Hot spots in TAFIa

See also Sanglas L, Arolas JL, Valnickova Z, Aviles FX, Enghild JJ, Gomis-Rüth FX. Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor. This issue, pp 1056–65. See also Sanglas L, Arolas JL, Valnickova Z, Aviles FX, Enghild JJ, Gomis-Rüth FX. Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor. This issue, pp 1056–65. In their paper in this issue of the Journal of Thrombosis and Haemostasis Sanglas et al. [1Sanglas L. Arolas J.L. Valnickova Z. Aviles F.X. Enghild J.J. Gomis-Ruth F.X. Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor, TAFIa.J Thromb Haemost. 2010; 8: 1056-65Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar] have studied the influence of protein inhibitors on activated human Thrombin Activatable Fibrinolysis Inhibitor (TAFIa) in order to identify the molecular determinants of TAFIa (in)stability. They found that two protein inhibitors, one derived from Hirudo medicinalis that is, leech carboxypeptidase inhibitor (LCI) and one derived from Rhipicephalus bursa that is, tick carboxypeptidase inhibitor (TCI), strongly reduce the instability and concomitantly the insolubility of human TAFIa through binding to dynamic regions. Through crystallization of the human TAFIa/TCI complex, they have elucidated the binding and subsequently the stabilization of human TAFIa by TCI. Binding is exerted by two interactions. The main interaction is exerted by the C-terminal domain of TCI. It interacts with protease segments Lβ3α2, Lα3α4, Lβ5β6, Lβ7α6, Lβ8α7 and with residues of the active site of TAFIa. Binding to these sites results in inhibition of TAFIa activity but increases also the overall stability and rigidity. The C-terminal end of TCI penetrates the TAFIa enzyme moiety. Through this insertion, the last residue [His75 (TCI)] is cleaved off allowing Leu74 (TCI) to bind to the catalytic zinc ion of TAFIa. The N-terminal part of TCI contacts only the beginning of the Lα3α4 region. The authors conclude that this second site is an exosite and that binding to this exosite may contribute to TAFIa stability. TCI was cloned, expressed and characterized in 2005 [2Arolas J.L. Lorenzo J. Rovira A. Castella J. Aviles F.X. Sommerhoff C.P. A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: isolation, cDNA cloning, recombinant expression, and characterization.J Biol Chem. 2005; 280: 3441-8Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. TCI inhibits human TAFIa with an inhibition constant (Ki) of 1.2 ± 0.4 nm but, similar to Potato Tuber Carboxypeptidase inhibitor (PCI), has no effect on Carboxypeptidase N (CPN). Depending on the concentration used, both small protein inhibitors (PCI and TCI) either prolong (low concentration) or shorten (high concentration) t-PA-induced clot lysis revealing a biphasic effect which has been explained for PCI by its effect on both the activity and the stability of TAFIa [3Walker J.B. Hughes B. James I. Haddock P. Kluft C. Bajzar L. Stabilization versus inhibition of TAFIa by competitive inhibitors in vitro.J Biol Chem. 2003; 278: 8913-21Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar]. Compared with PCI, TCI has only a minor anti-fibrinolytic effect at low concentrations, approximately 10-fold lower concentrations are sufficient to accelerate fibrinolysis and the maximal pro-fibrinolytic effect is more pronounced [2Arolas J.L. Lorenzo J. Rovira A. Castella J. Aviles F.X. Sommerhoff C.P. A carboxypeptidase inhibitor from the tick Rhipicephalus bursa: isolation, cDNA cloning, recombinant expression, and characterization.J Biol Chem. 2005; 280: 3441-8Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar]. In the present study, the authors identified two latent hot spots that is, regions in human TAFIa where conformational destabilization may begin. The first hot spot that was identified is the Lβ2β3 region. This GKEQAA region spans residues 143–148 in TAFI (TAFI ‘1–401’ numbering). Compared with human pancreatic procarboxypeptidase B1, it has an unique superficial Lys residue at position 144 and an unique one-residue insertion after residue Glu145 making the surface loop larger and thus more flexible. A number of studies have previously shown that this region is an important region. A polymorphism in the coding region of TAFI was first reported at position 147 (i.e. G to A substitution resulting in an Ala-to-Thr substitution). Biochemical comparison between both isoforms revealed no biochemical differences [4Zhao L. Morser J. Bajzar L. Nesheim M. Nagashima M. Identification and characterization of two thrombin-activatable fibrinolysis inhibitor isoforms.Thromb Haemost. 