Thrombin Activatable Fibrinolysis Inhibitor Activation Research Articles

Lys 42/43/44 and Arg 12 of thrombin-activable fibrinolysis inhibitor comprise a thrombomodulin exosite essential for its antifibrinolytic potential.

The thrombin-thrombomodulin (TM) complex activates thrombin-activable fibrinolysis inhibitor (TAFI) more efficiently than thrombin alone. The exosite on TAFI required for its TM-dependent activation by thrombin has not been identified. Based on previous work by us and others, we generated TAFI variants with one or more of residues Lys 42, Lys 43, Lys 44 and Arg 12 within the activation peptide mutated to alanine. Mutation of one, two, or three Lys residues or the Arg residue alone decreased the catalytic efficiency of TAFI activation by thrombin-TM by 2.4-, 3.2-, 4.7-, and 15.0-fold, respectively, and increased the TAFI concentrations required for half-maximal prolongation of clot lysis times (K1/2) by 3-, 4,- 15-, and 24-fold, respectively. Mutation of all four residues decreased the catalytic efficiency of TAFI activation by 45.0-fold, increased the K1/2 by 130-fold, and abolished antifibrinolytic activity in a clot lysis assay at physiologic levels of TAFI. Similar trends in the antifibrinolytic activity of the TAFI variants were observed when plasma clots were formed using HUVECs as the source of TM. When thrombin was used as the activator, mutation of all four residues reduced the rate of activation by 1.1-fold compared with wild-type TAFI, suggesting that these mutations only impacted activation kinetics in the presence of TM. Surface plasmon resonance data suggest that mutation of the four residues abrogates TM binding with or without thrombin. Therefore, Lys 42, Lys 43, Lys 44 and Arg 12 are critical for the interaction of TAFI with the thrombin-TM complex, which modulates its antifibrinolytic potential.

Fibrinolysis in patients with a mild-to-moderate bleeding tendency of unknown cause

In more than 50% of patients with a mild-to-moderate bleeding tendency, no underlying cause can be identified (bleeding of unknown cause, BUC). Data on parameters of fibrinolysis in BUC are scarce in the literature and reveal discrepant results. It was the aim of this study to investigate increased fibrinolysis as a possible mechanism of BUC. We included 270 patients (227 females, median age 44 years, 25–75th percentile 32–58) with BUC and 98 healthy controls (65 females, median age 47 years, 25–75thpercentile 39–55). Tissue plasminogen activator (tPA-) antigen and activity, plasminogen activator inhibitor type-1 (PAI-1), tPA-PAI-1 complexes, thrombin activatable fibrinolysis inhibitor (TAFI), α2-antiplasmin, and D-dimer were determined. While PAI-1 deficiency was equally frequent in patients with BUC and controls (91/270, 34%, and 33/98, 34%, p = 0.996), tPA activity levels were more often above the detection limit in patients than in controls (103/213, 48%, and 23/98, 23%, p < 0.0001). We found lower levels of tPA-PAI-1 complexes (6.86 (3.99–10.00) and 9.11 (7.17–13.12), p < 0.001) and higher activity of TAFI (18.61 (15.80–22.58) and 17.03 (14.02–20.02), p < 0.001) and α2-antiplasmin (102 (94–109) and 98 (90–106], p = 0.003) in patients compared to controls. Detectable tPA activity (OR 3.02, 95%CI 1.75–5.23, p < 0.0001), higher levels of TAFI (OR 2.57, 95%CI 1.48–4.46, p = 0.0008) and α2-antiplasmin (OR 1.03, 95%CI 1.01–1.05, p = 0.011), and lower levels of tPA-PAI-1 complexes (OR 0.90, 95%CI 0.86–0.95, p < 0.0001) were independently associated with BUC in sex-adjusted logistic regression analyses. We conclude that the fibrinolytic system can play an etiological role for bleeding in patients with BUC.

E-WE Thrombin (ProCase) Inhibits Thrombin Mediated TAFI Activation and Accelerates TPA-Induced Thrombolysis

