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Addition of a sequence from alpha2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis.

Sheffield WP, Eltringham-Smith LJ, Gataiance S, Bhakta V - BMC Biotechnol. (2009)

Bottom Line: The alpha2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native alpha2AP.Excess alpha2AP(13-42)-HSA competed with alpha2AP for cross-linking to chemical lysine donors more effectively than a synthetic alpha2AP(13-42) peptide, and reduced the alpha2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide.Native alpha2AP was found in in vivo clots in rabbits to a greater extent than alpha2AP(13-42), however.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada. sheffiel@mcmaster.ca

ABSTRACT

Background: The plasma protein alpha2-antiplasmin (alpha2AP) is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of alpha2-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein.

Results: Fusion protein alpha2AP(13-42)-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer alpha2AP moieties were inefficiently secreted and/or degraded. The alpha2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native alpha2AP. Excess alpha2AP(13-42)-HSA competed with alpha2AP for cross-linking to chemical lysine donors more effectively than a synthetic alpha2AP(13-42) peptide, and reduced the alpha2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native alpha2AP was found in in vivo clots in rabbits to a greater extent than alpha2AP(13-42), however.

Conclusion: In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein alpha2AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of alpha2-antiplasmin predicted to act in a clot-localized manner.

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Cross-linking of α2AP and α2AP(13-42)-HSA by fXIIIa to fibrinogen. FXIII was pre-activated to fXIIIa by thrombin, the thrombin inactivated with FPRck, and the fXIIIa then combined with fibrinogen and α2AP or α2AP(13-42)-HSA or α2AP(13-73)-HSA in transglutamination reactions. Reactions were terminated with SDS, DTT, and urea as described in "Methods". (A) depicts a Coomassie Blue-stained SDS-polyacrylamide gel highlighting the cross-linking of fibrinogen into γ-γ dimers (γ-γ) and α-polymers (α). (B) shows an anti-α2AP immunoblot of transglutamination reactions containing α2AP for the times identified above the lanes; the first lane contains α2AP alone. (C), right panel, is identical to B except that α2AP(13-42)-HSA was substituted for α2AP; (C), left panel, is identical to the right panel except an anti-HSA antibody was used. (D) is identical to (C), left panel, except that α2AP(13-73)-HSA was substituted for α2AP(13-42)-HSA. The position of molecular mass markers, is shown to the left or right of the panels.
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Figure 3: Cross-linking of α2AP and α2AP(13-42)-HSA by fXIIIa to fibrinogen. FXIII was pre-activated to fXIIIa by thrombin, the thrombin inactivated with FPRck, and the fXIIIa then combined with fibrinogen and α2AP or α2AP(13-42)-HSA or α2AP(13-73)-HSA in transglutamination reactions. Reactions were terminated with SDS, DTT, and urea as described in "Methods". (A) depicts a Coomassie Blue-stained SDS-polyacrylamide gel highlighting the cross-linking of fibrinogen into γ-γ dimers (γ-γ) and α-polymers (α). (B) shows an anti-α2AP immunoblot of transglutamination reactions containing α2AP for the times identified above the lanes; the first lane contains α2AP alone. (C), right panel, is identical to B except that α2AP(13-42)-HSA was substituted for α2AP; (C), left panel, is identical to the right panel except an anti-HSA antibody was used. (D) is identical to (C), left panel, except that α2AP(13-73)-HSA was substituted for α2AP(13-42)-HSA. The position of molecular mass markers, is shown to the left or right of the panels.

