Thrombin-activatable fibrinolysis inhibitor (TAFI) is a pro-metallocarboxypeptidase that can be proteolytically activated (TAFIa). TAFIa is unique among carboxypeptidases in that it spontaneously inactivates with a short half-life, a property that is crucial for its role in controlling blood clot lysis. We studied the intrinsic instability of TAFIa by solving crystal structures of TAFI, a TAFI inhibitor (GEMSA) complex and a quadruple TAFI mutant (70-fold more stable active enzyme). The crystal structures show that TAFIa stability is directly related to the dynamics of a 55-residue segment (residues 296-350) that includes residues of the active site wall. Dynamics of this flap are markedly reduced by the inhibitor GEMSA, a known stabilizer of TAFIa, and stabilizing mutations. Our data provide the structural basis for a model of TAFI auto-regulation: in zymogen TAFI the dynamic flap is stabilized by interactions with the activation peptide. Release of the activation peptide increases dynamic flap mobility and in time this leads to conformational changes that disrupt the catalytic site and expose a cryptic thrombincleavage site present at Arg302. This represents a novel mechanism of enzyme control that enables TAFI to regulate its activity in plasma in the absence of specific inhibitors. (Blood. 2008;112: 2803-2809) Introduction TAFI 1,2 is a pro-metallocarboxypeptidase that links the coagulation and fibrinolytic systems. TAFI is activated by thrombin, the thrombin-thrombomodulin complex or plasmin. 3 Activated TAFI (TAFIa) inhibits plasmin-mediated blood clot lysis by removing C-terminal lysine residues from partially degraded fibrin that are required for positive feedback in tissue plasminogen-activator dependent plasmin generation. In addition, TAFIa has been implicated in modulation of the inflammatory response by inactivating bradykinin and the anaphylatoxins C3a and C5a. 4,5 Although it is a powerful antifibrinolytic agent, there are no known physiologic inhibitors of TAFIa. Instead, the half-life of TAFIa activity is regulated by its intrinsic instability. The inactivation rate, 5 to 10 minutes at 37°C, is highly temperature-dependent, suggesting that inactivation involves a large conformational change. 6 This is also suggested by the susceptibility of the inactive enzyme, TAFIai to proteolytic cleavage by thrombin at Arg302, a site that is cryptic in TAFI and TAFIa. 6,7 The stability of TAFIa is an important determinant for its antifibrinolytic potential because TAFIa inhibits fibrinolysis through a threshold-dependent mechanism. [8][9][10] Full-length TAFI consists of 401 amino acids divided into 2 domains: the first 92 amino acids form the activation peptide; the next 309 amino acids form the catalytic domain. The activation peptide restricts substrate access to the catalytic cleft in the zymogen. TAFI is activated through cleavage at Arg92, which releases the activation peptide.TAFI is highly homologous to the pancreatic procarboxypeptidases with 42% sequence identity to human procarboxypeptidase B (pro...

To cite this article: Plug T, Meijers JCM. Structure-function relationships in thrombin-activatable fibrinolysis inhibitor. J Thromb Haemost 2016; 14: 633-44.Summary. Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance of coagulation and fibrinolysis. TAFI is a metallocarboxypeptidase that circulates in plasma as zymogen. Activated TAFI (TAFIa) cleaves C-terminal lysine or arginine residues from peptide substrates. The removal of C-terminal lysine residues from partially degraded fibrin leads to reduced plasmin formation and thus attenuation of fibrinolysis. TAFI also plays a role in inflammatory processes via the removal of C-terminal arginine or lysine residues from bradykinin, thrombin-cleaved osteopontin, C3a, C5a and chemerin. TAFI has been studied extensively over the past three decades and recent publications provide a wealth of information, including crystal structures, mutants and structural data obtained with antibodies and peptides. In this review, we combined and compared available data on structure/function relationships of TAFI.

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.

To cite this article: Plug T, Kramer G, Meijers JCM. A role for arginine-12 in thrombin-thrombomodulin-mediated activation of thrombinactivatable fibrinolysis inhibitor. J Thromb Haemost 2014; 12: 1717-25.Summary. Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a proenzyme that links coagulation and fibrinolysis. TAFI can be activated by thrombin, the thrombin-thrombomodulin complex and plasmin through cleavage of the first 92 amino acids from the enzyme. In silico analysis of the TAFI sequence revealed a potential thrombin cleavage site at Arg12. The aim of this study was to determine whether TAFI can be cleaved at Arg12 and whether this cleavage plays a role in TAFI activation. Methods: A peptide based on the first 18 amino acids of TAFI was used to determine whether thrombin was able to cleave at Arg12. Mass spectrometry was performed to determine whether the Arg12-cleaved peptide was released from full-length TAFI. Furthermore, a TAFI mutant in which Arg12 was replaced by a glutamine (TAFI-R12Q) was constructed and characterized with respect to its activation kinetics. Results: The peptide and mass spectrometry data showed that thrombin was able to cleave TAFI at Arg12, but with low efficiency in full-length TAFI. Characterization of TAFI-R12Q showed no difference in thrombin-mediated activation from wild-type TAFI. However, there was an approximately 60-fold impairment in activation of TAFI-R12Q by the thrombin-thrombomodulin complex. Conclusions: Arg12 of TAFI plays an important role in thrombomodulin-mediated TAFI activation by thrombin. Thrombin is able to cleave TAFI at Arg12, but it remains to be determined whether Arg12 is part of an exosite for thrombomodulin or whether cleavage at Arg12 accelerates thrombomodulin-mediated TAFI activation.