Thrombin-activatable fibrinolysis inhibitor (TAFI) activation during thrombolysis may limit the effectiveness of tissue plasminogen activator (tPA) treatment. Our data suggest that an antithrombotic human thrombin analog, E-WE thrombin (ProCase), which is a selective protein C activator under development for treating acute thrombotic emergencies, also promotes tPA-induced fibrinolysis. We found that E-WE thrombin is a poor activator of TAFI compared to wild-type (WT) thrombin. In the presence of thrombomodulin (TM), WT thrombin induced TAFI activation within 5 min. In contrast, detectable amounts of activated TAFI were not generated by E-WE thrombin in the presence of TM until 60 min. Further studies using a quantitative TAFI activity assay showed that E-WE thrombin inhibited thrombin/thrombomodulin-mediated TAFI activation in a concentration-dependent manner. The ability of E-WE to enhance tPA induced clot lysis was also evaluated in vitro. When both E-WE thrombin and tPA were incorporated within plasma clots, lysis was accelerated by up to 74% compared with the addition of tPA alone. We then tested the ability of E-WE thrombin to interrupt arterial-type experimental thrombus formation in baboons when combined with a standard interventional dose of tPA (1mg/kg). Thrombosis was initiated in the baboons by interposing 4mm internal diameter collagen coated ePTFE vascular grafts within an arterio/venous shunt. Thrombus formation was monitored by real-time gamma camera imaging of autologous 111In-labelled platelet accumulation in the grafts for a total of 90 min. Fibrin deposition was determined by direct endpoint measurement of incorporated 125I-labelled fibrinogen. Antithrombotic interventions were injected intravenously at 30 min after graft deployment into the shunt. Treatment with tPA (1mg/kg, iv) reduced fibrin deposition by 57%, but did not reduce graft-associated platelet accumulation compared with controls (n=8 and 9, respectively). E-WE thrombin, at doses ranging from 2-10µg/kg (n=8), interrupted thrombus growth within 10 min of treatment, and reduced platelet and fibrin deposition by 53-70% and 45-58% at 90 min, respectively, compared with controls. When E-WE thrombin (2µg/kg) was co-administered with tPA (1mg/kg), a profound 91% reduction in thrombus fibrin content was observed (n=1), with platelet deposition being reduced by 34%. Bleeding time and volume were also assessed during the studies, with E-WE treated animals showing no increased bleeding compared with controls. However, tPA caused ecchymoses that were not observed with E-WE thrombin treatment. The combination therapy showed no overt anti-hemostatic effects beyond tPA administration alone. These data suggest that E-WE thrombin can inhibit TAFI activation, and co-administration of E-WE thrombin with tPA may improve the efficacy of thrombolysis without additional hemostasis impairment. DisclosuresTucker:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Markway:Aronora, Inc: Employment, Equity Ownership. Wallisch:Aronora, Inc: Employment, Equity Ownership. Verbout:Aronora, Inc: Employment, Equity Ownership. Lorentz:Aronora, Inc: Employment, Equity Ownership. Carris:Aronora, Inc: Employment, Equity Ownership. Gruber:Aronora, Inc: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

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.

Defective TAFI Activation in Hemophilia Exacerbates Vascular Remodeling in Hemophilic Arthropathy