Mentions: Having demonstrated the potential superiority of α2AP(13-42)-HSA over α2AP(13-42) as a competitor of α2AP cross-linking of a chemical lysine donor, we next asked how effectively it was cross-linked to a natural polypeptide lysine donor, fibrinogen. While fibrin is thought to be the predominant physiological lysine donor for α2AP transglutamination, it has been demonstrated that α2AP can be cross-linked to circulating fibrinogen [36]. Fibrinogen is a more convenient experimental substrate in that it does not clot, and numerous groups have used it rather than fibrin to examine cross-linking (e.g. [37]). We therefore first examined the ability of α2AP(13-42)-HSA to be cross-linked to fibrinogen, utilising sub-physiological concentrations of both fibrinogen and α2AP, in order to compare fairly initial rates. As shown in Fig. 3A, fXIIIa catalyzed the formation of readily visualized γ-γ chains (migrating between the 85 and 100 kDa marker positions) and less abundant α polymers (migrating between the 150 kDa and 200 kDa markers) visible by Coomassie Blue staining of SDS-gels. α2AP-related protein cross-linking at the concentrations of these proteins that were employed required immunoblotting to detect. As shown in Fig. 3B, higher molecular-weight transglutamination products were detected when plasma-derived α2AP was combined with fibrinogen and fXIIIa, whose abundance increased over time and of which a product migrating near the 170 kDa mass marker was arguably the most abundant. Similar results were obtained with α2AP(13-42)-HSA, although it was more difficult to visualize this fusion protein than α2AP, likely because of the lesser reactivity of the polyclonal anti-α2AP with a fusion protein containing only 30 residues of α2AP (see Fig. 3C, right panel). When aliquots of the same reactions as those shown in Fig. 3C, right panel, were probed using anti-HSA, an identical pattern was obtained. In contrast, no cross-linked products were obtained when α2AP(13-73)-HSA was substituted for α2AP(13-42)-HSA in cross-linking reactions (Fig. 3D), even when incubations were prolonged to 5 minutes. Elimination of either fibrinogen or fXIIIa from cross-linking reactions abrogated complex formation (data not shown); antibody specificity, for instance the lack of cross-reaction of anti-HSA monoclonal antibody (Fig. 3D), was as expected. The similar size of the cross-linked products reflected the similar size of glycosylated plasma-derived α2AP and α2AP(13-42)-HSA; less abundant products of lesser mobility seen in Figs. 3B and 3C resembled those reported by others in similar reactions [31,38].


Addition of a sequence from alpha2-antiplasmin transforms human serum albumin into a blood clot component that speeds clot lysis.

Sheffield WP, Eltringham-Smith LJ, Gataiance S, Bhakta V - BMC Biotechnol. (2009)

Cross-linking of α2AP and α2AP(13-42)-HSA by fXIIIa to fibrinogen. FXIII was pre-activated to fXIIIa by thrombin, the thrombin inactivated with FPRck, and the fXIIIa then combined with fibrinogen and α2AP or α2AP(13-42)-HSA or α2AP(13-73)-HSA in transglutamination reactions. Reactions were terminated with SDS, DTT, and urea as described in "Methods". (A) depicts a Coomassie Blue-stained SDS-polyacrylamide gel highlighting the cross-linking of fibrinogen into γ-γ dimers (γ-γ) and α-polymers (α). (B) shows an anti-α2AP immunoblot of transglutamination reactions containing α2AP for the times identified above the lanes; the first lane contains α2AP alone. (C), right panel, is identical to B except that α2AP(13-42)-HSA was substituted for α2AP; (C), left panel, is identical to the right panel except an anti-HSA antibody was used. (D) is identical to (C), left panel, except that α2AP(13-73)-HSA was substituted for α2AP(13-42)-HSA. The position of molecular mass markers, is shown to the left or right of the panels.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654442&req=5

