Thrombin-activatable Fibrinolysis Inhibitor Deficiency Research Articles

A new pedigree with thrombomodulin‐associated coagulopathy in which delayed fibrinolysis is partially attenuated by co‐inherited TAFI deficiency

BackgroundThrombomodulin‐associated coagulopathy (TM‐AC) is a rare bleeding disorder in which a single reported p.Cys537* variant in the thrombomodulin gene THBD causes high plasma thrombomodulin (TM) levels. High TM levels attenuate thrombin generation and delay fibrinolysis. ObjectivesTo report the characteristics of pedigree with a novel THBD variant causing TM‐AC, and co‐inherited deficiency of thrombin‐activatable fibrinolysis inhibitor (TAFI). Patients/methodsIdentification of pathogenic variants in hemostasis genes by next‐generation sequencing and case recall for deep phenotyping. ResultsPedigree members with a previously reported THBD variant predicting p.Pro496Argfs*10 and chain truncation in TM transmembrane domain had abnormal bleeding and greatly increased plasma TM levels. Affected cases had attenuated thrombin generation and delayed fibrinolysis similar to previous reported TM_AC cases with THBD p.Cys537*. Coincidentally, some pedigree members also harbored a stop‐gain variant in CPB2 encoding TAFI. This reduced plasma TAFI levels but was asymptomatic. Pedigree members with TM‐AC caused by the p.Pro496Argfs*10 THBD variant and also TAFI deficiency had a partially attenuated delay in fibrinolysis, but no change in the defective thrombin generation. ConclusionsThese data extend the reported genetic repertoire of TM‐AC and establish a common molecular pathogenesis arising from high plasma levels of TM extra‐cellular domain. The data further confirm that the delay in fibrinolysis associated with TM‐AC is directly linked to increased TAFI activation. The combination of the rare variants in the pedigree members provides a unique genetic model to develop understanding of the thrombin‐TM system and its regulation of TAFI.

TAFI deficiency causes maladaptive vascular remodeling after hemophilic joint bleeding.

Excessive vascular remodeling is characteristic of hemophilic arthropathy (HA) and may contribute to joint bleeding and the progression of HA. Mechanisms for pathological vascular remodeling after hemophilic joint bleeding are unknown. In hemophilia, activation of thrombin-activatable fibrinolysis inhibitor (TAFI) is impaired, which contributes to joint bleeding and may also underlie the aberrant vascular remodeling. Here, hemophilia A (factor VIII-deficient; FVIII-deficient) mice or TAFI-deficient mice with transient (antibody-induced) hemophilia A were used to determine the role of FVIII and TAFI in vascular remodeling after joint bleeding. Excessive vascular remodeling and vessel enlargement persisted in FVIII-deficient and TAFI-deficient mice, but not in transient hemophilia WT mice, after similar joint bleeding. TAFI-overexpression in FVIII-deficient mice prevented abnormal vessel enlargement and vascular leakage. Age-related vascular changes were observed with FVIII or TAFI deficiency and correlated positively with bleeding severity after injury, supporting increased vascularity as a major contributor to joint bleeding. Antibody-mediated inhibition of uPA also prevented abnormal vascular remodeling, suggesting that TAFI's protective effects include inhibition of uPA-mediated plasminogen activation. In conclusion, the functional TAFI deficiency in hemophilia drives maladaptive vascular remodeling in the joints after bleeding. These mechanistic insights allow targeted development of potentially new strategies to normalize vascularity and control rebleeding in HA.