Introduction: Patients with hemophilia A or B develop hemophilic arthropathy (HA), characterized by inflammation, cell proliferation, and hypervascularity in the synovial joint tissue. The hypervascular response in HA results in aberrant vascular remodeling and the formation of enlarged, irregularly shaped and leaky vessels, which are prone to bleeding. Recently, we showed that after a single joint bleed in FVIII-/- mice changes in soft tissue proliferation, neo-angiogenesis, and cartilage destruction were reversible, whereas vascular remodeling in the joint became progressively worse over time (Bhat et al. 2015). Activated thrombin activatable fibrinolysis inhibitor (TAFIa) conveys anti-fibrinolytic and anti-inflammatory functions by removal of C-terminal (CT) Lys or Arg residues from fibrin and pro-inflammatory mediators, such as C3a, C5a, bradykinin, and thrombin-cleaved osteopontin that may be relevant to HA. Because the activation of TAFI is defective in hemophilia, we determined whether TAFI deficiency contributes to aberrant vascular remodeling in HA using a mouse knee injury model.Methods and Results: Joint bleeding was induced by a subpatellar needle puncture in WT and TAFI-/- mice after a single injection of a FVIII inhibiting antibody to evoke sufficient joint bleeding. Inhibitor-treated TAFI-/- mice bled to the same extent as FVIII-/- mice, however inhibitor-treated WT mice bled minimally. Combining both inhibitory anti-TAFI and anti-FVIII antibodies resulted in bleeding in WT mice comparable to that in FVIII-/- and inhibitor-treated TAFI-/-mice as indicated by decreased hematocrits 2 days post injury (27±9%, 28±10%, 24±7%) and a 4-fold impaired weight-bearing of the injured hind leg. The inhibitors were cleared after 4 days permitting analyses of TAFI-specific effects on long-term vascular remodeling after joint bleeding.At week 2 post injury, all mice showed evidence of increased vascularity in the injured joint. Power Doppler Ultrasound (PD) signals, indicating flow in the joint, were increased 3.5-fold in FVIII-/-, TAFI-/- and WT mice. Accordingly, Safranin O-Fast Green analysis indicated induction of neoangiogenesis as mean vessel count increased from 40 at baseline to 101, 119 and 71 in FVIII-/-, TAFI-/- and WT mice, respectively. At week 4 post injury, however, WT mice diverged from FVIII-/- and TAFI-/- mice. PD signals of FVIII-/- and TAFI-/- mice remained high (3.5-fold increase over baseline, p< 0.001), whereas those of WT mice were significantly reduced compared to week 2 (p< 0.05). Persistent vascular remodeling occurred in FVIII-/- and TAFI-/- mice, but not in WT mice. While total vessel counts decreased in all mice, the percentage of vessels ≥ 20 µm, considered the upper limit of normal vessel diameter in the joint, was increased 2-fold in FVIII-/- and TAFI-/- mice (p< 0.001) but was similar to baseline (11%) in WT mice. Similarly, a 2-fold increase of αSMA expression in the medial meniscal area in TAFI-/-mice support ongoing vascular remodeling in these mice (p< 0.01). Thus, the impaired TAFI function in HA promotes vascular remodeling.In search of proangiogenic mediators implicated in the pathogenesis of HA and susceptible to inactivation by TAFIa, we identified stromal cell-derived factor 1α (SDF1α; CXCL12), a well-known endothelial progenitor recruitment factor. SDF1α contains a functionally requisite CT Lys that is needed for CXCR4 activation. Histological analysis using a CT-Lys-specific anti-SDF1α revealed pronounced expression of intact SDF1α on the joint vessels in TAFI-/- mice at week 4 post injury. In vitro,TAFIa rapidly cleaved the CT-Lys of SDF1α as determined by a CT-Lys specific W-blot and ELISA. In addition, TAFIa-treated SDF1α failed to induce migration of endothelial cells in a transwell cell migration assay.Conclusion: Vascular remodeling after joint bleeding was perpetuated in TAFI-/- mice to the same extent as in FVIII-/- mice. Based on the identification of SDF1α as a novel substrate for TAFIa, we propose that the defective activation of TAFI in hemophilia results in impaired inactivation of SDF1α, allowing functional SDF1α to accumulate on synovial vessels in the injured joint thereby exacerbating the recruitment of endothelial progenitor cells to the joint. This in combination with the ongoing angiogenesis in the joint ultimately results in irreversible vascular remodeling that underlies the development of HA. Disclosuresvon Drygalski:Baxalta/Shire: Consultancy, Honoraria, Speakers Bureau; Bayer: Consultancy, Honoraria, Speakers Bureau; Biogen: Consultancy, Honoraria, Speakers Bureau; CSL-Behring: Consultancy, Honoraria, Speakers Bureau; Novo Nordisk: Consultancy, Honoraria, Speakers Bureau; Pfizer: Consultancy, Honoraria, Speakers Bureau; Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees. Mosnier:Hematherix LLC: Membership on an entity’s Board of Directors or advisory committees; The Scripps Research Institute: Patents & Royalties; Bayer: Honoraria, Speakers Bureau; Baxalta: Honoraria, Speakers Bureau.

Protein C Activation Is the Critical Thrombomodulin Function in Embryonic and Adult Survival in Mice

