Structure of activated thrombin-activatable fibrinolysis inhibitor, a molecular link between coagulation and fibrinolysis.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a metallocarboxypeptidase (MCP) that links blood coagulation and fibrinolysis. TAFI hampers fibrin-clot lysis and is a pharmacological target for the treatment of thrombotic conditions. TAFI is transformed through removal of its prodomain by thrombin-thrombomodulin into TAFIa, which is intrinsically unstable and has a short half-life in vivo. Here we show that purified bovine TAFI activated in the presence of a proteinaceous inhibitor renders a stable enzyme-inhibitor complex. Its crystal structure reveals that TAFIa conforms to the alpha/beta-hydrolase fold of MCPs and displays two unique flexible loops on the molecular surface, accounting for structural instability and susceptibility to proteolysis. In addition, point mutations reported to enhance protein stability in vivo are mainly located in the first loop and in another surface region, which is a potential heparin-binding site. The protein inhibitor contacts both the TAFIa active site and an exosite, thus contributing to high inhibitory efficiency.

Thrombin activatable fibrinolysis inhibitor: a putative target to enhance fibrinolysis.

Thrombin activatable fibrinolysis inhibitor (TAFI) was discovered two decades ago consequent to the identification of an unstable carboxypeptidase (CPU) formed upon thrombin activation of its proenzyme. The antifibrinolytic effects of the activated form (TAFIa, CPU) are linked with its capacity to remove C-terminal lysines from the surface of the fibrin clot. A distinctive characteristic of TAFIa is its temperature-dependent conformational instability: TAFIa activity spontaneously decays with an apparent half-life of 8 to 15 minutes at 37°C. A variety of studies has demonstrated a role for TAFI/TAFIa in venous and arterial diseases. In addition, a role for TAFI/TAFIa in inflammation and cell migration has also been shown. Because TAFI/TAFIa is a potential risk factor for thrombotic disorders, many inhibitors, both at the level of activation or at the level of activity, have been developed and were proven to exhibit a profibrinolytic effect in animal models. Pharmacologically active inhibitors of the TAFI/TAFIa system may open new ways for the prevention of thrombotic diseases or for the establishment of adjunctive treatments during thrombolytic therapy.

The antifibrinolytic function of factor XIII is exclusively expressed through α2-antiplasmin cross-linking.

Factor XIII (FXIII) generates fibrin-fibrin and fibrin-inhibitor cross-links. Our flow model, which is sensitive to cross-linking, was used to assess the effects of FXIII and the fibrinolytic inhibitor, α2-antiplasmin (α2AP) on fibrinolysis. Plasma model thrombi formed from FXIII or α2AP depleted plasma lysed at strikingly similar rates, 9-fold faster than pooled normal plasma (PNP). In contrast, no change was observed on depletion of PAI-1 or thrombin activatable fibrinolysis inhibitor (TAFI). Inhibition of FXIII did not further enhance lysis of α2AP depleted thrombi. Addition of PNP to FXIII or α2AP depleted plasmas normalized lysis. Lysis rate was strongly inversely correlated with total cross-linked α2AP in plasma thrombi. Reconstitution of FXIII into depleted plasma stabilized plasma thrombi and normalized γ-dimers and α-polymers formation. However, the presence of a neutralizing antibody to α2AP abolished this stabilization. Our data show that the antifibrinolytic function of FXIII is independent of fibrin-fibrin cross-linking and is expressed exclusively through α2AP.

Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites.

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 µm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 µm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 µm(-1) s(-1). Interestingly, this value increases to 3.85 µm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.

Flexibility of the thrombin-activatable fibrinolysis inhibitor pro-domain enables productive binding of protein substrates.

We have previously reported that thrombin-activatable fibrinolysis inhibitor (TAFI) exhibits intrinsic proteolytic activity toward large peptides. The structural basis for this observation was clarified by the crystal structures of human and bovine TAFI. These structures evinced a significant rotation of the pro-domain away from the catalytic moiety when compared with other pro-carboxypeptidases, thus enabling access of large peptide substrates to the active site cleft. Here, we further investigated the flexible nature of the pro-domain and demonstrated that TAFI forms productive complexes with protein carboxypeptidase inhibitors from potato, leech, and tick (PCI, LCI, and TCI, respectively). We determined the crystal structure of the bovine TAFI-TCI complex, revealing that the pro-domain was completely displaced from the position observed in the TAFI structure. It protruded into the bulk solvent and was disordered, whereas TCI occupied the position previously held by the pro-domain. The authentic nature of the presently studied TAFI-inhibitor complexes was supported by the trimming of the C-terminal residues from the three inhibitors upon complex formation. This finding suggests that the inhibitors interact with the active site of TAFI in a substrate-like manner. Taken together, these data show for the first time that TAFI is able to form a bona fide complex with protein carboxypeptidase inhibitors. This underlines the unusually flexible nature of the pro-domain and implies a possible mechanism for regulation of TAFI intrinsic proteolytic activity in vivo.

