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Blood, 1 April 2006, Vol. 107, No. 7, pp. 3011-3012.
CORRESPONDENCE To the editor:
Fibrinogen containing
A recently published paper by Siebenlist et al1 presents data that apparently contradicts results published previously by our laboratory2 and others.3 Siebenlist et al's data show that factor XIII activation is more rapid in the presence of | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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A/' fibrinogen was added to factor XIII. Therefore, the reason for the discrepancies between our results and those of Siebenlist et al1 is still unclear. One possibility is that Siebenlist et al1 measured the rate of factor XIIIa-subunit activation peptide cleavage, whereas our laboratory measured the appearance of factor XIIIa activity. These two assays do not, in fact, measure the same thing. While factor XIII activation peptide cleavage is necessary for factor XIII activation under physiologic conditions, it is not sufficient. A second step that must occur prior to activation is the dissociation of the b-subunits of factor XIII.7 The paper by Siebenlist et al1 measured the first step in factor XIII activation, activation peptide cleavage, whereas our paper measured the last step in factor XIII activation, the expression of catalytic activity. Our hypothesis is that A/' fibrin(ogen), by virtue of its binding to factor XIIIb-subunits through the ' chain,4 increases the rate of factor XIII activation by increasing the rate of factor XIIIb-subunit dissociation from the catalytic a'-subunits.
Another possibility for the discrepancy in results is that subtle differences in fibrinogen purification methods among different laboratories may result in different amounts of contaminants or different amounts of fibrinogen degradation that may influence these assays. Such differences could potentially account for the discrepancies between the paper by Siebenlist et al1 and our paper, and possibly the discrepancies between their paper and a previous paper by Cooper et al.3 The data of Cooper et al3 indicate that fibrinopeptide A release from A/A and A/' fibrinogen is identical but that fibrinopeptide B release is delayed from A/' fibrinogen. In contrast, the data presented by Siebenlist et al shows that fibrinopeptide A release from A/' fibrinogen is delayed compared to A/A fibrinogen, as well as fibrinopeptide B release. The reason for these discrepancies is still unclear.
Correspondence: Department of Pathology, L113, School of Medicine, Oregon Health & Science University, 3181 S.W. Sam Jackson Park Road, Portland, OR, 97239-3098; e-mail: farrelld{at}ohsu.edu.
References
' chains. Blood. Prepublished on July 7, 2005, as DOI 10.1182/blood-2005-01-0240. (Now available as Blood. 2005;106:2730-2736.)A/' fibrinogen in plasma factor XIII activation. J Biol Chem. 2000;275: 32135-32140.-chain splice variant ' alters fibrin formation and structure. Blood. 2003;102: 535-540.' chains. Biochemistry. 1996;35: 10448-10453.[CrossRef][Medline]
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' chains; is the assay measuring what Farrell's group expects?
A-') was slower than with fibrinogen 1 (A-A). To explain the differences, we suggested that the fibrinogen 2 used in his experiments was likely to have contained fXIII that had coeluted with the fibrinogen 2.As far as we can determine, Farrell's group did not process the fibrinogen 2 in a manner that would have removed the fXIII present in chromatographically isolated fibrinogen 2.3 Contrary to Dr Farrell's assertion, we very carefully redescribed our method for rendering fibrinogen 2 fXIII free and how the product was analyzed and shown to be fXIII free.1,3 Farrell asserted that his "activity" assay for fXIII activation was more appropriate than activation peptide cleavage from fXIII. Activation peptide release is an accepted and often-used surrogate for measuring fXIII activation. Furthermore, activation peptide release is the rate limiting step of fXIII activation, not the Ca++-mediated dissociation of A* from the B subunits.
To support the contention that the fibrinogen 2 Farrell's group used was depleted of fXIII, he included some unpublished experiments in his letter. The methodological design of these experiments raises more questions than they answer. First, since chromatographic separation of fibrinogen 1 from fibrinogen 2 results in fXIII-free fibrinogen 1, why was transglutaminase activity observed with fibrinogen 1 alone? Second, we previously demonstrated that "unactivated" fXIII, such as is present in chromatographically purified fibrinogen 2 and in the activation mixtures that Farrell measured in his assay, cross-links fibrin almost as rapidly as thrombin-activated factor XIIIa. Factor XIII also cross-links fibrinogen and artificial substrates, but at slower rates.4 Since only one (ie, biotinylated pentylamine incorporation into dimethylcasein) of several reactions (eg, fibrin(ogen) cross-linking, dimethylamine incorporation into fibrin(ogen), biotinylated pentylamine incorporation into fibrin(ogen)) was measured in his assay, we wonder what the effect of competing fXIII substrate(s), namely fibrin(ogen), was on the pentylamine incorporation into dimethylcasein?
Dr Farrell also posited that the discrepancies in FPA release between our paper and one by Cooper et al5 might be explained by contaminants or variable degrees of fibrinogen degradation in our preparations. Our explanation is simpler. Differences between our results and those of Cooper et al5 were due to the lower levels of thrombin we employed, and those lower concentrations enabled us to measure differences in FPA release between fibrinogen 2 and fibrinogen 1. Other experiments in our paper, showing down-regulation of thrombin catalytic activity (kcat) when bound to fibrin 2 ' chains, provide more than adequate support for our explanation and also for the slower release of activation peptide from fXIII in the presence of fibrinogen 2.
Correspondence: Department of Biomedical Sciences, College of Health Sciences, Marquette University, PO Box 1881, Milwaukee, WI 53233-1881; e-mail: kevin.siebenlist{at}marquette.edu.
References
' chains. Blood. Prepublished on July 7, 2005, as DOI 10.1182/blood-2005-01-0240. (Now available as Blood. 2005;106:2730-2736.)A/' fibrinogen in plasma factor XIII activation. J Biol Chem. 2000;275: 32135-32140.' chains. Biochemistry. 1996;35: 10448-10453.[CrossRef][Medline]
[Order article via Infotrieve]-chain splice variant ' alters fibrin formation and structure. Blood. 2003;102: 535-540.
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' chains
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| Copyright © 2006 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-thrombin in 100 µL buffer containing 1 mM CaCl2, and 1 mM GPRP peptide to prevent fibrin polymerization,
), 86 nM 
), 86 nM 
), or 86 nM 
). Thrombin was inactivated at the indicated times with 0.5 mM PPACK. Factor XIIIa activity was measured by the incorporation of 0.5 mM 5-(biotinamido)pentylamine into immobilized N, N'-dimethylcasein, and detected at 405 nm by P-nitrophenyl phosphate hydrolysis following incubation with streptavidin-conjugated alkaline phosphatase.