BACKGROUND AND APPROACH:Thrombomodulin (Thbd) is a multifunctional transmembrane molecule expressed on endothelial, hematopoietic, placental trophoblast, and various other cells. Thbd facilitates protein C and TAFI activation by thrombin, but also exerts thrombin- and protein C-independent functions to regulate complement activity and inflammatory signaling. Complete Thbd deficiency causes embryonic death through as yet unclear mechanisms, while ablation of the lectin-like Thbd domain, reduction of Thbd cofactor activity for protein C (pC) activation, or truncation of the cytoplasmic domain do not impair embryonic development1-3. Here we applied in vivo structure-function and genetic complementation analyses to differentiate between pC-dependent and -independent Thbd functions.RESULTS:Three mouse strains with various function-selective Thbd mutations were generated, i.e. ThbdPro/ΔCS, ThbdEGF4-6, and ThbdLT (Table 1). Of these, only the latter mutant with defective thrombin-binding reproduced the embryonic lethal phenotype of complete Thbd deficiency. To examine the developmental role of Thbd expression in cell types other than placental trophoblast, Thbd LoxP mice carrying a conditional Thbd allele were crossed with mice expressing Cre recombinase under control of the endogenous Meox2 gene promotor, resulting in selective ablation of Thbd in the embryo proper, but preservation in extra-embryonic placental tissue. This yielded a normal number of Thbd-deficient embryos at term pregnancy. Preservation of Thbd function in the embryonic placenta therefore was sufficient to prevent intrauterine lethality until birth. Immunohistochemistry of term embryos and adult mice (week 10-16) confirmed persistent absence of Thbd expression. Half of Thbd-null mice were lost prior to weaning (3 weeks). Surviving mice were smaller, yet exhibited normal weight gain thereafter. By 4 weeks of age, 19 of 28 Thbd-deficient animals developed grossly observable lesions, i.e. purple discoloration and swelling of tail segments, followed by, edematic swelling and discoloration of the tail and hind limb digits, distal tail necrosis and/or uni- or bilateral eye degeneration. More than half of the Thbd-null mice expired within the first 10 weeks of life, and only 2 of 28 mice survived to week 40. Neither gross anatomical inspection nor histological surveys of internal organs revealed evidence of severe thrombotic organ damage, and except for elevated IL-6, cytokine plasma levels were normal. Tamoxifen-induced Thbd LoxP gene ablation in adult mice (10-12 week old) reproduced the phenotype of Meox2Cre-Thbd-null mice. Genetic supplementation of activated PC by transgenic expression of a partially Thbd-independent murine pC zymogen variant (“hyperactivatable” protein C4), prevented perinatal lethality, partially restored normal birth weight, and largely prevented the phenotype of Thbd-null mice. However, Thbd-null females expressing the pC transgene exhibited pregnancy-induced morbidity and mortality with complete penetrance. When mated to wild type males (embryonic placenta expresses Thbd), 8 of 8 pregnancies ended in death or severe distress during gestation or postpartum.CONCLUSIONS:These findings indicate that the lack of protein C activation by Thbd-bound thrombin is the sole cause of lethality of embryonic and adult Thbd-null mice; and that Thbd expression in extra-embryonic placental tissue is sufficient to prevent intra-uterine lethality. The mortality of pregnant Thbd-null females expressing the protein C transgene constitutes a novel model for pregnancy-specific complications associated with defects in the function of natural anticoagulant pathways.REFERENCES:1. Isermann B et al. Nat Med. 2003;9(3):331-337.2. Conway EM et al. Blood. 1999;93(10):3442-3450.3. Conway EM et al. J Exp Med. 2002;196(5):565-577.4. Isermann B et al. Nat Med. 2007;13(11):1349-1358. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Novel Class of Direct Thrombin Inhibitors Prevent Thrombosis By Inhibiting Fibrinogen Cleavage While Preserving Platelet Function

We have developed a new class of direct thrombin inhibitors (VE-DTIs) that act through reversible covalent enzyme inhibition. These compounds inhibit thrombin with high potency (IC50 < 10 nM) and with better than 100× selectivity against related serine proteases. In rodents, VE-DTIs show dose-dependent efficacy in preventing arterial and venous thromboses respectively in arteriovenous shunt thrombosis and venous stasis thrombosis models. They also prevent thrombin-induced pulmonary embolism in rodents. Despite their strong anticoagulation properties, VE-DTIs are associated with significantly lower bleeding times than other approved anticoagulants dabigatran, argatroban or apixaban in rodent bleeding models (tail bleeding time and saphenous vein bleeding time).We compared the effect of VE-DTIs and the DTIs argatroban and dabigatran on the coagulation cascade. In vitro, VE-DTIs inhibited thrombin-mediated fibrinogen cleavage with similar potency. Inhibition of thrombin/thrombomodulin complex-mediated activation of TAFI and protein C by VE-DTIs were also comparable. In contrast, VE-DTIs were more than 1000-fold less potent in inhibiting thrombin-mediated platelet activation than the other DTIs. Additionally, and also in contrast to argatroban and dabigatran, VE-DTIs protect rodents from the effects of thrombin-induced pulmonary embolism without significantly inhibiting platelet activation. The ability of VE-DTIs to prevent thrombosis without significantly impacting platelet function could lead to a new anticoagulant therapy with lower bleeding risk. DisclosuresSivaraja:Verseon Corporation: Employment, Equity Ownership. Sizikov:Verseon Corporation: Employment, Equity Ownership. Sandoval:Verseon Corporation: Employment, Equity Ownership. Lin:Verseon Corporation: Employment, Equity Ownership. Igoudin:Verseon Corporation: Employment, Equity Ownership. Dash:Verseon Corporation: Employment, Equity Ownership. Xu:Verseon Corporation: Employment, Equity Ownership. Clemens:Verseon Corporation: Employment, Equity Ownership. Estiarte:Verseon Corporation: Employment, Equity Ownership. Shiau:Verseon Corporation: Employment, Equity Ownership. Short:Verseon Corporation: Employment, Equity Ownership. Williams:Verseon Corporation: Employment, Equity Ownership. Datta:Verseon Corporation: Employment, Equity Ownership. Kita:Verseon Corporation: Employment, Equity Ownership.

Sulfamide as Zinc Binding Motif in Small Molecule Inhibitors of Activated Thrombin Activatable Fibrinolysis Inhibitor (TAFIa).