Thrombin activatable fibrinolysis inhibitor.

Thrombin activatable fibrinolysis inhibitor (TAFI) was discovered two decades ago as a consequence of the identification of an unstable carboxypeptidase (CPU), which was formed upon thrombin activation of the respective pro-enzyme (proCPU). The antifibrinolytic function of the activated form (TAFIa, CPU) is directly linked to its capacity to remove C-terminal lysines from the surface of the fibrin clot. No endogenous inhibitors have been identified, but TAFIa activity is regulated by its intrinsic temperature-dependent instability with a half-life of 8 to 15 min at 37 °C. A variety of studies have demonstrated a role for TAFI/TAFIa in venous and arterial diseases. In addition, a role in inflammation and cell migration has been shown. Since an elevated level of TAFIa it is a potential risk factor for thrombotic disorders, many inhibitors, both at the level of activation or at the level of activity, have been developed and were proven to exhibit a profibrinolytic effect in animal models. Pharmacologically active inhibitors of the TAFI/TAFIa system may open new ways for the prevention of thrombotic diseases or for the establishment of adjunctive treatments during thrombolytic therapy.

Crystal structures of TAFI elucidate the inactivation mechanism of activated TAFI: a novel mechanism for enzyme autoregulation.

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 thrombin-cleavage 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.

The effects of hyperglycaemia on thrombin-activatable fibrinolysis inhibitor.

Epidemiological studies have shown a strong association between type 2 diabetes mellitus and cardiovascular diseases, and hypofibrinolysis may contribute to this phenomenon. The aim of this study was to determine the effect of hyperglycaemia on thrombin-activatable fibrinolysis inhibitor (TAFI). Hyperglycaemia was mimicked in vitro by incubation of TAFI with glyceraldehyde and in vivo by hyperglycaemic clamping of healthy volunteers. The effects of long-term hyperglycaemia in vivo on TAFI were investigated by comparing TAFI from poorly regulated and tightly regulated patients with type 2 diabetes. In vitro glycated TAFI showed an altered migration pattern on SDS-PAGE due to aggregation. Glycated TAFI showed decreased activity after activation by thrombin-thrombomodulin in a glyceraldehyde-dose-dependent manner and a reduced anti-fibrinolytic potential. In vivo, no differences in TAFI parameters were found after hyperglycaemic clamping of healthy volunteers and between tightly and poorly regulated patients with type 2 diabetes. Moreover, TAFI purified from poorly regulated and tightly regulated patients with type 2 diabetes migrated similarly on SDS-PAGE, indicating little or no glycation of the protein. Despite the deleterious effects of glycation of TAFI in vitro on its function, TAFI was neither affected by hyperglycaemic clamping, nor by long-term hyperglycaemia in patients with type 2 diabetes. This is in contrast to fibrinolytic factors as plasminogen-activator inhibitor I and tissue-type plasminogen activator, which are affected. We therefore hypothesise that a normally functioning TAFI under hyperglycaemic conditions may tip the haemostatic balance towards hypofibrinolysis, which may contribute to the development of cardiovascular diseases in type 2 diabetic patients.

Recent developments in thrombin-activatable fibrinolysis inhibitor research.

Thrombin-activatable fibrinolysis inhibitor (TAFI) provides an important molecular link between the coagulation and fibrinolytic systems. In this review, recent major advances in TAFI research, including the elucidation of crystal structures, the development of small inhibitors and the role of TAFI in systems other than hemostasis, are described and discussed.

Biochemical characterization of bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI).