1998; 80: 949-55Crossref PubMed Scopus (72) Google Scholar]. This region harbors an Arg at position 147 in rat (GKEHRV) and mouse (GKEQRI) TAFI. It was previously shown that this Arg147 is a primary cleavage site for plasmin resulting in a non-active 32-kDa TAFI form [5Hillmayer K. Macovei A. Pauwels D. Compernolle G. Declerck P.J. Gils A. Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI) - a comparative study assessing the biological equivalence of rat, murine and human TAFI.J Thromb Haemost. 2006; 4: 2470-7Crossref PubMed Scopus (19) Google Scholar]. The second hot spot that was identified is the α5-Lα5β7-β7 region spanning residues 307–336 (TAFI ‘1–401’ numbering). Again, a number of studies have previously shown that this region determines the stability of TAFIa. Boffa et al. [6Boffa M.B. Bell R. Stevens W.K. Nesheim M.E. Roles of thermal instability and proteolytic cleavage in regulation of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2000; 275: 12868-78Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar] have demonstrated that replacement of Arg302, Arg320 and Arg330 by Gln decreases TAFIa activity. A second polymorphism in the coding region of TAFI was reported at position 325 (i.e. C to T substitution resulting in a Thr-to-Ile substitution) [7Brouwers G.J. Vos H.L. Leebeek F.W. Bulk S. Schneider M. Boffa M. Koschinsky M. Van-Tilburg N.H. Nesheim M.E. Bertina R.M. Gomez-Garcia E.B. A novel, possibly functional, single nucleotide polymorphism in the coding region of the thrombin-activatable fibrinolysis inhibitor (TAFI) gene is also associated with TAFI levels.Blood. 2001; 98: 1992-3Crossref PubMed Scopus (105) Google Scholar]. Biochemical comparison between both isoforms revealed that Ile at position 325 extends the half-life 2-fold and this is associated with a 60% higher anti-fibrinolytic effect [8Schneider M. Boffa M.B. Stewart R. Rahman M. Koschinsky M. Nesheim M. Two naturally occurring variants of TAFI (Thr-325 and Ile-325) differ substantially with respect to thermal stability and antifibrinolytic activity of the enzyme.J Biol Chem. 2002; 277: 1021-30Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar]. Marx et al. [9Marx P.F. Havik S.R. Marquart J.A. Bouma B.N. Meijers J.C. Generation and characterization of a highly stable form of activated thrombin-activable fibrinolysis inhibitor.J Biol Chem. 2004; 279: 6620-8Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar] created a TAFI/CPB chimera in which the 293–401 amino acid region of TAFI was substituted with the corresponding residues in pCPB. Although this chimera revealed a higher stability, it lost most of its antifibrinolytic potential. In 2007, Ceresa et al. combined the stabilizing mutations that were identified previously by Ceresa et al. [10Ceresa E. Van De Borne K. Peeters M. Lijnen H.R. Declerck P.J. Gils A. Generation of a stable activated thrombin activable fibrinolysis inhibitor variant.J Biol Chem. 2006; 281: 15878-83Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar] and by Knecht et al. [11Knecht W. Willemse J. Stenhamre H. Andersson M. Berntsson P. Furebring C. Harrysson A. Hager A.C. Wissing B.M. Hendriks D. Cronet P. Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.FEBS J. 2006; 273: 778-92Crossref PubMed Scopus (39) Google Scholar] into an extremely stable TAFI mutant that is, the Cys305-Ile325-Ile329-Tyr333-Gln335-TAFI mutant with a 180-fold stable half-life and concomitantly a high anti-fibrinolytic effect [12Ceresa E. De Maeyer M. Jonckheer A. Peeters M. Engelborghs Y. Declerck P.J. Gils A. Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability.J Thromb Haemost. 2007; 5: 2105-12Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar]. Previously, the authors elucidated the crystal structure of bovine TAFIa [13Sanglas L. Valnickova Z. Arolas J.L. Pallares I. Guevara T. Sola M. Kristensen T. Enghild J.J. Aviles F.X. Gomis-Ruth F.X. Structure of activated thrombin-activatable fibrinolysis inhibitor, a molecular link between coagulation and fibrinolysis.Mol Cell. 2008; 31: 598-606Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar]. Both human and bovine TAFIa–TCI complexes fit well to each other after optimal superposition. Two segments (i.e. Lα2β4and Lβ2β3) were not defined in the bovine structure but could be defined in the human structure. In contrast to human TAFIa, bovine TAFIa shows a peak of flexibility at Lα2β4. This additional latent hot spot region is potentially important for inactivation of bovine TAFIa (as well as rat and mouse TAFI) due to the presence of Lys182 (TAFI ‘1–401’ numbering) which is freely exposed to solvent and targetable by trypsin/plasmin. The presence of three (bovine) versus two (human) latent hot spots highlights the differences in TAFIa inactivation mechanism among species whereas the availability of the crystal structure of human TAFIa makes structure-based drug design feasible. Furthermore, the authors state that the two latent hot spots reported here in human TAFIa are also present in human TAFI but hypothesize that the prodomain may provide sufficient stabilization and solubility to guarantee protein persistence in vivo. When the pro-domain is removed in vitro, the free TAFIa becomes a labile enzyme that is inactivated by an unspecified conformational change into TAFIai in a temperature dependent manner. Marx et al. have previously shown in vitro that TAFIai but not TAFIa is prone to aggregation and precipitation. They hypothesize that the primary roles of the activation peptide are to shield the catalytic center from physiological substrates and to restrict the movements of the dynamic segment, ensuring structural integrity of the enzyme moiety [14Marx P.F. Plug T. Havik S.R. Morgelin M. Meijers J.C. The activation peptide of thrombin-activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme?.J Thromb Haemost. 2009; 7: 445-52Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar]. Both studies [1Sanglas L. Arolas J.L. Valnickova Z. Aviles F.X. Enghild J.J. Gomis-Ruth F.X. Insights into the molecular inactivation mechanism of human activated thrombin-activatable fibrinolysis inhibitor, TAFIa.J Thromb Haemost. 2010; 8: 1056-65Abstract Full Text Full Text PDF PubMed Scopus (12) Google Scholar, 14Marx P.F. Plug T. Havik S.R. Morgelin M. Meijers J.C. The activation peptide of thrombin-activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme?.J Thromb Haemost. 2009; 7: 445-52Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar] provided evidence that in some conditions (in vitro) the activation peptide is totally released from the enzyme moiety upon cleavage. But what happens in vivo? Does TAFIai also aggregate and precipitate in the circulation? Most likely the inactive TAFIai is prone to further proteolytic cleavage by trypsin like enzymes at sites that become accessible in TAFIai. From these data, it can be concluded that measuring the extent of TAFI activation either through measurement of TAFIa or TAFIai is very difficult as a result of the short half-life of both forms. During activation of TAFI to TAFIa, Marx et al. separated the soluble from the insoluble fraction and demonstrated that in contrast to TAFIai that was present in the insoluble fraction, the activation peptide remained soluble. Determination of the amount of activation peptide seems, at this moment, the best method to measure the extent of TAFI activation [15Ceresa E. Brouwers E. Peeters M. Jern C. Declerck P.J. Gils A. Development of ELISAs measuring the extent of TAFI activation.Arterioscler Thromb Vasc Biol. 2006; 26: 423-8Crossref PubMed Scopus (36) Google Scholar]. The author states that she has no conflict of interest.