Generation and characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor

Buelens, K.; Hassanzadeh-Ghassabeh, Gholamreza; Muyldermans, Serge; Gils, Ann; Declerck, Paul

doi:10.1111/j.1538-7836.2010.03816.x

Cited by 39 publications

(71 citation statements)

References 50 publications

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“…TAFI is a procarboxypeptidase activated by plasmin, the thrombin-TM complex, and trypsin-like enzymes to remove exposed terminal lysines from fibrin which are needed for plasmin formation. A recent study from Buelens et al (51) provides a novel approach to inhibiting TAFIa and promoting fibrinolysis using nanobodies. A nanobody is a singlechain monomeric antigen-binding immunoglobulin, naturally devoid of light chains, found uniquely in camelids (llama, alpaca, and Bactrian camels) (150).…”

Section: B Targeting Coagulopathy In Sepsismentioning

confidence: 98%

“…A nanobody is a singlechain monomeric antigen-binding immunoglobulin, naturally devoid of light chains, found uniquely in camelids (llama, alpaca, and Bactrian camels) (150). Nanobodies from an alpaca immunized with TAFIa showed broad specificity and sensitivity for functional inhibition of TAFI activation and good penetration into the clot (51). The small size of nanobodies (15 kDa) with relatively low immunoge- .…”

Section: B Targeting Coagulopathy In Sepsismentioning

confidence: 99%

See 1 more Smart Citation

Sepsis: Multiple Abnormalities, Heterogeneous Responses, and Evolving Understanding

Iskander

Osuchowski

Stearns-Kurosawa

et al. 2013

Physiological Reviews

357

335

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Sepsis represents the host's systemic inflammatory response to a severe infection. It causes substantial human morbidity resulting in hundreds of thousands of deaths each year. Despite decades of intense research, the basic mechanisms still remain elusive. In either experimental animal models of sepsis or human patients, there are substantial physiological changes, many of which may result in subsequent organ injury. Variations in age, gender, and medical comorbidities including diabetes and renal failure create additional complexity that influence the outcomes in septic patients. Specific system-based alterations, such as the coagulopathy observed in sepsis, offer both potential insight and possible therapeutic targets. Intracellular stress induces changes in the endoplasmic reticulum yielding misfolded proteins that contribute to the underlying pathophysiological changes. With these multiple changes it is difficult to precisely classify an individual's response in sepsis as proinflammatory or immunosuppressed. This heterogeneity also may explain why most therapeutic interventions have not improved survival. Given the complexity of sepsis, biomarkers and mathematical models offer potential guidance once they have been carefully validated. This review discusses each of these important factors to provide a framework for understanding the complex and current challenges of managing the septic patient. Clinical trial failures and the therapeutic interventions that have proven successful are also discussed.

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Section: B Targeting Coagulopathy In Sepsismentioning

confidence: 98%

Section: B Targeting Coagulopathy In Sepsismentioning

confidence: 99%

Sepsis: Multiple Abnormalities, Heterogeneous Responses, and Evolving Understanding

Iskander

Osuchowski

Stearns-Kurosawa

et al. 2013

Physiological Reviews

357

335

View full text Add to dashboard Cite

show abstract

“…An alternative anti-TAFI approach by Buelens et al created a panel of inhibitory nanobodies effective against the various modes of TAFI activation and activity. Nanobodies are single domain antibodies from the sera of members of the Camilidae family which have advantageous properties such as low immunogenicity and high affinity, solubility and stability [150]. Two nanobodies showed a potent profibrinolytic effect in an in vitro clot lysis assay and their interaction with TAFI was later characterised using X-ray crystallography.…”

Section: Current Approaches To Reduce Hypofibrinolysis In Diabetesmentioning

confidence: 99%

Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk

Kearney

Tomlinson

Smith

et al. 2017

Cardiovasc Diabetol

104

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An enhanced thrombotic environment and premature atherosclerosis are key factors for the increased cardiovascular risk in diabetes. The occlusive vascular thrombus, formed secondary to interactions between platelets and coagulation proteins, is composed of a skeleton of fibrin fibres with cellular elements embedded in this network. Diabetes is characterised by quantitative and qualitative changes in coagulation proteins, which collectively increase resistance to fibrinolysis, consequently augmenting thrombosis risk. Current long-term therapies to prevent arterial occlusion in diabetes are focussed on anti-platelet agents, a strategy that fails to address the contribution of coagulation proteins to the enhanced thrombotic milieu. Moreover, antiplatelet treatment is associated with bleeding complications, particularly with newer agents and more aggressive combination therapies, questioning the safety of this approach. Therefore, to safely control thrombosis risk in diabetes, an alternative approach is required with the fibrin network representing a credible therapeutic target. In the current review, we address diabetes-specific mechanistic pathways responsible for hypofibrinolysis including the role of clot structure, defects in the fibrinolytic system and increased incorporation of anti-fibrinolytic proteins into the clot. Future anti-thrombotic therapeutic options are discussed with special emphasis on the potential advantages of modulating incorporation of the anti-fibrinolytic proteins into fibrin networks. This latter approach carries theoretical advantages, including specificity for diabetes, ability to target a particular protein with a possible favourable risk of bleeding. The development of alternative treatment strategies to better control residual thrombosis risk in diabetes will help to reduce vascular events, which remain the main cause of mortality in this condition.

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“…A TAFIa activity assay demonstrated that VHH‐i83 inhibited TAFIa activity in a dose‐dependent manner (Fig. C) ). However, this observation could not be readily explained by the structure of TAFI–VHH‐i83, as VHH‐i83 mainly interacts with the AP and does not bind close to the active site cleft in the CM (Fig.…”

Section: Resultsmentioning

confidence: 91%

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

Zhou

Weeks

Ameloot

et al. 2016

Journal of Thrombosis and Haemostasis

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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.

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Generation and characterization of inhibitory nanobodies towards thrombin activatable fibrinolysis inhibitor

Cited by 39 publications

References 50 publications

Sepsis: Multiple Abnormalities, Heterogeneous Responses, and Evolving Understanding

Sepsis: Multiple Abnormalities, Heterogeneous Responses, and Evolving Understanding

Hypofibrinolysis in diabetes: a therapeutic target for the reduction of cardiovascular risk

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

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