Defective TAFI Activation in Hemophilia Worsens Joint Bleeding

Background: Joint bleeds are common in hemophilia A or B and cause hemophilic arthropathy. It is clinically well recognized that patients with acquired hemophilia generally do not suffer from joint bleeding, but the molecular mechanisms responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia are ill defined. FVIII deficiency causes defective thrombin generation, impaired coagulation, and increased fibrinolysis. The latter is caused by impaired activation of thrombin activatable fibrinolysis inhibitor (TAFI). Our previous plasma-based analyses showed that clotting and thrombin generation were readily inhibited by an anti-FVIII antibody, whereas a 10-fold higher antibody concentration was required to inhibit thrombin-mediated TAFI activation. We hypothesize that residual TAFI activation occurring in acquired hemophilia, but not in congenital hemophilia, protects against joint bleeding. Here, we determine whether TAFI activation prevents joint bleeding in a mouse model of acquired hemophilia.Methods and results: A transient (anti-FVIII) acquired hemophilia A model was set up to compare joint bleeding in wild type (WT) vs. TAFI-/- mice. Joint bleeding was induced by a subpatellar needle puncture in the right knee. This model caused considerable joint bleeding in FVIII-/- mice as evidenced by the decreased hematocrit (Hct) 2 days post injury (D2 Hct) (D2 Hct= 29 ± 11 % (n= 9) vs. baseline Hct (46 ± 2 %); p< 0.0001). A single injection of the FVIII inhibiting antibody (GMA-8015; 0.25 mg/kg) in WT mice caused acquired hemophilia for up to 72 hours as evident from increased tail bleeding similar to that observed in FVIII-/- mice. Consistent with clinical findings, only minimal joint bleeding was observed in inhibitor-treated WT mice (D2 Hct= 44 ± 4 % (n= 15) for BALB/c and 40 ± 4 % (n= 17) for C57Bl/6J). Significant joint bleeding (D2 Hct= 36 ± 9% (n= 12) for C57Bl/6J; p< 0.05) could be induced by a higher dose of inhibitor (1 mg/kg), however bleeding remained considerably less severe than that observed in FVIII-/-mice.In vitro, the FVIII inhibitor readily inhibited thrombin generation but was relatively ineffective in inhibiting TAFI activation. Therefore, we tested our hypothesis that continued TAFI activation prevented severe joint bleeding in the inhibitor-treated WT mice. Indeed, administration of the FVIII inhibitor (0.25 mg/kg) in TAFI-/-mice resulted in excessive joint bleeding (D2 Hct= 25 ± 8 %; n= 14; p< 0.0001). Similarly, joint bleeding in WT mice was increased significantly when the FVIII inhibitor was co-administered with an inhibitory antibody against TAFI (D2 Hct= 34 ± 7 %; n= 13; p< 0.01). In contrast, TAFI deficiency did not increase tail bleeding with or without FVIII inhibitor, as determined by acute blood loss, 24-hour mortality, and Hct of the survivors at 24 hours post tail resection. These data clearly demonstrate that different vascular beds empower different mechanisms to curb bleeding and suggest that the protective effects of TAFI are specifically relevant for the vascular beds of the synovial joint.Activated TAFI (TAFIa) conveys multiple functions, including anti-fibrinolytic effects and numerous anti-inflammatory activities. Interestingly, tranexamic acid (TXA), a Lys analogue and potent anti-fibrinolytic agent, added at 50 mg/ml to the drinking water, did not reduce joint bleeding in FVIII-/- mice or TAFI-/- mice with the FVIII inhibitor, whereas TXA did correct tail bleeding in these mice. This suggests that the protective effects of TAFI on joint bleeding were independent of its anti-fibrinolytic effects and may result from its anti-inflammatory activities. This is supported by histological analysis at day 7 showing increased stromal proliferation and inflammatory cell recruitment in the joints of TAFI-/-mice.Conclusions:TAFI activation is impaired in congenital hemophilia but not in acquired hemophilia. Abrogation of TAFIa activity, either genetically or pharmaceutically, increased joint bleeding in mice with acquired hemophilia, indicating that TAFI may be responsible for the difference in joint bleeding tendency between acquired and congenital hemophilia. Protective effects of TAFI were vascular bed specific and independent of its anti-fibrinolytic effects, suggesting that one or more of TAFIa's other substrates promote hemophilic joint bleeding. Disclosuresvon Drygalski:Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Pfizer: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau. Mosnier:The Scripps Research Institute: Patents & Royalties; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.

TAFI deficiency promotes liver damage in murine models of liver failure through defective down-regulation of hepatic inflammation

