RESUMO
ABSTRACT: von Willebrand factor (VWF) is an essential contributor to microvascular thrombosis. Physiological cleavage by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) limits its prothrombotic properties, explaining why ADAMTS13 deficiency leads to attacks of microthrombosis in patients with thrombotic thrombocytopenic purpura (TTP). We previously reported that plasminogen activation takes place during TTP attacks in these patients. Furthermore, stimulation of plasminogen activation attenuates pathogenesis in preclinical TTP models in vivo. This suggests that plasmin is an endogenous regulator of VWF thrombogenicity, in particular when ADAMTS13 falls short to prevent microvascular occlusions. VWF cleavage by plasmin is biochemically distinct from cleavage by ADAMTS13. We hypothesized that plasmin-cleaved VWF (cVWF) holds value as a biomarker of microvascular thrombosis. Here, we describe the development of a variable domain of heavy-chain-only antibody (VHH)-based bioassay that can distinguish cVWF from intact and ADAMTS13-cleaved VWF in plasma. We validate this assay by tracking cVWF release during degradation of microthombi in vitro. We demonstrate that endogenous cVWF formation takes place in patients with TTP during acute attacks of thrombotic microangiopathy but not in those in remission. Finally, we show that therapeutic plasminogen activation in a mouse model of TTP amplifies cVWF formation, which is accompanied by VWF clearance. Our combined findings indicate that cVWF is released from microthrombi in the context of microvascular occlusion.
Assuntos
Biomarcadores , Fibrinolisina , Púrpura Trombocitopênica Trombótica , Fator de von Willebrand , Animais , Feminino , Humanos , Camundongos , Proteína ADAMTS13/metabolismo , Proteína ADAMTS13/sangue , Biomarcadores/sangue , Biomarcadores/metabolismo , Fibrinolisina/metabolismo , Púrpura Trombocitopênica Trombótica/metabolismo , Púrpura Trombocitopênica Trombótica/sangue , Púrpura Trombocitopênica Trombótica/diagnóstico , Trombose/metabolismo , Trombose/sangue , Trombose/patologia , Microangiopatias Trombóticas/metabolismo , Microangiopatias Trombóticas/sangue , Fator de von Willebrand/metabolismoRESUMO
Thrombotic microangiopathies are hallmarked by attacks of disseminated microvascular thrombosis. In thrombotic thrombocytopenic purpura (TTP), this is caused by a rise in thrombogenic ultra-large von Willebrand factor (VWF) multimers because of ADAMTS13 deficiency. We previously reported that systemic plasminogen activation is therapeutic in a TTP mouse model. In contrast to its natural activators (ie, tissue plasminogen activator and urokinase plasminogen activator [uPA]), plasminogen can directly bind to VWF. For optimal efficacy and safety, we aimed to focus and accelerate plasminogen activation at sites of microvascular occlusion. We here describe the development and characterization of Microlyse, a fusion protein consisting of a high-affinity VHH targeting the CT/CK domain of VWF and the protease domain of uPA, for localized plasminogen activation on microthrombi. Microlyse triggers targeted destruction of platelet-VWF complexes by plasmin on activated endothelial cells and in agglutination studies. At equal molar concentrations, Microlyse degrades microthrombi sevenfold more rapidly than blockade of platelet-VWF interactions with a bivalent humanized VHH (caplacizumab*). Finally, Microlyse attenuates thrombocytopenia and tissue damage (reflected by increased plasma lactate dehydrogenase activity, as well as PAI-1 and fibrinogen levels) more efficiently than caplacizumab* in an ADAMTS13-/- mouse model of TTP, without affecting hemostasis in a tail-clip bleeding model. These findings show that targeted thrombolysis of VWF by Microlyse is an effective strategy for the treatment of TTP and might hold value for other forms of VWF-driven thrombotic disease.
Assuntos
Fibrinolíticos/uso terapêutico , Microangiopatias Trombóticas/tratamento farmacológico , Fator de von Willebrand/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Púrpura Trombocitopênica Trombótica/tratamento farmacológico , Púrpura Trombocitopênica Trombótica/metabolismo , Proteínas Recombinantes de Fusão/uso terapêutico , Microangiopatias Trombóticas/metabolismoRESUMO
BACKGROUND: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (EGF-1) domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis. OBJECTIVES: To unravel and prevent EGF-1-mediated FXII surface-binding with a variable domain of heavy chain-only antibody (VHH). METHODS: FXII variants with glutamine substitutions of 2 positively charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain-specific VHHs were raised to inhibit EGF-1-mediated FXII contact activation while preserving quiescence. RESULTS: Two unique, positively charged patches in the EGF-1 domain were identified (upstream, 73K74K76K78H81K82H; downstream, 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1-specific VHHs were developed that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective VHH "F2" binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. CONCLUSION: The 2 unique, positively charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding, making both patches crucial for contact activation. Targeting these with FXII EGF-1-specific VHHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable targets in preventing medical device-induced thrombosis.
