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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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Localization of radiolabeled proteins in rabbit jugular vein clots in vivo. The radioactivity remaining in rabbit jugular vein clots allowed to polymerize in clamped-off vessels in situ for 30 minutes in the anesthetized animal, then to age with circulation restored for 60 minutes, prior to clot recovery and γ-counting is shown. Clots formed in the presence of 125I-fibrinogen and 131I-labelled plasma-derived α2AP or recombinant α2AP(13-42)-HSA or recombinant HSA. Individual data points are the mean of values for both left and right jugular veins, shown as the mean of 6 such means ± SD). Asterisk indicates the only comparison in the group significant (p < 0.05) by paired t-tests.
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Figure 5: Localization of radiolabeled proteins in rabbit jugular vein clots in vivo. The radioactivity remaining in rabbit jugular vein clots allowed to polymerize in clamped-off vessels in situ for 30 minutes in the anesthetized animal, then to age with circulation restored for 60 minutes, prior to clot recovery and γ-counting is shown. Clots formed in the presence of 125I-fibrinogen and 131I-labelled plasma-derived α2AP or recombinant α2AP(13-42)-HSA or recombinant HSA. Individual data points are the mean of values for both left and right jugular veins, shown as the mean of 6 such means ± SD). Asterisk indicates the only comparison in the group significant (p < 0.05) by paired t-tests.

Mentions: While the results of both the transglutamination assays and the fibrinolysis resistance experiments suggested successful transfer to albumin of substrate status associated with fusion of α2AP(13-42), they also suggested that the fusion protein was a less effective substrate than α2AP. We sought to test this conclusion in vivo using a Wessler-type model. In this protocol, coagulation is initiated in autologous rabbit blood that is then rapidly introduced into a clamped-off major blood vessel. Clots are allowed to form in situ, blood flow is restored, and after aging, the clots are recovered and analysed. 125I-fibrinogen was co-injected with a single 131I-labelled protein from a group comprised of plasma-derived α2AP, recombinant α2AP(13-42)-HSA, and recombinant HSA. Figure 5 shows the quantification of the protein-bound radioactivity remaining in the clot at the end of the procedure, normalized to the amount of fibrin(ogen) remaining. The most retained protein was α2AP, followed by α2AP(13-42)-HSA and HSA. While significantly more α2AP was found in the clot than HSA, the amounts of α2AP(13-42)-HSA and HSA that were retained were not statistically different. The amount of protein retained in the clot is expected to be the sum of that fraction that is non-covalently trapped and that fraction that is cross-linked. The results support the concept that α2AP(13-42)-HSA is a less effective substrate for cross-linking than α2AP, not only in vitro but also in vivo, explaining the need for excess fusion protein to compete the effects of the natural antiplasmin.


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)

Localization of radiolabeled proteins in rabbit jugular vein clots in vivo. The radioactivity remaining in rabbit jugular vein clots allowed to polymerize in clamped-off vessels in situ for 30 minutes in the anesthetized animal, then to age with circulation restored for 60 minutes, prior to clot recovery and γ-counting is shown. Clots formed in the presence of 125I-fibrinogen and 131I-labelled plasma-derived α2AP or recombinant α2AP(13-42)-HSA or recombinant HSA. Individual data points are the mean of values for both left and right jugular veins, shown as the mean of 6 such means ± SD). Asterisk indicates the only comparison in the group significant (p < 0.05) by paired t-tests.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Localization of radiolabeled proteins in rabbit jugular vein clots in vivo. The radioactivity remaining in rabbit jugular vein clots allowed to polymerize in clamped-off vessels in situ for 30 minutes in the anesthetized animal, then to age with circulation restored for 60 minutes, prior to clot recovery and γ-counting is shown. Clots formed in the presence of 125I-fibrinogen and 131I-labelled plasma-derived α2AP or recombinant α2AP(13-42)-HSA or recombinant HSA. Individual data points are the mean of values for both left and right jugular veins, shown as the mean of 6 such means ± SD). Asterisk indicates the only comparison in the group significant (p < 0.05) by paired t-tests.
Mentions: While the results of both the transglutamination assays and the fibrinolysis resistance experiments suggested successful transfer to albumin of substrate status associated with fusion of α2AP(13-42), they also suggested that the fusion protein was a less effective substrate than α2AP. We sought to test this conclusion in vivo using a Wessler-type model. In this protocol, coagulation is initiated in autologous rabbit blood that is then rapidly introduced into a clamped-off major blood vessel. Clots are allowed to form in situ, blood flow is restored, and after aging, the clots are recovered and analysed. 125I-fibrinogen was co-injected with a single 131I-labelled protein from a group comprised of plasma-derived α2AP, recombinant α2AP(13-42)-HSA, and recombinant HSA. Figure 5 shows the quantification of the protein-bound radioactivity remaining in the clot at the end of the procedure, normalized to the amount of fibrin(ogen) remaining. The most retained protein was α2AP, followed by α2AP(13-42)-HSA and HSA. While significantly more α2AP was found in the clot than HSA, the amounts of α2AP(13-42)-HSA and HSA that were retained were not statistically different. The amount of protein retained in the clot is expected to be the sum of that fraction that is non-covalently trapped and that fraction that is cross-linked. The results support the concept that α2AP(13-42)-HSA is a less effective substrate for cross-linking than α2AP, not only in vitro but also in vivo, explaining the need for excess fusion protein to compete the effects of the natural antiplasmin.

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