The activation peptide of thrombin‐activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme?

Abstract

Summary. 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°C. So far, it is unknown whether the activation peptide is released or remains attached to the catalytic domain, and whether it influences TAFIaÕs 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.

Prognostic Value of Plasma Fibrinolysis Activation Markers in Cardiovascular Disease

“…Because of the relatively long plasma half-life of PAI-1 activity (approximately 1 h), antigen and activity measurements might go hand in hand in healthy subjects. However, the poor correlation between measurements of TAFI-Ag and activity might be because activated TAFI is quickly inactivated by a rapid spontaneous conversion to the latent form with a half-life of approximately 10 min (83). Therefore, the very short half-life of TAFIa presents a practical measurement problem, which is difficult to overcome in clinical settings (84).…”

New clues regarding the mysterious mechanism of activated thrombin-activatable fibrinolysis inhibitor self-destruction

“…However, this was challenged by Marx et al . , who separated TAFIa from the activation peptide, and demonstrated that the activation peptide had no effect on TAFIa's activity or stability. The deletion mutant constructed by Zhou et al .…”

“…It was suggested that this interaction might affect TAFIa activity. However, this was challenged by Marx et al [18], who separated TAFIa from the activation peptide, and demonstrated that the activation peptide had no effect on TAFIa's activity or stability. The deletion mutant constructed by Zhou et al lacks most of the activation peptide, and makes it ideal for this debate.…”

Selective modulation of thrombin‐activatable fibrinolysis inhibitor (TAFI) activation by thrombin or the thrombin‐thrombomodulin complex using TAFI‐derived peptides

“…Immediately after TAFI activation, H‐D‐Phe‐Pro‐Arg‐chloromethylketone (PPACK, 1 μ m ) was added to inhibit thrombin activity and TAFIa activity was measured using the method developed by Willemse et al . as previously described . The Michaelis‐Menten constants were determined by using non‐linear regression in Graphpad Prism version 5.01 (Graphpad, San Diego, CA, USA).…”

“…For the determination of the Michaelis-Menten constants (V max , K M and V max /K M ), TAFI (31-2000 nM) was added to a premix of thrombin (8 nM), thrombomodulin (16 nM) and CaCl 2 (5 mM) in the absence or presence of pure peptide 2 (50 lM), peptide 18 (250 lM), peptide 19 (700 lM), peptide 29 (700 lM) or peptide 34 (10 lM) for 1 min at RT. Immediately after TAFI activation, H-D-Phe-Pro-Arg-chloromethylketone (PPACK, 1 lM) was added to inhibit thrombin activity and TAFIa activity was measured using the method developed by Willemse et al [16] as previously described [15]. The Michaelis-Menten constants were determined by using non-linear regression in Graphpad Prism version 5.01 (Graphpad, San Diego, CA, USA).…”