansylcadaverine into the glutamine acceptor dimethylcasein in an uncompetitive manner with respect to dimethylcasein utilization and competitively with respect to dansylcadaverine. Uncompetitive inhibition was also observed with the synthetic glutamine substrate, LGPGQSKVIG. Theoretically, uncompetitive inhibition arises from preferential interaction of the inhibitor with the enzyme-substrate complex but is also found to inhibit ␥-chain cross-linking. The conjunction of the uncompetitive and competitive modes of inhibition indicates in theory that this bireactant system involves an ordered reaction in which docking of the glutamine substrate precedes the amine exchange. The presence of substrate enhanced binding of 5A2 to XIIIa, an interaction deemed to occur through a C-terminal segment of the XIIIa A-chain (643-658, GSDMTVTVQFT-NPLKE), 55% of which comprises sequences occurring in the fibrinogen epitope A␣-(529 -540) (GSESGIFTNTKE). Removal of the C-terminal domain from XIIIa abolishes the inhibitory effect of 5A2 on activity. Crystallographic studies on recombinant XIIIa place the segment 643-658 in the region of the groove through which glutamine substrates access the active site and have predicted that for catalysis, a conformational change may accompany glutamine-substrate binding. The uncompetitive inhibition and the substrate-dependent binding of 5A2 provide evidence for the conformational change.

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“…Hence, a sampling methodology rather than a continuous rate assay was decided upon. Several The first step in the procedure is the activation of 0.1 ml of purified FSF (10 "cross-linking equivalents" [14]) with 0. Fig.…”

Section: B a Coupled System For Measuring The Concen-tration Of Fibrmentioning

confidence: 99%

“…Dilution of the factor to a threshold level of activity provided the basis for empirical quantitation. In its most recently developed version (14), a multistage assay consisted of (a) the activation of fibrin-stabilizing factor by thrombin, followed (b) by the quenching of thrombin (unless water-insoluble enzyme was used (3), which could be removed by filtration); (c) the activated factor was then mixed with isolated fibrin and (d) cross-linking was allowed to proceed. Finally, (e) 1% monochloroacetic acid was added to test for the extent of cross-linking either by visual inspection or by quantitatively analyzing the acidinsoluble residue.…”

Section: Introductionmentioning

confidence: 99%

Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation

Lóránd¹,

Urayama²,

Kiewiet³

et al. 1969

J. Clin. Invest.

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210

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A B S T R A C T Fibrinoligase, the fibrin cross-linking enzyme, transiently appearing during the course of coagulation in normal blood, was shown to catalyze the incorporation of a fluorescent amine, monodansylcadaverine [or N-( 5-aminopentyl) -5-dimethylamino-1-naphthalenesulfonamide] into casein. The reaction provided the basis of a sensitive fluorimetric method for measuring the activity of the enzyme (and also of similar other transpeptidases, such as transglutaminase).In tests involving plasma, certain difficulties had to be overcome which were mainly due to the fact that the enzyme itself does not occur in citrated plasma. Only its precursor (fibrin-stabilizing factor or factor XIII) is present, still requiring limited proteolytic activation by thrombin. Thus, in order to measure amine incorporation with plasma as a source of the factor, thrombin must be added. This necessitated a differential desensitization of the intrinsic fibrinogen so that the latter could not clot and could not thereby interfere with amine incorporation. Also, the thrombin-inactivating capacity of plasma had to be saturated to enable full conversion of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of amine incorporation. A linear relationship with regard to plasma concentration could be obtained only under such conditions. No similar assay is presently available for quantitatively evaluating fibrin-stabilizing factor levels in plasma.

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Transpeptidation Mechanism in Blood Clotting

Cited by 118 publications

References 7 publications

Barnacle cement: a polymerization model based on evolutionary concepts

Barnacle cement: a polymerization model based on evolutionary concepts

Coagulation Factor XIIIa Undergoes a Conformational Change Evoked by Glutamine Substrate

Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation

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