BACKGROUND: TAFI is a plasma protein assumed to be an important link between coagulation and fibrinolysis. The three-dimensional crystal structures of authentic mature bovine TAFI (TAFIa) in complex with tick carboxypeptidase inhibitor, authentic full lenght bovine plasma thrombin-activatable fibrinolysis inhibitor (TAFI), and recombinant human TAFI have recently been solved. In light of these recent advances, we have characterized authentic bovine TAFI biochemically and compared it to human TAFI. RESULTS: The four N-linked glycosylation sequons within the activation peptide were all occupied in bovine TAFI, similar to human TAFI, while the sequon located within the enzyme moiety of the bovine protein was non-glycosylated. The enzymatic stability and the kinetic constants of TAFIa differed somewhat between the two proteins, as did the isoelectric point of TAFI, but not TAFIa. Equivalent to human TAFI, bovine TAFI was a substrate for transglutaminases and could be proteolytically cleaved by trypsin or thrombin/solulin complex, although small differences in the fragmentation patterns were observed. Furthermore, bovine TAFI exhibited intrinsic activity and TAFIa attenuated tPA-mediated fibrinolysis similar to the human protein. CONCLUSION: The findings presented here suggest that the properties of these two orthologous proteins are similar and that conclusions reached using the bovine TAFI may be extrapolated to the human protein.

Identification, Molecular Characterization, and In Silico Structural Analysis of Carboxypeptidase B2 of Anopheles stephensi.

Malaria is a vector-borne infectious disease that is considered a priority of the World Health Organization due to its enormous impacts on global health. Plasmodium spp. (Haemosporida: Plasmodiidae), Anopheles spp. (Diptera: Culicidae), and a suitable host are the key elements for malaria transmission. To disrupt the parasitic life cycle of malaria or prevent its transmission, these three key elements should be targeted by effective control strategies. Development of vaccines that interrupt malaria transmission is one of the solutions that has been recommended to the countries that aim to eliminate malaria. With respect to the important role of Anopheles stephensi in malaria transmission and involvement of Anopheles carboxypeptidase B1 in sexual parasite development, we characterized the second member of cpb gene family (cpbAs2) of An. Stephensi to provide some basic information and evaluate significance of cpbAs2's role in complementing sexual plasmodium development role of cpbAs1. The cpbAs2 mRNA sequence was characterized by 3' and 5' RACE and the structural features of its coded protein were studied by in silico modeling. The coding sequence and gene structure of cpbAs2 were determined empirically and compared with the in silico predictions from the An. stephensi genome sequencing project. Furthermore, homology modeling revealed that its structure is very similar to the structurally important domains of procarboxypeptidase B2 in humans. This study provides basic molecular and structural information about another member of the cpb gene family of An. stephensi. The reported results are informative and necessary for evaluation of the role of this gene in sexual parasite development by future studies.

Comparative evaluation of stable TAFIa variants: importance of alpha-helix 9 and beta-sheet 11 for TAFIa (in)stability.

BACKGROUND: Activated thrombin activatable fibrinolysis inhibitor (TAFIa) plays a pivotal role in fibrinolysis. TAFIa activity is regulated by a temperature-dependent instability. This instability has not only complicated the study of structure-function relationships of TAFIa but has also prevented the crystallization of TAFIa. Furthermore, the TAFIa instability has severely compromised the development of activity inhibiting monoclonal antibodies. Recently, we combined all known stabilizing mutations (i.e. S305C, T325I, T329I, H333Y and H335Q) resulting in a synergistic (one hundred and eightyfold) stabilization of TAFIa at 37 degrees C. All these residues are located in an amino acid region (AA297-335) consisting of alpha-helix 9 and beta-sheet 11. OBJECTIVES: To provide a comparative evaluation of the characteristics of a panel of stable TAFIa mutants and an energy-minimized model of the most stable TAFI variant. RESULTS: The catalytic efficiency for activation of TAFI by thrombin/thrombomodulin was higher for all TAFI mutants compared with TAFI-wild type (wt). Except for TAFI variants carrying T325I-T329I, S305C-T325I or S305C-T325I-T329I mutations, the catalytic efficiency for Hip-Arg hydrolysis by TAFIa was similar for the TAFI mutants compared with the wild type. All TAFIa variants were equally well inhibited by potato tuber carboxypeptidase inhibitor (PTCI) and showed a significantly increased antifibrinolytic potential in accordance with their increased stability. Based on the intrinsic fluorescence decay of TAFIa, two independent structural transitions were found to be associated with the loss of functional activity. CONCLUSIONS: Using molecular dynamic calculations on both TAFI-wt and TAFI-S305C-T325I-T329I-H333Y-H335Q models, we were able to identify the molecular interactions that contribute to the increased stability of the mutants.