Emerging evidence indicates that various haemostatic components can regulate the progression of liver disease. Thrombin-activatable fibrinolysis inhibitor (TAFI) possesses anti-inflammatory properties besides its anti-fibrinolytic function. Here, we investigated the contribution of TAFI to the progression of disease in murine models of chronic and acute liver failure. Chronic carbon tetrachloride (CCL4) administration induced liver damage and fibrosis both in TAFI knockout (TAFI-/-) mice and wild-type controls. Smooth muscle actin-α (α-SMA) content of liver tissue was significantly increased after 1 and 3 weeks, and pro-collagen α1 expression was significantly increased after 3 and 6 weeks in TAFI-/- mice. TAFI-/- mice showed significantly elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) after 3 weeks of CCL4. Neutrophil influx was significantly increased in TAFI-/- mice after 6 weeks of CCL4. No difference in hepatic fibrin deposition between TAFI-/- and wild-types was observed. After acetaminophen intoxication, necrosis was significantly increased in TAFI-/- mice at 24 hours (h) after injection. AST and ALT levels were decreased at 2 and 6 h after acetaminophen injection in TAFI-/- mice, but were significantly higher in the TAFI-/- mice at 24 h. Similarly, hepatic fibrin deposition was decreased at 6 h in TAFI-/- mice, but was comparable to wild-types at 24 h after injection. In conclusion, TAFI deficiency results in accelerated fibrogenesis and increased liver damage in murine models of chronic and acute liver disease, which may be related to increased inflammation.

The hyperfibrinolytic state of mice with combined thrombin‐activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor‐1 gene deficiency is critically dependent on TAFI deficiency

Mice with single gene deficiency of thrombin-activatable fibrinolysis inhibitor (TAFI) or plasminogen activator inhibitor-1 (PAI-1) have an enhanced fibrinolytic capacity. To unravel the function and relevance of both antifibrinolytic proteins through the generation and characterization of mice with combined TAFI and PAI-1 gene deficiency. Mating of TAFI knockout (KO) mice with PAI-1 KO mice resulted in the production of TAFI/PAI-1 double-KO mice that were viable, were fertile, and developed normally. In a tail vein bleeding model, the bleeding time and hemoglobin content of the TAFI/PAI-1 double-KO mice did not deviate significantly from those of the single-KO mice or of the wild-type (WT) counterparts. Interestingly, in ex vivo rotational thromboelastometry measurements with whole blood samples, TAFI KO mice and TAFI/PAI-1 double-KO mice were more sensitive to fibrinolytic activation with tissue-type plasminogen activator than WT or PAI-1 KO mice. This enhanced fibrinolytic capacity was confirmed in vivo in a mouse thromboembolism model, as shown by decreased fibrin deposition in the lungs of TAFI KO mice and TAFI/PAI-1 double-KO mice as compared with WT or PAI-1 KO mice. TAFI gene inactivation predominantly contributes to the increased fibrinolytic capacity of TAFI and PAI-1 double-gene-deficient mice, as observed in some basic thrombosis models.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice are susceptible to intracerebral thrombosis and ischemic stroke.

BackgroundThrombus formation is a key step in the pathophysiology of acute ischemic stroke and results from the activation of the coagulation cascade. Thrombin plays a central role in this coagulation system and contributes to thrombus stability via activation of thrombin-activatable fibrinolysis inhibitor (TAFIa). TAFIa counteracts endogenous fibrinolysis at different stages and elevated TAFI levels are a risk factor for thrombotic events including ischemic stroke. Although substantial in vitro data on the influence of TAFI on the coagulation-fibrinolysis-system exist, investigations on the consequences of TAFI inhibition in animal models of cerebral ischemia are still lacking. In the present study we analyzed stroke development and post stroke functional outcome in TAFI-/- mice.Methodology/Principal Findings TAFI-/- mice and wild-type controls were subjected to 60 min transient middle cerebral artery occlusion (tMCAO) using the intraluminal filament method. After 24 hours, functional outcome scores were assessed and infarct volumes were measured from 2,3,5-Triphenyltetrazoliumchloride (TTC)-stained brain slices. Hematoxylin and eosin (H&E) staining was used to estimate the extent of neuronal cell damage. Thrombus formation within the infarcted brain areas was analyzed by immunoblot. Infarct volumes and functional outcomes did not significantly differ between TAFI-/- mice and controls (p>0.05). Histology revealed extensive ischemic neuronal damage regularly including the cortex and the basal ganglia in both groups. TAFI deficiency also had no influence on intracerebral fibrin(ogen) formation after tMCAO.ConclusionOur study shows that TAFI does not play a major role for thrombus formation and neuronal degeneration after ischemic brain challenge.

What has been learnt from the thrombin-activatable fibrinolysis inhibitor-deficient mouse?

