The activation peptide of thrombin-activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme?
BACKGROUND
Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 56-kDa procarboxypeptidase. Proteolytic enzymes activate TAFI into TAFIa, an inhibitor of fibrinolysis, by cleaving off the N-terminal activation peptide (amino acids 1-92), from the enzyme moiety. Activated TAFI is unstable, with a half-life of approximately 10 min at 37 degrees C. So far, it is unknown whether the activation peptide is released or remains attached to the catalytic domain, and whether it influences TAFIa's properties. The current study was performed to clarify these issues.


METHODS
TAFI was activated, and the activity and half-life of the enzyme were determined in the presence and absence of the activation peptide.


RESULTS
TAFIa was active both before and after removal of the activation peptide, and the half-life of TAFIa was identical in the two preparations. Furthermore, we observed that intrinsically inactivated TAFIa (TAFIai) aggregated into large, insoluble complexes that could be removed by centrifugation.


CONCLUSIONS
The data presented in this article show that the activation peptide of TAFI is not required for TAFIa activity and that the activation peptide has no effect on the stability of the enzyme. These results are in favour of a model in which the activation peptide solely stabilizes the structure of the proenzyme. After activation of TAFI and subsequent breakage of interactions between the activation peptide and the catalytic domain, the activation peptide is no longer capable of performing this stabilizing task, and the integrity of the catalytic domain is lost rapidly. The resulting TAFIai is more prone to proteolysis and aggregation.
Marx
P F
PF
Department of Vascular Medicine, Academic Medical Centre, University of Amsterdam, Amsterdam, The Netherlands. P.F.Marx@amc.uva.nl
Plug
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Havik
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Mörgelin
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Meijers
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D025901:Carboxypeptidase B2 / Q000378:metabolism*; D004789:Enzyme Activation; D020536:Enzyme Activators / Q000378:metabolism*; D004795:Enzyme Stability; D006207:Half-Life; D006801:Humans; D006868:Hydrolysis; D010447:Peptide Hydrolases / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D012995:Solubility
D020536:Enzyme Activators; D010455:Peptides; D010447:Peptide Hydrolases; D025901:Carboxypeptidase B2