Previously disclosed TAFIa inhibitors having a urea zinc-binding motif were used as the starting point for the development of a novel class of highly potent inhibitors having a sulfamide zinc-binding motif. High-resolution X-ray cocrystal structures were used to optimize the structures and reveal a highly unusual sulfamide configuration. A selected sulfamide was profiled in vitro and in vivo and displayed a promising ADMET profile.

Altered fibrinolysis in autosomal dominant thrombomodulin-associated coagulopathy

AbstractThrombomodulin-associated coagulopathy (TM-AC) is a newly recognized dominant bleeding disorder in which a p.Cys537Stop variant in the thrombomodulin (TM) gene THBD, results in high plasma TM levels and protein C-mediated suppression of thrombin generation. Thrombin in complex with TM also activates thrombin-activatable fibrinolysis inhibitor (TAFI). However, the effect of the high plasma TM on fibrinolysis in TM-AC is unknown. Plasma from TM-AC cases and high-TM model control samples spiked with recombinant soluble TM showed reduced tissue factor–induced thrombin generation. Lysis of plasma clots from TM-AC cases was significantly delayed compared with controls but was completely restored when TM/thrombin-mediated TAFI activation was inhibited. Clots formed in blood from TM-AC cases had the same viscoelastic strength as controls but also showed a TAFI-dependent delay in fibrinolysis. Delayed fibrinolysis was reproduced in high-TM model plasma and blood samples. Partial restoration of thrombin generation with recombinant activated factor VII or activated prothrombin complex concentrate did not alter the delayed fibrinolysis in high-TM model blood. Our finding of a previously unrecognized fibrinolytic phenotype indicates that bleeding in TM-AC has a complex pathogenesis and highlights the pivotal role of TM as a regulator of hemostasis.

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.

Stimulation of thrombin- and plasmin-mediated activation of thrombin-activatable fibrinolysis inhibitor by anionic molecules

BackgroundThrombin-activatable fibrinolysis inhibitor (TAFI) is a proenzyme that, once activated, attenuates fibrinolysis by removing C-terminal lysine residues from partially degraded fibrin. TAFI can be activated by thrombin or plasmin via a cleavage at Arg92 that removes the activation peptide from the enzyme, TAFIa. Thrombomodulin enhances thrombin-mediated TAFI activation and glycosaminoglycans enhance plasmin-mediated TAFI activation. The aim of this study was to investigate whether there are other anionic molecules that function as a cofactor for thrombin- or plasmin-mediated TAFI activation. MethodsTAFI activation by thrombin or plasmin was studied in the presence of physiological anionic molecules (polyphosphate, heparin, hyaluronan, DNA and dermatan sulfate) and the non-physiological sodium dodecyl sulfate (SDS). Additionally, the effect of these molecules on TAFIa stability and on thrombin-mediated protein C activation was determined. ResultsUnfractioned heparin, calcium-saturated polyphosphate with an average chain length of 100 monomers (Ca-PolyP100) and SDS significantly enhanced TAFI activation by thrombin and plasmin. Dermatan sulfate and polyphosphates with sodium as counter ion (Na-PolyP700, Na-PolyP100 and Na-PolyP70) enhanced plasmin-mediated but not thrombin-mediated TAFI activation. Additionally, unfractioned heparin, Ca-PolyP100 and SDS enhanced thrombin-mediated protein C activation. The different nature of anionic molecules capable of enhancing TAFI and protein C activation suggests a general mechanism. ConclusionsSeveral anionic molecules function as (potent) cofactors for thrombin- and plasmin-mediated TAFI activation and thrombin-mediated protein C activation. This may imply that thrombin and plasmin activity is regulated in the vasculature by more cofactors than currently appreciated.

Is resistance futile? The role of activated thrombin‐activatable fibrinolysis inhibitor resistance in bleeding in factor XI deficiency

Is resistance futile? The role of activated thrombin‐activatable fibrinolysis inhibitor resistance in bleeding in factor XI deficiency

Elucidation of the molecular mechanisms of two nanobodies that inhibit thrombin‐activatable fibrinolysis inhibitor activation and activated thrombin‐activatable fibrinolysis inhibitor activity