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating zymogen that is activated physiologically by the thrombin/thrombomodulin complex to activated TAFI (TAFIa) which is a basic carboxypeptidase. Substrates include fibrin, leading to a reduction in rate of plasmin generation, and several proinflammatory mediators such as bradykinin, thrombin-cleaved osteopontin and complement factor C5a. TAFI-deficient mice have no phenotype without being challenged and TAFIa appears to play a limited role in physiological fibrinolysis in vivo. In several disease models, the TAFI-deficient mice have different outcomes from the wild type (WT), but whether the difference is beneficial or an exacerbation of the disease depends on the model. The consequences of TAFI deficiency include increased plasmin as a result of enhanced incorporation of plasminogen and tissue plasminogen activator into the fibrin clot, but also loss of its ability to degrade other substrates, with the resultant up-regulation of several proinflammatory mediators, including C5a. Criteria are recommended to demonstrate that a substrate is a physiological substrate of TAFIa.

Pulmonary hypertension is ameliorated in mice deficient in thrombin-activatable fibrinolysis inhibitor.

The fibrinolytic system has been implicated in the pathogenesis of pulmonary hypertension (PH). Thrombin-activatable fibrinolysis inhibitor (TAFI) inhibits fibrinolysis and therefore its absence would be expected to increase fibrinolysis and ameliorate PH. The objective of the present study was to evaluate the effect of TAFI deficiency on pulmonary hypertension in the mouse. PH was induced in C57/Bl6 wild-type (WT) or TAFI-deficient (KO) mice by weekly subcutaneous treatment with 600 mg kg(-1) monocrotaline (MCT) for 8 weeks. PH was inferred from right heart hypertrophy measured using the ratio of right ventricle-to-left ventricle-plus-septum weight [RV/(LV+S)]. Pulmonary vascular remodeling was analyzed by morphometry. TAFI-deficient MCT-treated and wild-type MCT-treated mice suffered similar weight loss. TAFI-deficient MCT-treated mice had reduced levels of total protein and tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta) and monocyte chemoattractant protein-1 (MCP-1) in bronchial alveolar lavage compared with wild-type MCT-treated mice. The ratio of RV to (LV+S) weight was significantly higher in WT/MCT than in KO/MCT mice. The pulmonary artery wall area and vascular stenosis were both greater in MCT-treated WT mice compared with MCT-treated TAFI-deficient mice. TAFI-deficient MCT-treated mice had less pulmonary hypertension, vascular remodeling and reduced levels of cytokines compared with MCT-treated WT animals, possibly as a result of reduced coagulation activation.

Assessment of thrombin-activatable fibrinolysis inhibitor (TAFI) activation in acquired hemostatic dysfunction: a diagnostic challenge