Limited mutagenesis increases the stability of human carboxypeptidase U (TAFIa) and demonstrates the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

Procarboxypeptidase U [proCPU, thrombin-activatable fibrinolysis inhibitor (TAFI), EC 3.4.17.20] belongs to the metallocarboxypeptidase family and is a zymogen found in human plasma. ProCPU has been proposed to be a molecular link between coagulation and fibrinolysis. Upon activation of proCPU, the active enzyme (CPU) rapidly becomes inactive due to its intrinsic instability. The inherent instability of CPU is likely to be of major importance for the in vivo down-regulation of its activity, but the underlying structural mechanisms of this fast and spontaneous loss of activity of CPU have not yet been explained, and they severely inhibit the structural characterization of CPU. In this study, we screened for more thermostable versions of CPU to increase our understanding of the mechanism underlying the instability of CPU's activity. We have shown that single as well as a few 2-4 mutations in human CPU can prolong the half-life of CPU's activity at 37 degrees C from 0.2 h of wild-type CPU to 0.5-5.5 h for the mutants. We provide evidence that the gain in stable activity is accompanied by a gain in thermostability of the enzyme and increased resistance to proteolytic digest by trypsin. Using one of the stable mutants, we demonstrate the importance of CPU stability over proCPU concentration in down-regulating fibrinolysis.

Characterization of rat thrombin-activatable fibrinolysis inhibitor (TAFI)--a comparative study assessing the biological equivalence of rat, murine and human TAFI.

BACKGROUND AND OBJECTIVES: Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis. Although rat models to study the role of TAFI are available, the biochemical properties of rat TAFI are not well investigated and immunologic tools are lacking. Therefore, we have characterized recombinant rat TAFI-6His and compared its properties with those of human TAFI as well as of murine TAFI-V5-6His. METHODS AND RESULTS: TAFI from all three species is activatable by the thrombin-thrombomodulin complex, generating a highly unstable protein (TAFIa). Half-lives at 37 degrees C are 8.5+/-0.6 min, 3.4+/-0.4 min and 2.2+/-0.2 min for human, rat and murine TAFIa, respectively. The 50% clot lysis times are 6+/-1 min for TAFI-depleted rat plasma and 137+/-34 min, 62+/-9 min and 50+/-8 min when TAFI-depleted rat plasma is supplemented with 0.02 U of human, rat or murine TAFIa, respectively, which correlates with their half-lives. Upon incubation with the thrombin-thrombomodulin complex, the 36-kDa fragment of rat and murine TAFI is not cleaved into 25-kDa and 11-kDa fragments. Upon incubation of rat TAFI and murine TAFI with plasmin, a 32-kDa fragment is formed due to cleavage at Arg147, in contrast to the formation of a 36-kDa fragment for human TAFI. Concomitantly, activity levels upon plasmin incubation are drastically reduced for rat and murine TAFI. CONCLUSIONS: Recombinant human, rat and murine TAFI have similar but not identical biochemical characteristics, suggesting a similar role during fibrinolysis in vivo.

[Basic carboxypeptidases of blood: significance for coagulology].

This review considers the basic metallocarboxypeptidases of human blood and their role in coagulologic disorders. In includes information on the history of the discovery and biological characteristics of potential enzymes-regulators of the fibrinolytic process: carboxypeptidase U and carboxypeptidase N. Certain attention is paid to the biochemical mechanisms and the main modern concepts of the antifibrinolytic effects of these enzymes.

Identification of a thrombomodulin interaction site on thrombin-activatable fibrinolysis inhibitor that mediates accelerated activation by thrombin.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a human plasma zymogen that provides a molecular connection between coagulation and fibrinolysis. TAFI is activated through proteolytic cleavage by thrombin, thrombin in complex with the endothelial cell cofactor thrombomodulin (TM) or plasmin. Evidence from several studies suggests that TM and TAFI make direct contact at sites remote from the activating cleavage site to facilitate acceleration of thrombin-mediated TAFI activation. The elements of TAFI structure that allow accelerated activation of thrombin by TM are incompletely defined. OBJECTIVES: To identify TM interaction regions on TAFI that mediate acceleration of activation by thrombin and therefore indicate TM binding sites on TAFI. METHODS: We mutated selected surface-exposed charged residues on TAFI to alanine in order to identify sites that mediate acceleration of activation by TM. The kinetics of activation of the mutants by thrombin in the presence or absence of TM, as well as their thermal stabilities and antifibrinolytic potentials, were determined. RESULTS: TAFI variants R15A, E28A, K59A, D75A/E77A/D78A, E99A and E106A all exhibited moderately reduced catalytic efficiencies of activation by thrombin-TM. TAFI variants R377A and, particularly, R12A and R12A/R15A exhibited severely reduced activation by thrombin-TM that was not explained by differences in activation by thrombin alone. CONCLUSIONS: We have identified R12 as a critical residue for the activation of TAFI by thrombin-TM, extending a previous report that identified a role for this residue. R12 is likely to directly bind to TM while another key residue, R377, may affect the thrombin-TAFI interaction specifically in the presence of TM.