Coagulation factor XI is activated by thrombin or factor XIIa resulting in a conformational change that converts the catalytic domain into its active form and exposing exosites for factor IX on the apple domains. Although crystal structures of the zymogen factor XI and the catalytic domain of the protease are available, the structure of the apple domains and hence the interactions with the catalytic domain in factor XIa are unknown. We now used chemical footprinting to identify lysine residue containing regions that undergo a conformational change following activation of factor XI. To this end, we employed tandem mass tag in conjunction with mass spectrometry. Fifty-two unique peptides were identified, covering 37 of the 41 lysine residues present in factor XI. Two identified lysine residues that showed altered flexibility upon activation were mutated to study their contribution in factor XI stability or enzymatic activity. Lys357, part of the connecting loop between A4 and the catalytic domain, was more reactive in factor XIa but mutation of this lysine residue did not impact on factor XIa activity. Lys516 and its possible interactor Glu380 are located in the catalytic domain and are covered by the activation loop of factor XIa. Mutating Glu380 enhanced Arg369 cleavage and thrombin generation in plasma. In conclusion, we have identified novel regions that undergo a conformational change following activation. This information improves knowledge about factor XI and will contribute to development of novel inhibitors or activators for this coagulation protein.

Fibrin glue combined with 500 kunits/ml aprotinin resulted in reversible PVE in 80 per cent of animals, with a hypertrophy response comparable to that achieved with permanent embolization material. Surgical relevance Portal vein embolization (PVE) is used to increase future remnant liver volume in patients scheduled for major liver resection who have insufficient future remnant liver size to perform a safe resection. The current standard is PVE with permanent embolization materials, which renders patients found to have unresectable disease prone to complications owing to the permanently deportalized liver segments. Absorbable embolization might prevent the PVE-associated morbidity and lower the threshold for its application. In this study, PVE using fibrin glue and aprotinin resulted in an adequate hypertrophy response with 80 per cent recanalization after 42 days. Considering the minor histological changes following recanalization of embolized segments and potentially preserved function, reversible PVE might also be applied in living donor liver transplantation.

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To cite this article: Plug T, Marquart JA, Marx PF, Meijers JCM. Selective modulation of thrombin-activatable fibrinolysis inhibitor (TAFI) activation by thrombin or the thrombin-thrombomodulin complex using TAFI-derived peptides. J Thromb Haemost 2015; 13: 2093-101.Summary. Background: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for coronary heart disease. TAFI is proteolytically activated by thrombin, the thrombin-thrombomodulin complex and plasmin. Once active, it dampens fibrinolysis and inflammation. The aim of this study was to generate TAFI-derived peptides that specifically modulate TAFI activation and activity. Methods: Thirty-four overlapping TAFI peptides, and modifications thereof, were synthesized. The effects of these peptides on TAFI activation and TAFIa activity were determined. In addition, the binding of the peptides to thrombin were determined. Results: Four peptides (peptides 2, 18, 19 and 34) inhibited TAFI activation and two peptides (peptides 14 and 24) inhibited TAFIa activity directly. Peptide 2 (Arg12-Glu28) and peptide 34 (Cys383-Val401) inhibited TAFI activation by the thrombin-thrombomodulin complex with IC 50 values of 7.3 AE 1.8 and 6.1 AE 0.9 lM, respectively. However, no inhibition was observed in the absence of thrombomodulin. This suggests that the regions Arg12-Glu28 and Cys383-Val401 in TAFI are involved in thrombomodulin-mediated TAFI activation. Peptide 18 (Gly205-Ser221) and peptide 19 (Arg214-Asp232) inhibited TAFI activation by thrombin and the thrombin-thrombomodulin complex. Furthermore, these peptides bound to thrombin (K D : 1.5 AE 0.4 and 0.52 AE 0.07 lM for peptides 18 and 19, respectively), suggesting that Gly205-Asp232 of TAFI is involved in binding to thrombin. Peptide 14 (His159-His175) inhibited TAFIa activity.The inhibition was TAFIa specific, because no effect on the homologous enzyme carboxypeptidase B was observed. Conclusions: Thrombin-activatable fibrinolysis inhibitor-derived peptides show promise as new tools to modulate TAFI activation and TAFIa activity. Furthermore, these peptides revealed potential binding sites on TAFI for thrombin and the thrombin-thrombomodulin complex.