Limits...
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.

Show MeSH

Related in: MedlinePlus

Effects of α2AP and derivatives on plasma clot formation and lysis. (A) Clot formation and lysis was followed by monitoring turbidity (absorbance at 340 nm) every 30 seconds for 4 hours using a plate reader, of clots formed using diluted α2AP-deficient plasma containing both 5 nM thrombin and 0.125 nM tPA, and taking the area under the turbidity versus time curve (AUC). Reactions were supplemented with increasing concentrations of purified plasma-derived α2AP, and the AUC relative to that of reactions lacking tPA reported as a percentage. Results of a single experiment are shown. (B) shows turbidity plots for reactions similar to those shown in A, under 7 conditions described in the + or - table in panel C; for instance, (1) shows stable clot formation in the absence of tPA, (3) shows clot formation and rapid lysis in the presence of tPA, (4) shows attenuation of clot lysis in the presence of 1.0 μM α2AP and (6) and (7) show competition of the α2AP effect by 14 μM α2AP(13-42) synthetic peptide (Pep) or α2AP(13-42)-HSA fusion protein (FP), respectively. C shows the results of quantification of the experiment shown in B and repeated 4 times (n = 3 to 11 ± SD), under the conditions summarized below the graph, as indicated below the lanes. A control peptide unrelated to α2AP corresponding to residues 54–75 of heparin cofactor II was used at 14 μM (HCII Pep). Asterisks indicate significant differences between groups compared between the horizontal lines (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654442&req=5

Figure 4: Effects of α2AP and derivatives on plasma clot formation and lysis. (A) Clot formation and lysis was followed by monitoring turbidity (absorbance at 340 nm) every 30 seconds for 4 hours using a plate reader, of clots formed using diluted α2AP-deficient plasma containing both 5 nM thrombin and 0.125 nM tPA, and taking the area under the turbidity versus time curve (AUC). Reactions were supplemented with increasing concentrations of purified plasma-derived α2AP, and the AUC relative to that of reactions lacking tPA reported as a percentage. Results of a single experiment are shown. (B) shows turbidity plots for reactions similar to those shown in A, under 7 conditions described in the + or - table in panel C; for instance, (1) shows stable clot formation in the absence of tPA, (3) shows clot formation and rapid lysis in the presence of tPA, (4) shows attenuation of clot lysis in the presence of 1.0 μM α2AP and (6) and (7) show competition of the α2AP effect by 14 μM α2AP(13-42) synthetic peptide (Pep) or α2AP(13-42)-HSA fusion protein (FP), respectively. C shows the results of quantification of the experiment shown in B and repeated 4 times (n = 3 to 11 ± SD), under the conditions summarized below the graph, as indicated below the lanes. A control peptide unrelated to α2AP corresponding to residues 54–75 of heparin cofactor II was used at 14 μM (HCII Pep). Asterisks indicate significant differences between groups compared between the horizontal lines (p < 0.05).

Mentions: We next sought to determine if α2AP(13-42)-HSA could compete with a physiologically and clinically relevant property of α2AP, that of providing resistance to fibrinolysis in clots in which it is cross-linked to fibrin. To ensure the specificity of the effect, we obtained human plasma deficient in α2AP. When this anticoagulated plasma was clotted with thrombin, in the presence of calcium ions and low concentrations of tPA, clots formed and then lysed rapidly under the influence of plasmin, whereas if tPA was left out of the reaction, clots were stable for hours (compare turbidity profiles 3 and 1 in Fig. 4B). This difference can be quantified as the area under the turbidity curve. As shown in Fig. 4A, the addition of increasing amounts of purified plasma-derived α2AP to this system progressively delayed clot lysis, as has been reported by others, for instance by measuring clot lysis times [22]. Having shown the dependence of this effect on α2AP, we added the protein back to its physiological concentration of 1.0 μM, noting that fibrinolysis still occurred, as indicated by the downward slope of turbidity profiles 4 and 5 in Fig. 4B, but at greatly attenuated rates. When a 14-fold molar excess of unrelated peptide HCII 54–75 was added with the α2AP, there was no change in the resistance to fibrinolysis. In contrast, use of an identical excess of either α2AP(13-42) peptide or α2AP(13-42)-HSA fusion protein had a dramatic effect in accelerating clot lysis (compare profiles 6 and 7 to 4 in Fig. 4B). The effect was reproducible and statistically significant (see Fig. 4C), although the greater effect of the fusion protein over the peptide did not reach statistical significance. Similarly, lesser excesses of peptide or fusion protein reduced the mean area under the curve in a dose-dependent manner, but one which did not become significant until 14-fold, the largest amount of fusion protein we could add without re-concentration of our stock solution (data not shown).