Human thrombin activatable fibrinolysis inhibitor (TAFI) is a carboxypeptidase zymogen that was first described in 1995 [1]. It had been previously reported under different names such as carboxypeptidase basic (B), unstable (U), and arginine (R) [2]. The mature TAFI protein contains 401 amino acids, and after glycosylation, the molecular weight becomes 60 kDa. TAFI in its plasma zymogen form can be activated by thrombin, the thrombin-thrombomodulin complex, or plasmin. The activated TAFI (TAFIa) decays spontaneously into the inactive form of TAFI (TAFIai) through a temperature-dependent conformational change. TAFIa plays a role in the inhibition of fibrinolysis by cleaving the carboxyl-terminal lysine residues from partially degraded fibrin. In the coagulation cascade, the generated thrombin can activate TAFI, and the resulting TAFIa inhibits fibrinolysis, finally resulting in the formation of more stable clots [3]. Therefore, a deficiency of the coagulation factors might reduce the TAFIa levels via reduced thrombin generation, resulting in the formation of an unstable clot. In an animal study, TAFI was activated during a thrombotic event, and the inhibition of TAFIa activity enhanced the efficacy of thrombolytic therapy, thereby confirming its role as an inhibitor of fibrinolysis [4-6]. TAFIa also plays a role in the modulation of inflammation through down-regulation of pericellular plasminogen activation and inactivation of the inflammatory peptides bradykinin and anaphylatoxins C3a and C5a. In mice with Escherichia coli-induced sepsis, TAFI deficiency prevented liver damage via inactivation of C3a and C5a [7]. The plasma concentration of TAFI appears to be under the control of genetic factors and non-genetic factors such as sex hormones, cytokines, and lipids [2]. Several diseases, including diabetes, kidney transplantation, hypertension, nephritic syndrome, insulin resistance, obesity, and inflammatory bowel disease, have been shown to be positively associated with plasma TAFI levels [2]. Because TAFI is expressed in adipocytes, an increased TAFI level can be expected in metabolic disturbance. The TAFI levels were also elevated in thrombotic diseases. In keeping with the role of TAFI as an antifibrinolytic factor, high plasma level of TAFI is a risk factor for thrombotic diseases. Meanwhile, TAFI levels are reduced in acquired hemostatic dysfunctions, such as post-partum hemorrhage and sepsis accompanying disseminated intravascular coagulation [3], because the enhanced generation of thrombin or plasmin may result in enhanced TAFI activation and consequent consumption of the TAFI zymogen. Many different methods for measuring TAFI by using ELISA, enzymological methods, or functional methods have been described. All the methods have their inherent advantages and disadvantages, and to date, there is no reference standard for TAFI. Most studies have used the total antigen level of TAFI, but this has yielded ambiguous results, because of the existence of different isoforms and various degrees of activation. Ceres et al. [8] have reported the use of an ELISA that specifically detects TAFI activation. Since substantial TAFI activation occurs in acquired hemostatic dysfunction, ELISA that measures the extent of TAFI activation is potentially useful. In this issue of the Korean Journal of Hematology, Park et al. [9] reported high levels of TAFI activation in sepsis patients by measuring TAFIa/ai levels using an ELISA that they developed in-house. In the in-house ELISA, potato tuber carboxypeptidase inhibitor was used as a capture agent that specifically captured the activated and inactivated TAFI isoforms and did not capture the intact TAFI zymogen. Although only a few septic patients (n=25) were enrolled in the study, the activation status of TAFI could clearly differentiate septic patients from healthy controls. This result is in agreement with the results of a recent study [10] that reported a significant elevation of TAFI activation in meningococcal infection. Since decreases in total TAFI level are associated with increases of TAFI activation level, simultaneous determination of total TAFI and TAFI activation marker is required to interpret the role of TAFI in acquired hemostatic dysfunctions including sepsis. Although the principal physiologic role of TAFI is still a matter of debate, TAFI is reported to be an important protein in the fibrinolytic system. The advent of more accurate assays for measuring plasma TAFI levels may help validate the mechanism underlying TAFI activation in acquired hemostatic dysfunction. This information may help elucidate the contribution of the TAFI pathway to acquired hemostatic dysfunction and will form the basis of potential therapeutic strategies to manipulate the TAFI pathway for acquired hemostatic dysfunction.

Immune complex-mediated glomerulonephritis is ameliorated by thrombin-activatable fibrinolysis inhibitor deficiency

The activity of plasmin plays a critical role in the development of chronic glomerulonephritis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a potent inhibitor of plasmin generation. We hypothesized that TAFI is involved in the pathogenesis of glomerulonephritis because it inhibits plasmin generation. To demonstrate this hypothesis, we compared the development of immune complex-mediated glomerulonephritis in wild-type and TAFI-deficient mice. After six weeks of treatment with horse spleen apoferritin and lipoplysaccharide to induce glomerulonephritis, mice deficient in TAFI had significantly better renal function as shown by lower concentrations of albumin in urine and blood urea nitrogen compared to wild-type mice. In addition, the activity of plasmin and matrix metalloproteinases was significantly increased, and mesangial matrix expansion and the deposition of collagen and fibrin in kidney tissues were significantly decreased in TAFI-knockout mice as compared to their wild-type counterparts. Depletion of fibrinogen by batroxobin (Defibrase) treatment led to equalization of the renal function and the amount of collagen deposition in the kidneys of TAFI-knockout and wild-type mice with immune complex-mediated glomerulonephritis. Together these observations suggest that TAFI-mediated inhibition of plasmin generation plays a role in the pathogenesis of glomerulonephritis, and that it may constitute a novel molecular target for the therapy of this disease.