Essentials Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for cardiovascular disorders. TAFI inhibitory nanobodies represent a promising step in developing profibrinolytic therapeutics. We have solved three crystal structures of TAFI in complex with inhibitory nanobodies. Nanobodies inhibit TAFI through distinct mechanisms and represent novel profibrinolytic leads. Background Thrombin-activatable fibrinolysis inhibitor (TAFI) is converted to activated TAFI (TAFIa) by thrombin, plasmin, or the thrombin-thrombomodulin complex (T/TM). TAFIa is antifibrinolytic, and high levels of TAFIa are associated with an increased risk for cardiovascular disorders. TAFI-inhibitory nanobodies represent a promising approach for developing profibrinolytic therapeutics. Objective To elucidate the molecular mechanisms of inhibition of TAFI activation and TAFIa activity by nanobodies with the use of X-ray crystallography and biochemical characterization. Methods and results We selected two nanobodies for cocrystallization with TAFI. VHH-a204 interferes with all TAFI activation modes, whereas VHH-i83 interferes with T/TM-mediated activation and also inhibits TAFIa activity. The 3.05-Å-resolution crystal structure of TAFI-VHH-a204 reveals that the VHH-a204 epitope is localized to the catalytic moiety (CM) in close proximity to the TAFI activation site at Arg92, indicating that VHH-a204 inhibits TAFI activation by steric hindrance. The 2.85-Å-resolution crystal structure of TAFI-VHH-i83 reveals that the VHH-i83 epitope is located close to the presumptive thrombomodulin-binding site in the activation peptide (AP). The structure and supporting biochemical assays suggest that VHH-i83 inhibits TAFIa by bridging the AP to the CM following TAFI activation. In addition, the 3.00-Å-resolution crystal structure of the triple TAFI-VHH-a204-VHH-i83 complex demonstrates that the two nanobodies can simultaneously bind to TAFI. Conclusions This study provides detailed insights into the molecular mechanisms of TAFI inhibition, and reveals a novel mode of TAFIa inhibition. VHH-a204 and VHH-i83 merit further evaluation as potential profibrinolytic therapeutics.

Reduced fibrinolytic resistance in patients with factor XI deficiency. Evidence of a thrombin‐independent impairment of the thrombin‐activatable fibrinolysis inhibitor pathway

Essentials Plasma of factor XI-deficient patients (FXI-dp) displays enhanced fibrinolysis. We investigated the role of thrombin activatable fibrinolysis inhibitor (TAFI) in 18 FXI-dp. FXI-dp generated less activated TAFI (TAFIa) on clotting challenge and were resistant to TAFIa. TAFI activation and TAFIa resistance correlated with bleeding score and bleeding phenotype. Background Factor XI (FXI) deficiency, a rare disorder with unpredictable bleeding, has been associated with reduced fibrinolytic resistance as a result of abnormal fibrin density. Objective We investigated the involvement of thrombin-activatable fibrinolysis inhibitor (TAFI) in the increased lysability of FXI-deficient (FXI-def) clots and the role of thrombin. Patients/Methods Eighteen patients with FXI deficiency (1-58%) and 17 matched controls were investigated for fibrinolytic resistance to t-PA, thrombin generation, TAFI activation and response to TAFIa. Results When clotting was induced by 0.5 pm tissue factor (TF), FXI-def plasmas displayed less thrombin and TAFIa generation and shorter lysis time than controls. A 100-fold higher TF concentration (to bypass FXI) abolished the difference in thrombin generation but not in lysis time between patients and controls. Normalization of FXI levels by a FXI concentrate increased thrombin generation but had no effect on the lysis time of FXI-def plasma. Moreover, when clots were induced by purified thrombin and high concentrations of FXa inhibitor, FXI-def plasma still generated less TAFIa and displayed a shorter lysis time than controls. Finally, upon TAFIa addition, the lysis time of FXI-def plasma was prolonged significantly less than that of control plasma, suggesting a TAFIa resistance. TAFIa generation and TAFIa resistance were correlated with the bleeding score, displaying a considerable capacity to discriminate between patients with and without bleeding. Conclusions TAFI pathway impairment, largely caused by a hitherto unknown TAFIa resistance, appears to be one main cause of decreased fibrinolytic resistance in FXI deficiency and might be clinically useful for assessing the bleeding risk of FXI-def patients.

Decreased Plasma Fibrinolytic Potential As a Risk for Venous and Arterial Thrombosis

It has been well established that inherited or acquired hypercoagulability is a risk factor for venous thrombosis. In addition, hypercoagulability may contribute to the risk of arterial events. Much less is known regarding the role of the fibrinolytic system in the risk of thrombotic disease, which partly relates to the lack of validated assays. A plasma-based global fibrinolysis assay, which is sensitive to plasma levels of plasminogen, regulators of fibrinolysis, and proteins involved in coagulation, has been used in large epidemiological studies to assess the role of fibrinolysis in thrombosis. It has been demonstrated that a hypofibrinolytic state increases the risk of a first venous thrombosis, but not of a recurrence. This increased risk of venous thrombosis associated with plasma hypofibrinolysis appears primarily driven by elevated plasma levels of thrombin-activatable fibrinolysis inhibitor and plasminogen activator inhibitor type 1. The combination of hypercoagulability and hypofibrinolysis synergistically enhances the risk of a first venous event. Plasma hypofibrinolysis may constitute a risk factor for the postthrombotic syndrome. Decreased fibrinolytic potential is also associated with an increased risk of arterial thrombosis, but only in individuals younger than 55 years. The association between hypofibrinolysis and myocardial infarction appears primarily driven by elevated levels of α2-antiplasmin. Although recent studies have clearly demonstrated a role of the fibrinolytic system in thrombotic disease, the clinical utility of plasma-based clot lysis assays is probably limited.