Figure 3: Cross-linking of α2AP and α2AP(13-42)-HSA by fXIIIa to fibrinogen. FXIII was pre-activated to fXIIIa by thrombin, the thrombin inactivated with FPRck, and the fXIIIa then combined with fibrinogen and α2AP or α2AP(13-42)-HSA or α2AP(13-73)-HSA in transglutamination reactions. Reactions were terminated with SDS, DTT, and urea as described in "Methods". (A) depicts a Coomassie Blue-stained SDS-polyacrylamide gel highlighting the cross-linking of fibrinogen into γ-γ dimers (γ-γ) and α-polymers (α). (B) shows an anti-α2AP immunoblot of transglutamination reactions containing α2AP for the times identified above the lanes; the first lane contains α2AP alone. (C), right panel, is identical to B except that α2AP(13-42)-HSA was substituted for α2AP; (C), left panel, is identical to the right panel except an anti-HSA antibody was used. (D) is identical to (C), left panel, except that α2AP(13-73)-HSA was substituted for α2AP(13-42)-HSA. The position of molecular mass markers, is shown to the left or right of the panels.
Mentions: Having demonstrated the potential superiority of α2AP(13-42)-HSA over α2AP(13-42) as a competitor of α2AP cross-linking of a chemical lysine donor, we next asked how effectively it was cross-linked to a natural polypeptide lysine donor, fibrinogen. While fibrin is thought to be the predominant physiological lysine donor for α2AP transglutamination, it has been demonstrated that α2AP can be cross-linked to circulating fibrinogen [36]. Fibrinogen is a more convenient experimental substrate in that it does not clot, and numerous groups have used it rather than fibrin to examine cross-linking (e.g. [37]). We therefore first examined the ability of α2AP(13-42)-HSA to be cross-linked to fibrinogen, utilising sub-physiological concentrations of both fibrinogen and α2AP, in order to compare fairly initial rates. As shown in Fig. 3A, fXIIIa catalyzed the formation of readily visualized γ-γ chains (migrating between the 85 and 100 kDa marker positions) and less abundant α polymers (migrating between the 150 kDa and 200 kDa markers) visible by Coomassie Blue staining of SDS-gels. α2AP-related protein cross-linking at the concentrations of these proteins that were employed required immunoblotting to detect. As shown in Fig. 3B, higher molecular-weight transglutamination products were detected when plasma-derived α2AP was combined with fibrinogen and fXIIIa, whose abundance increased over time and of which a product migrating near the 170 kDa mass marker was arguably the most abundant. Similar results were obtained with α2AP(13-42)-HSA, although it was more difficult to visualize this fusion protein than α2AP, likely because of the lesser reactivity of the polyclonal anti-α2AP with a fusion protein containing only 30 residues of α2AP (see Fig. 3C, right panel). When aliquots of the same reactions as those shown in Fig. 3C, right panel, were probed using anti-HSA, an identical pattern was obtained. In contrast, no cross-linked products were obtained when α2AP(13-73)-HSA was substituted for α2AP(13-42)-HSA in cross-linking reactions (Fig. 3D), even when incubations were prolonged to 5 minutes. Elimination of either fibrinogen or fXIIIa from cross-linking reactions abrogated complex formation (data not shown); antibody specificity, for instance the lack of cross-reaction of anti-HSA monoclonal antibody (Fig. 3D), was as expected. The similar size of the cross-linked products reflected the similar size of glycosylated plasma-derived α2AP and α2AP(13-42)-HSA; less abundant products of lesser mobility seen in Figs. 3B and 3C resembled those reported by others in similar reactions [31,38].

Bottom Line: The alpha2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native alpha2AP.Excess alpha2AP(13-42)-HSA competed with alpha2AP for cross-linking to chemical lysine donors more effectively than a synthetic alpha2AP(13-42) peptide, and reduced the alpha2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide.Native alpha2AP was found in in vivo clots in rabbits to a greater extent than alpha2AP(13-42), however.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology and Molecular Medicine, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada. sheffiel@mcmaster.ca

ABSTRACT

Background: The plasma protein alpha2-antiplasmin (alpha2AP) is cross-linked to fibrin in blood clots by the transglutaminase factor XIIIa, and in that location retards clot lysis. Competition for this effect could be clinically useful in patients with thrombosis. We hypothesized that fusion of N-terminal portions of alpha2-antiplasmin to human serum albumin (HSA) and production of the chimeric proteins in Pichia pastoris yeast would produce a stable and effective competitor protein.

Results: Fusion protein alpha2AP(13-42)-HSA was efficiently secreted from transformed yeast and purified preparations contained within a mixed population the full-length intact form, while fusions with longer alpha2AP moieties were inefficiently secreted and/or degraded. The alpha2AP(13-42)-HSA protein, but not recombinant HSA, was cross-linked to both chemical lysine donors and fibrin or fibrinogen by factor XIIIa, although with less rapid kinetics than native alpha2AP. Excess alpha2AP(13-42)-HSA competed with alpha2AP for cross-linking to chemical lysine donors more effectively than a synthetic alpha2AP(13-42) peptide, and reduced the alpha2AP-dependent resistance to fibrinolysis of plasma clots equally effectively as the peptide. Native alpha2AP was found in in vivo clots in rabbits to a greater extent than alpha2AP(13-42), however.

Conclusion: In this first report of transfer of transglutamination substrate status from one plasma protein to another, fusion protein alpha2AP(13-42)-HSA was shown to satisfy initial requirements for a long-lasting, well-tolerated competitive inhibitor of alpha2-antiplasmin predicted to act in a clot-localized manner.

Show MeSH
Related in: MedlinePlus