TAFI deficiency in liver cirrhosis: Relation with plasma fibrinolysis and survival

Introduction TAFI (thrombin-activatable fibrinolysis inhibitor) is a potent anti-fibrinolytic and anti-inflammatory factor of liver origin. It is markedly reduced in liver cirrhosis but its effect on fibrinolysis remains controversial and no data are available on its prognostic value. We evaluated the relationship of TAFI level with plasma fibrinolysis and survival in cirrhotic patients. Patients and methods Sixty-five patients with liver cirrhosis were studied. TAFI antigen, plasma fibrinolysis and other laboratory variables were assayed at study entry and their association with mortality was assessed during a 3-year follow-up. Results TAFI level and fibrinolysis time were markedly reduced in liver cirrhosis as compared to healthy subjects ( p < 0.0001) and TAFI deficiency was strongly correlated with fibrinolysis time ( p = 0.0002). TAFI level at entry, but not fibrinolysis time, was significantly lower in non-survivors ( n = 25) than in survivors ( n = 40, p = 0.0001). By Cox regression analysis, after adjustment for possible confounding factors, TAFI, but not fibrinolysis time, was identified as an independent predictor of mortality. TAFI assay, moreover, showed a clinically relevant accuracy in assessing patients' survival (ROC curve analysis, p < 0.0001) achieving a sensitivity of 92%, a specificity of 55%, and a negative predictive value of 91.7%. Conclusions Our data indicate that TAFI deficiency in liver cirrhosis is associated with enhanced plasma fibrinolysis. Moreover, they suggest that TAFI, but not fibrinolysis time, is a strong predictor of survival and thus TAFI assay might prove useful to select candidates for liver transplantation.

Deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI) protected mice from ferric chloride-induced vena cava thrombosis

Thrombin-activatable fibrinolysis inhibitor (TAFI) is a plasma carboxypeptidase that renders a fibrin-containing thrombus less sensitive to lysis. Since the role of TAFI in thrombus formation is still controversial in mice, our present study was designed to evaluate mice deficient in TAFI (TAFI(-/-)) on FeCl(3)-induced vena cava and carotid artery thrombosis. Parallel studies were carried out in wild-type mice using a potato carboxypeptidase inhibitor (PCI), a selective inhibitor of activated TAFI (TAFIa). Significant reduction in thrombus formation was observed in TAFI(-/-) mice (n = 8, P < 0.05 compared to wild-type littermates) but not in heterozygous (TAFI(+/-)) mice in 3.5% FeCl(3)-induced vena cava thrombosis. A similar effect was observed following treatment with 5 mg/kg bolus plus 5 mg/kg/h PCI in the same venous thrombosis model in C57BL/6 mice (n = 8, P < 0.01 compared to vehicle). No compositional difference was observed for the venous thrombi in TAFI(-/-) and wild-type littermates with or without PCI treatment using histological assessment. In contrast, neither TAFI deficiency nor treatment with PCI showed antithrombotic efficacy in the 3.5% FeCl(3)-induced carotid artery thrombosis model. In a tail transection bleeding time model, both TAFI deficiency and PCI treatment increased bleeding time up to 4.5 and 3.5 times, respectively, over controls (P < 0.05, n = 8). Similar ex vivo fibrinolytic activities were demonstrated for both TAFI deficiency and PCI treatment as enhanced lysis of thrombin-induced plasma clots and lysis of whole blood clot in a thrombelastograph. These data provide direct evidence for the role of TAFIa in vena cava thrombosis without the addition of exogenous thrombolytic in mice. The strong ex vivo fibrinolytic activity of TAFI deficiency or TAFIa inhibition by PCI provides a biomarker of TAFIa inhibition that tracks in vivo antithrombotic efficacy.

TAFI: regulating the cross talk between coagulation and fibrinolysis TAFI: Regulierung der Wechselwirkung zwischen Gerinnung und Fibrinolyse

Abstract Thrombin activates thrombin-activatable fibrinolysis inhibitor (TAFI) that removes C-terminal lysines or arginines. The activity of active TAFI (TAFIa) is lost rapidly. TAFIa is not inactivated by proteolysis but converted to an inactive state by a conformational transition. Numerous polymorphisms were identified in the TAFI gene. The TAFI-325Ile variant seems to influence TAFI levels. TAFIa retards plasmin formation and makes plasmin more susceptible to inhibition by antiplasmin; it prevents the conversion of the fibrin fragment DD(E) to fragment DD that impairs fibrin polymerization. The complement-derived factors C3a and C5a as well as bradykinin are further substrates for TAFIa. Elevated TAFI levels were associated with an increased risk of venous thrombosis. TAFI deficiency has been shown to be associated with an enhanced leucocyte migration. The absence of TAFI results in delayed wound healing with disturbed keratinocyte migration. Pro-inflammatory properties of osteopontin are downregulated by TAFIa. Hence, TAFI plays a role – besides in regulation of fibrinolysis – in wound healing, angiogenesis, and inflammation.