Inhibition of Thrombin-Activatable Fibrinolysis Inhibitor and Plasminogen Activator Inhibitor-1 Reduces Ischemic Brain Damage in Mice.

Cerebral ischemia and reperfusion is associated with activation of the coagulation cascade and fibrin deposition in cerebral microvessels. Both thrombin-activatable fibrinolysis inhibitor (TAFI) and plasminogen activator inhibitor-1 (PAI-1) attenuate fibrinolysis and are therefore attractive targets for the treatment of ischemic stroke. TAFI and PAI-1 were inhibited by monoclonal antibodies in a mouse model of transient middle cerebral artery occlusion. Twenty-four hours after stroke, mice were neurologically scored, cerebral thrombotic burden was assessed, and brain infarct sizes were calculated. Inhibition of TAFI or PAI-1 significantly decreased cerebral infarct sizes by 50% 24 hours after stroke. This reduction in cerebral damage was associated with a significant decrease in fibrin(ogen) deposition in the ischemic brain. Concurrently, functional recovery of the animals was improved. Interestingly, combined targeting of TAFI and PAI-1 using low, and by themselves inactive, doses of antibodies improved cerebral blood flow and reduced cerebral fibrin(ogen) deposition and infarct sizes by 50%. When dual treatment was delayed to 1 hour after the start of reperfusion, it still reduced brain injury; however, this was not statistically significant. Targeting of PAI-1 and TAFI is protective in an ischemic stroke model by attenuating fibrin(ogen) deposition, thereby improving reperfusion. Combined inhibition has a co-operative effect that could become useful in ischemic stroke therapy.

Changes in Activated Thrombin-Activatable Fibrinolysis Inhibitor Levels Following Thrombolytic Therapy in Ischemic Stroke Patients Correlate with Clinical Outcome

Background and Purpose: Thrombin-activatable fibrinolysis inhibitor (TAFI) activation following thrombolysis may affect thrombolysis effectiveness in acute ischemic stroke (AIS). To support this hypothesis, we propose to study the relationship between TAFI consumption, activated/inactivated TAFI (TAFIa/ai) and stroke severity and outcome in 2 groups of AIS patients, one treated and one untreated with intravenous recombinant tissue type plasminogen activator (rt-PA). Methods: In this prospective, longitudinal, multicenter, observational study, we aimed to study the association between TAFIa/ai and stroke outcome. TAFI levels were sequentially measured in patients treated with intravenous rt-PA thrombolysis (T), and in patients not given any thrombolytic therapy (NT). Baseline reference values were established in healthy subjects matched for age and gender. The National Institutes of Health Stroke Scale (NIHSS) score assessed at baseline and on day 2 was dichotomized into 2 severity groups (0-7 vs. >7). The modified Rankin Scale (mRS) score at day 90 was dichotomized for favorable (0-1) and unfavorable (2-6) outcomes. Results: A total of 109 patients were included, with 41 receiving rt-PA. At admission, patients had higher TAFIa/ai levels than reference. A significant increase in TAFIa/ai levels was observed at the end of thrombolysis (mean change from baseline of 963%) and lasted up to 4 h (191%). Higher TAFIa/ai levels were associated with a more severe day 2 NIHSS score (p = 0.0098 at T2h post thrombolysis) and an unfavorable mRS score from T48h (p = 0.0417) to day 90 (p = 0.0046). In NT patients, higher TAFIa/ai levels at admission were associated with a more severe stroke, as assessed by day 2 NIHSS score (p = 0.0026) and mRS score (p = 0.0003). Conclusion: These data demonstrate a consistent relationship between TAFI levels and early clinical severity during rt-PA treatment.

Laboratory evaluation of patients with undiagnosed bleeding disorders.