Assuntos
Coagulação Sanguínea , Fator de Crescimento Epidérmico , Fator XII , Ligação Proteica , Anticorpos de Domínio Único , Humanos , Fator XII/metabolismo , Coagulação Sanguínea/efeitos dos fármacos , Anticorpos de Domínio Único/imunologia , Sítios de Ligação , Fator de Crescimento Epidérmico/metabolismo , Domínios Proteicos , Trombose/prevenção & controle , Trombose/imunologia , Trombose/sangue , Relação Estrutura-AtividadeRESUMO
Medical device associated thrombosis is an important clinical problem. This type of thrombosis can result from Factor XII (FXII) binding to non-natural surface materials and subsequent activation of the contact pathway. This drives the development of new therapeutic strategies to block this pathway and information on the structural properties of FXII should catalyse this quest. Presently, there is no publicly available crystal structure of full-length FXII. However, the AlphaFold Protein Structure Database provides a model structure. We here explore this model in combination with previous structure-function studies to identify opportunities for selective pharmacological blockade of the contribution of FXII in medical device associated thrombosis. Previous studies demonstrated that FXII activation is dependent on molecular cleavage after R353. We subsequently proposed that protein conformation protects this cleavage site to ensure zymogen quiescence and prevent inappropriate FXII activation. The AlphaFold model shows that a small loop containing R353 indeed is buried in the globular molecule. This is the result of intra-molecular interactions between the (N-terminal) Fibronectin type II domain, (central) kringle and (C-terminal) protease domain, in a structure that resembles a three-point harness. Furthermore, this interaction pushes the intermediate domains, as well as the flexible proline-rich region (PRR), outward while encapsulating R353 in the molecule. The outward directed positively charged patches are likely to be involved in binding to anionic surfaces. The binding of FXII to surfaces (and several monoclonal antibodies) acccelerates its activation by inducing conformational changes. For prevention of medical device associated thrombosis, it is therefore important to target the surface binding sites of FXII without causing structural changes.
Assuntos
Fator XII , Trombose , Humanos , Fator XII/metabolismo , Coagulação Sanguínea , Precursores Enzimáticos/química , Sítios de Ligação , Fator XIIa/metabolismoRESUMO
BACKGROUND: Occlusive thrombi are not homogeneous in composition. The core of a thrombus is rich in activated platelets and fibrin while the outer shell contains resting platelets. This core is inaccessible to plasma proteins. We produced a fusion protein (targeted SERPIN-TaSER), consisting of a function-blocking VH H against glycoprotein Ibα (GPIbα) and a thrombin-inhibiting serine protease inhibitor (SERPIN; α1-antitrypsin 355 AIAR358 ) to interfere with platelet-driven thrombin formation. AIM: To evaluate the antithrombotic properties of TaSER. METHODS: Besides TaSER, we generated three analogous control variants with either a wild-type antitrypsin subunit, a non-targeting control VH H, or their combination. We investigated TaSER and controls in protease activity assays, (platelet-dependent) thrombin generation assays, and by western blotting. The effects of TaSER on platelet activation and von Willebrand factor (VWF) binding were studied by fluorescence-activated cell sorting, in agglutination studies, and in ATP secretion experiments. We studied the influence of TaSER in whole blood (1) on platelet adhesion on VWF, (2) aggregate formation on collagen, and (3) thrombus formation (after recalcification) on collagen and tissue factor. RESULTS: TaSER binds platelets and inhibits thrombin activity on the platelet surface. It blocks VWF binding and disassembles platelet agglutinates. TaSER delays tissue factor-triggered thrombin generation and ATP secretion in platelet-rich plasma in a targeted manner. In flow studies, TaSER interferes with platelet adhesion and aggregate formation due to GPIbα blockade and limits thrombus formation due to targeted inhibition of platelet-dependent thrombin activity. CONCLUSION: The synergy between the individual properties of TaSER makes it a highly effective antithrombotic agent with possible clinical implications.