Tumor growth and metastasis are not affected in thrombin‐activatable fibrinolysis inhibitor‐deficient mice

Many studies have indicated that the plasminogen activation system may have a prominent role in cancer. Activation of the zymogen plasminogen into the serine protease plasmin by plasminogen activator is mediated by carboxyterminal basic amino acids in fibrin, including lysines and arginines. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a circulating carboxypeptidase B-type proenzyme that, after activation, removes carboxyterminal lysine or arginine residues in fibrin, resulting in decreased plasminogen activation and attenuated fibrinolysis. To determine directly whether TAFI is involved in primary tumor growth and metastasis formation, we examined the effects of TAFI deficiency on subcutaneous growth and experimentally or spontaneously induced pulmonary metastasis formation of different tumor cell types in mice. In all tumor models TAFI deficiency did not affect the formation and growth of primary and metastasized tumors.

Impaired healing of cutaneous wounds and colonic anastomoses in mice lacking thrombin-activatable fibrinolysis inhibitor

Plasmin and other components of the plasminogen activation system play an important role in tissue repair by regulating extracellular matrix remodeling, including fibrin degradation. Thrombin-activatable fibrinolysis inhibitor (TAFI) is a procarboxypeptidase that, after activation, can attenuate plasmin-mediated fibrin degradation by removing the C-terminal lysine residues from fibrin, which play a role in the binding and activation of plasminogen. To test the hypothesis that TAFI is an important determinant in the control of tissue repair, we investigated the effect of TAFI deficiency on the healing of cutaneous wounds and colonic anastomoses. Histological examination revealed inappropriate organization of skin wound closure in the TAFI knockout mice, including an altered pattern of epithelial migration. The time required to completely heal the cutaneous wounds was slightly delayed in TAFI-deficient mice. Healing of colonic anastomoses was also impaired, as reflected by decreased strength of the tissue at the site of the suture, and by bleeding complications in 3 of 14 animals. Together, these abnormalities resulted in increased mortality in TAFI-deficient mice after colonic anastomoses. Although our study shows that tissue repair, including re-epithelialization and scar formation, occurs in TAFI-deficient mice, TAFI appears to be important for appropriate organization of the healing process.

Deficiency of thrombin activatable fibrinolysis inhibitor in cirrhosis is associated with increased plasma fibrinolysis

Hyperfibrinolysis is thought to contribute to bleeding associated with advanced cirrhosis. Thrombin activatable fibrinolysis inhibitor (TAFI) is a plasma precursor of a carboxypeptidase (TAFIa) with antifibrinolytic activity and was recently shown to be reduced in cirrhosis. In this study, we evaluated the influence of TAFI deficiency on in vitro fibrinolysis in cirrhotic patients. Fifty-three patients with cirrhosis and 43 healthy controls were studied. TAFI antigen was measured by enzyme-linked immunosorbent assay and TAFI activity by chromogenic assay. Fibrinolysis was evaluated as tissue plasminogen activator-induced plasma clot lysis time in the absence and in the presence of a specific inhibitor of TAFIa. TAFI antigen and activity levels were markedly reduced in cirrhotic patients ( P < .0001). In these patients, the lysis time of plasma clots was shorter than in controls (median, interquartile range: 25 minutes, 21-36 minutes vs. 48 minutes, 40-60 minutes, respectively; P < .0001) and was poorly influenced by the TAFIa inhibitor. Accordingly, TAFIa and thrombin activity, generated in cirrhotic samples during clot lysis, were significantly lower than in control samples. Addition of purified TAFI to cirrhotic plasma prolonged the lysis time and enhanced the response to TAFIa inhibitor in a dose-dependent manner. In conclusion, our results indicate that in vitro plasma hyperfibrinolysis in cirrhosis is largely due to a defective TAFIa generation resulting from low TAFI levels and probably from impaired thrombin generation. Impairment of the antifibrinolytic TAFI pathway might contribute to bleeding associated with this disease. (H epatology 2003;38:230-237.)

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficiency is compatible with murine life.