The evaluation of patients with a bleeding tendency represents a challenge as the routinely available tests for evaluating bleeding disorders are limited, complicating the laboratory determination of the clinically observed bleeding tendency. As a result, some bleeding disorders remain undiagnosed. The aim of the study was to evaluate whether global coagulation tests would contribute to the laboratory analysis of patients with undiagnosed bleeding disorders. Patients were evaluated for coagulation and fibrinolysis activities by thrombin generation test and euglobulin lysis time. In addition, plasma activity of factor XIII, plasminogen, α-2 antiplasmin, plasminogen activator inhibitor-1, and thrombin-activatable fibrinolysis inhibitor was also obtained. Forty-five patients were included. Eight per cent presented a mild bleeding disorder and 20% a moderate bleeding disorder. The thrombin generation test results were similar between patients and controls. Euglobulin lysis time results, however, were lower in patients than in controls, both before (median 175 vs. 250 min, respectively; P = 0.003) and after (median 145 vs. 115 min, respectively; P ≤ 0.001) arm constriction, suggesting that they were experiencing hyperfibrinolysis. Interestingly, patients' median thrombin-activatable fibrinolysis inhibitor activity was higher than in controls (21.2 vs. 19.46 μg/ml; P = 0.016). However, plasminogen, α-2 antiplasmin, plasminogen activator inhibitor-1, and factor XIII activities did not differ between the groups. Global coagulation and fibrinolysis tests proved to be limited in detecting the hemostatic disorders in some patients with a relevant bleeding tendency and may not be adequate to address their bleeding risk. Bleeding scores are currently the available medical approach for the evaluation of these patients.

Increased fibrin formation and impaired fibrinolytic capacity in severe chronic kidney disease.

Chronic kidney disease (CKD) is associated with a concurrent increased risk of thrombosis and bleeding. We aimed to investigate whether CKD is associated with increased fibrin formation, impaired fibrin degradation, or both. Twenty-one patients with CKD stage 4 (CKD 4), 15 haemodialysis patients, and 13 controls (C) without kidney disease were studied. We used a global assay to determine fibrin formation and degradation in plasma. Fibrin turbidity was measured over time to obtain a value of the coagulation activation profile (Cp) and the fibrinolysis activation profile (Fp), and the amount of fibrin formed, termed fibrin optical density sum (fibrin OD-sum). We used scanning electron microscopy (SEM) to visualize the fibrin network. Plasminogen activator inhibitor type-1 antigen, thrombin-activatable fibrinolysis inhibitor activity, fibrinogen, von Willebrand factor, antithrombin, albumin, and C-reactive protein were measured in plasma. Fibrin OD-sum was significantly elevated in haemodialysis patients [312 a.u.; 278-435 (median; interquartile range); P < 0.0013] and in CKD 4 (293 a.u.; 169-434; P = 0.0119) compared with controls (115 a.u.; 82-234). SEM showed a tight fibrin network in haemodialysis and CKD 4 patients. Fp was lower in the haemodialysis group than in controls (P = 0.030). Plasminogen activator inhibitor type-1 was lower in haemodialysis patients (P = 0.034). Thrombin-activatable fibrinolysis inhibitor activity, Cp, antithrombin, and C-reactive protein did not differ between groups. Fibrinogen was significantly elevated and albumin decreased in both haemodialysis and CKD 4 patients compared with controls. Von Willebrand factor was elevated in haemodialysis patients compared with controls (P = 0.010). The prothrombotic state in severe CKD is characterized by impaired fibrinolysis in association with increased fibrin formation despite normal levels of endogenous fibrinolysis inhibitors.

Activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates breast cancer cell metastatic behaviors through inhibition of plasminogen activation and extracellular proteolysis.

BackgroundThrombin activatable fibrinolysis inhibitor (TAFI) is a plasma zymogen, which can be converted to activated TAFI (TAFIa) through proteolytic cleavage by thrombin, plasmin, and most effectively thrombin in complex with the endothelial cofactor thrombomodulin (TM). TAFIa is a carboxypeptidase that cleaves carboxyl terminal lysine and arginine residues from protein and peptide substrates, including plasminogen-binding sites on cell surface receptors. Carboxyl terminal lysine residues play a pivotal role in enhancing cell surface plasminogen activation to plasmin. Plasmin has many critical functions including cleaving components of the extracellular matrix (ECM), which enhances invasion and migration of cancer cells. We therefore hypothesized that TAFIa could act to attenuate metastasis.MethodsTo assess the role of TAFIa in breast cancer metastasis, in vitro migration and invasion assays, live cell proteolysis and cell proliferation using MDA-MB-231 and SUM149 cells were carried out in the presence of a TAFIa inhibitor, recombinant TAFI variants, or soluble TM.ResultsInhibition of TAFIa with potato tuber carboxypeptidase inhibitor increased cell invasion, migration and proteolysis of both cell lines, whereas addition of TM resulted in a decrease in all these parameters. A stable variant of TAFIa, TAFIa-CIIYQ, showed enhanced inhibitory effects on cell invasion, migration and proteolysis. Furthermore, pericellular plasminogen activation was significantly decreased on the surface of MDA-MB-231 and SUM149 cells following treatment with various concentrations of TAFIa.ConclusionsTaken together, these results indicate a vital role for TAFIa in regulating pericellular plasminogen activation and ultimately ECM proteolysis in the breast cancer microenvironment. Enhancement of TAFI activation in this microenvironment may be a therapeutic strategy to inhibit invasion and prevent metastasis of breast cancer cells.