Isolation, Co-Crystallization and Structure-Based Characterization of Anabaenopeptins as Highly Potent Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa).

Mature thrombin activatable fibrinolysis inhibitor (TAFIa) is a carboxypeptidase that stabilizes fibrin clots by removing C-terminal arginines and lysines from partially degraded fibrin. Inhibition of TAFIa stimulates the degradation of fibrin clots and may help to prevent thrombosis. Applying a lead finding approach based on literature-mining, we discovered that anabaenopeptins, cyclic peptides produced by cyanobacteria, were potent inhibitors of TAFIa with IC50 values as low as 1.5 nM. We describe the isolation and structure elucidation of 20 anabaenopeptins, including 13 novel congeners, as well as their pronounced structure-activity relationships (SAR) with respect to inhibition of TAFIa. Crystal structures of the anabaenopeptins B, C and F bound to the surrogate protease carboxypeptidase B revealed the binding modes of these large (~850 Da) compounds in detail and explained the observed SAR, i.e. the strong dependence of the potency on a basic (Arg, Lys) exocyclic residue that addressed the S1' binding pocket, and a broad tolerance towards substitutions in the pentacyclic ring that acted as a plug of the active site.

Role of isoleucine residues 182 and 183 in thrombin-activatable fibrinolysis inhibitor.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, once activated, can attenuate fibrinolysis. The active form, TAFIa, is a labile enzyme, with a half-life of a few minutes at 37 degrees C. Understanding the molecular mechanisms of TAFIa inactivation will allow the development of compounds that modulate TAFIa activity. Based on their three-dimensional model of TAFI, Barbosa Pereira et al. [J Mol Biol (2002), vol. 321, pp. 537-547] suggested that Ile182 and Ile183 were involved in the instability of TAFIa. However, these carboxypeptidases are, unlike TAFIa, stable proteases. Therefore, we constructed, expressed and characterized a TAFI mutant in which Ile182 and Ile183 were changed into the residues found in pancreas carboxypeptidase B at corresponding positions, Arg and Glu. The active form of the mutant, TAFIa-I182R-I183E, had a similar half-life as wild-type TAFIa, showing that Ile182 and Ile183 were not involved in the regulation of TAFIa stability. Remarkably, however, TAFI-I182R-I183E was activated at a lower rate by thrombin-thrombomodulin (mutant: 45 +/- 2 U L(-1) s(-1) and wild type: 103 +/- 3 U L(-1) s(-1)), thrombin (mutant: 1 +/-0.1 U L(-1) s(-1) and wild type 3 +/- 0.2 U L(-1) s(-1)) and plasmin (mutant: 0.8 +/- 0.04 U L(-1) s(-1) and wild type: 5.0 +/-0.2 U L(-1) s(-1)) compared with wild-type TAFI. Accordingly, it had a sixfold reduced antifibrinolytic potential. In conclusion, analysis of TAFI-I182R-I183E showed that I182 and I183 are not involved in TAFIa inactivation by conformational instability but that these residues may be involved in the activation of TAFI and stabilization of the fibrin clot.

Rational structure-based design of a novel carboxypeptidase R inhibitor.

A novel carboxypeptidase R (CPR) inhibitor, related to potato carboxypeptidase inhibitor (PCI), was designed using rational structure-based strategies, incorporating two principle facts: CPR has a strong affinity for basic amino acids, and the two lysine and arginine residues of PCI are orientated in the same direction and held in close spatial proximity by three disulfide bonds. Initially, a disulfide-bonded fragment of PCI was synthesized showing weak competitive inhibitory activity against CPR. Subsequently, a smaller linear 9-mer peptide, designated CPI-2KR, was designed/synthesized and found to be a more efficient competitive inhibitor of CPR, without affecting the activity of the other plasma carboxypeptidase, carboxypeptidase N. In vitro studies showed that, together with tissue plasminogen activator, CPI-2KR synergistically accelerated fibrinolysis, representing a lead compound for the design of smaller organic molecules for use in thrombolytic therapy.

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

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 IC50 values of 7.3 ± 1.8 and 6.1 ± 0.9 µm, 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 (KD : 1.5 ± 0.4 and 0.52 ± 0.07 µm 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.