To investigate the consequence of deficiency in thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI-deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice produced offspring in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI-deficient mice developed normally, reached adulthood, and were fertile. No gross physical abnormalities were observed up to 24 months of age. Hematological analysis of TAFI-deficient mice did not show any major differences including plasma fibrinogen level, prothrombin time, and activated partial thromboplastin time. TAFI-deficient mice did not suffer from excess bleeding as determined by blood loss following tail transection, although their plasma failed to prolong clot lysis time in vitro. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous injury model. TAFI deficiency did not improve survival rate compared with the wild-type in thrombin-induced thromboembolism, factor X coagulant protein-induced thrombosis, and endotoxin-induced disseminated intravascular coagulation. Furthermore, TAFI deficiency did not alter kaolin-induced writhing response, implying that TAFI does not play a major role in bradykinin catabolism. The current study demonstrates that TAFI deficiency does not change normal responses to acute challenges.

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice

In order to examine the physiological role of thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice showed that TAFI mice were born in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI deficient mice developed normally and reached adulthood. No physical abnormalities were observed. They were fertile and pregnancies were carried to full term. Hematological analysis of TAFI deficient mice did not show any major differences compared with their wild type littermates, including plasma fibrinogen level, PT and aPTT. Prolongation of lysis time upon activation of TAFI was observed only with plasma from wild type and heterozygous mice in an in vitro clot lysis assay. TAFI deficiency did not lead to increased bleeding as determined by blood loss following tail transection. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous thrombosis model. The effects of TAFI deficiency were also investigated in thrombin-induced pulmonary thromboembolism, Factor X coagulant protein-induced thrombosis and endotoxin-induced disseminated intravascular coagulation models. In these models, TAFI deficiency did not improve the morbidity or mortality. Based on the kaolin-induced writhing test, TAFI did not play a major role in bradykinin degradation under normal conditions. These studies demonstrate that TAFI deficiency is compatible with murine life

Thrombin-activatable fibrinolysis inhibitor (TAFI) deficient mice.

In order to examine the physiological role of thrombin-activatable fibrinolysis inhibitor (TAFI), we generated homozygous TAFI deficient mice by targeted gene disruption. Intercrossing of heterozygous TAFI mice showed that TAFI mice were born in the expected Mendelian ratio, indicating that transmission of the mutant TAFI allele did not lead to embryonic lethality. TAFI deficient mice developed normally and reached adulthood. No physical abnormalities were observed. They were fertile and pregnancies were carried to full term. Hematological analysis of TAFI deficient mice did not show any major differences compared with their wild type littermates, including plasma fibrinogen level, PT and aPTT. Prolongation of lysis time upon activation of TAFI was observed only with plasma from wild type and heterozygous mice in an in vitro clot lysis assay. TAFI deficiency did not lead to increased bleeding as determined by blood loss following tail transection. In vivo, TAFI deficiency did not influence occlusion time in either an arterial or a venous thrombosis model. The effects of TAFI deficiency were also investigated in thrombin-induced pulmonary thromboembolism, Factor X coagulant protein-induced thrombosis and endotoxin-induced disseminated intravascular coagulation models. In these models, TAFI deficiency did not improve the morbidity or mortality. Based on the kaolin-induced writhing test, TAFI did not play a major role in bradykinin degradation under normal conditions. These studies demonstrate that TAFI deficiency is compatible with murine life

Reduced activity of TAFI (thrombin-activatable fibrinolysis inhibitor) in acute promyelocytic leukaemia.

Acute promyelocytic leukaemia (APL) is a disease that is distinguished from other leukaemias by the high potential for early haemorrhagic death. Several processes are involved, such as disseminated intravascular coagulation and hyperfibrinolysis. Recently, TAFI (thrombin-activatable fibrinolysis inhibitor) was identified as a link between coagulation and fibrinolysis. TAFI can be activated by thrombin, and in its activated form potently attenuates fibrinolysis by removing C-terminal lysine and arginine residues that are important for the binding and activation of plasminogen. Activation of TAFI by the coagulation system results in a down-regulation of fibrinolytic activity and, thereby, prevents a rapid dissolution of the fibrin clot. To establish whether TAFI was involved in the severity of the bleeding complications in APL, the TAFI antigen and activity levels were determined in a group of 15 patients. The TAFI antigen concentration was normal, but the activity of TAFI was severely reduced in APL by approximately 60%. The reduction of TAFI activity was most probably caused by the action of plasmin on TAFI because in vitro experiments revealed that plasmin slightly reduced antigen levels but severely reduced TAFI activity. The acquired functional TAFI deficiency in APL may contribute to the severity of the haemorrhagic diathesis because of the impaired capacity of the coagulation system to protect the fibrin clot from fibrinolysis.