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)

Effects of α2AP and derivatives on plasma clot formation and lysis. (A) Clot formation and lysis was followed by monitoring turbidity (absorbance at 340 nm) every 30 seconds for 4 hours using a plate reader, of clots formed using diluted α2AP-deficient plasma containing both 5 nM thrombin and 0.125 nM tPA, and taking the area under the turbidity versus time curve (AUC). Reactions were supplemented with increasing concentrations of purified plasma-derived α2AP, and the AUC relative to that of reactions lacking tPA reported as a percentage. Results of a single experiment are shown. (B) shows turbidity plots for reactions similar to those shown in A, under 7 conditions described in the + or - table in panel C; for instance, (1) shows stable clot formation in the absence of tPA, (3) shows clot formation and rapid lysis in the presence of tPA, (4) shows attenuation of clot lysis in the presence of 1.0 μM α2AP and (6) and (7) show competition of the α2AP effect by 14 μM α2AP(13-42) synthetic peptide (Pep) or α2AP(13-42)-HSA fusion protein (FP), respectively. C shows the results of quantification of the experiment shown in B and repeated 4 times (n = 3 to 11 ± SD), under the conditions summarized below the graph, as indicated below the lanes. A control peptide unrelated to α2AP corresponding to residues 54–75 of heparin cofactor II was used at 14 μM (HCII Pep). Asterisks indicate significant differences between groups compared between the horizontal lines (p < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Effects of α2AP and derivatives on plasma clot formation and lysis. (A) Clot formation and lysis was followed by monitoring turbidity (absorbance at 340 nm) every 30 seconds for 4 hours using a plate reader, of clots formed using diluted α2AP-deficient plasma containing both 5 nM thrombin and 0.125 nM tPA, and taking the area under the turbidity versus time curve (AUC). Reactions were supplemented with increasing concentrations of purified plasma-derived α2AP, and the AUC relative to that of reactions lacking tPA reported as a percentage. Results of a single experiment are shown. (B) shows turbidity plots for reactions similar to those shown in A, under 7 conditions described in the + or - table in panel C; for instance, (1) shows stable clot formation in the absence of tPA, (3) shows clot formation and rapid lysis in the presence of tPA, (4) shows attenuation of clot lysis in the presence of 1.0 μM α2AP and (6) and (7) show competition of the α2AP effect by 14 μM α2AP(13-42) synthetic peptide (Pep) or α2AP(13-42)-HSA fusion protein (FP), respectively. C shows the results of quantification of the experiment shown in B and repeated 4 times (n = 3 to 11 ± SD), under the conditions summarized below the graph, as indicated below the lanes. A control peptide unrelated to α2AP corresponding to residues 54–75 of heparin cofactor II was used at 14 μM (HCII Pep). Asterisks indicate significant differences between groups compared between the horizontal lines (p < 0.05).
Mentions: We next sought to determine if α2AP(13-42)-HSA could compete with a physiologically and clinically relevant property of α2AP, that of providing resistance to fibrinolysis in clots in which it is cross-linked to fibrin. To ensure the specificity of the effect, we obtained human plasma deficient in α2AP. When this anticoagulated plasma was clotted with thrombin, in the presence of calcium ions and low concentrations of tPA, clots formed and then lysed rapidly under the influence of plasmin, whereas if tPA was left out of the reaction, clots were stable for hours (compare turbidity profiles 3 and 1 in Fig. 4B). This difference can be quantified as the area under the turbidity curve. As shown in Fig. 4A, the addition of increasing amounts of purified plasma-derived α2AP to this system progressively delayed clot lysis, as has been reported by others, for instance by measuring clot lysis times [22]. Having shown the dependence of this effect on α2AP, we added the protein back to its physiological concentration of 1.0 μM, noting that fibrinolysis still occurred, as indicated by the downward slope of turbidity profiles 4 and 5 in Fig. 4B, but at greatly attenuated rates. When a 14-fold molar excess of unrelated peptide HCII 54–75 was added with the α2AP, there was no change in the resistance to fibrinolysis. In contrast, use of an identical excess of either α2AP(13-42) peptide or α2AP(13-42)-HSA fusion protein had a dramatic effect in accelerating clot lysis (compare profiles 6 and 7 to 4 in Fig. 4B). The effect was reproducible and statistically significant (see Fig. 4C), although the greater effect of the fusion protein over the peptide did not reach statistical significance. Similarly, lesser excesses of peptide or fusion protein reduced the mean area under the curve in a dose-dependent manner, but one which did not become significant until 14-fold, the largest amount of fusion protein we could add without re-concentration of our stock solution (data not shown).

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