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1.
Blood ; 143(15): 1465-1475, 2024 Apr 11.
Artículo en Inglés | MEDLINE | ID: mdl-38142404

RESUMEN

ABSTRACT: Direct oral anticoagulants (DOACs) that inhibit the coagulation proteases thrombin or factor Xa (FXa) have replaced warfarin and other vitamin K antagonists (VKAs) for most indications requiring long-term anticoagulation. In many clinical situations, DOACs are as effective as VKAs, cause less bleeding, and do not require laboratory monitoring. However, because DOACs target proteases that are required for hemostasis, their use increases the risk of serious bleeding. Concerns over therapy-related bleeding undoubtedly contribute to undertreatment of many patients who would benefit from anticoagulation therapy. There is considerable interest in the plasma zymogen factor XI (FXI) and its protease form factor XIa (FXIa) as drug targets for treating and preventing thrombosis. Laboratory and epidemiologic studies support the conclusion that FXI contributes to venous and arterial thrombosis. Based on 70 years of clinical observations of patients lacking FXI, it is anticipated that drugs targeting this protein will cause less severe bleeding than warfarin or DOACs. In phase 2 studies, drugs that inhibit FXI or FXIa prevent venous thromboembolism after total knee arthroplasty as well as, or better than, low molecular weight heparin. Patients with heart disease on FXI or FXIa inhibitors experienced less bleeding than patients taking DOACs. Based on these early results, phase 3 trials have been initiated that compare drugs targeting FXI and FXIa to standard treatments or placebo. Here, we review the contributions of FXI to normal and abnormal coagulation and discuss results from preclinical, nonclinical, and clinical studies of FXI and FXIa inhibitors.


Asunto(s)
Factor XI , Trombosis , Humanos , Factor XIa/farmacología , Warfarina/uso terapéutico , Warfarina/farmacología , Trombosis/tratamiento farmacológico , Trombosis/etiología , Trombosis/prevención & control , Coagulación Sanguínea , Anticoagulantes/efectos adversos , Hemorragia/inducido químicamente , Fibrinolíticos/uso terapéutico
2.
Blood ; 143(7): 641-650, 2024 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-37992228

RESUMEN

ABSTRACT: Hereditary angioedema (HAE) is associated with episodic kinin-induced swelling of the skin and mucosal membranes. Most patients with HAE have low plasma C1-inhibitor activity, leading to increased generation of the protease plasma kallikrein (PKa) and excessive release of the nanopeptide bradykinin from high-molecular-weight kininogen (HK). However, disease-causing mutations in at least 10% of patients with HAE appear to involve genes for proteins other than C1-inhibitor. A point mutation in the Kng1 gene encoding HK and low-molecular weight kininogen (LK) was identified recently in a family with HAE. The mutation changes a methionine (Met379) to lysine (Lys379) in both proteins. Met379 is adjacent to the Lys380-Arg381 cleavage site at the N-terminus of the bradykinin peptide. Recombinant wild-type (Met379) and variant (Lys379) versions of HK and LK were expressed in HEK293 cells. PKa-catalyzed kinin release from HK and LK was not affected by the Lys379 substitutions. However, kinin release from HK-Lys379 and LK-Lys379 catalyzed by the fibrinolytic protease plasmin was substantially greater than from wild-type HK-Met379 and LK-Met379. Increased kinin release was evident when fibrinolysis was induced in plasma containing HK-Lys379 or LK-Lys379 compared with plasma containing wild-type HK or LK. Mass spectrometry revealed that the kinin released from wild-type and variant kininogens by PKa is bradykinin. Plasmin also released bradykinin from wild-type kininogens but cleaved HK-Lys379 and LK-Lys379 after Lys379 rather than Lys380, releasing the decapeptide Lys-bradykinin (kallidin). The Met379Lys substitutions make HK and LK better plasmin substrates, reinforcing the relationship between fibrinolysis and kinin generation.


Asunto(s)
Angioedemas Hereditarios , Bradiquinina , Humanos , Lisina , Angioedemas Hereditarios/genética , Fibrinolisina , Metionina , Células HEK293 , Quininógenos , Calicreínas/genética , Racemetionina
3.
Blood ; 144(17): 1821-1833, 2024 Oct 24.
Artículo en Inglés | MEDLINE | ID: mdl-39158072

RESUMEN

ABSTRACT: Loss of endothelial barrier function contributes to the pathophysiology of many inflammatory diseases. Coagulation factor XI (FXI) plays a regulatory role in inflammation. Although activation of FXI increases vascular permeability in vivo, the mechanism by which FXI or its activated form FXIa disrupts endothelial barrier function is unknown. We investigated the role of FXIa in human umbilical vein endothelial cell (HUVEC) or human aortic endothelial cell (HAEC) permeability. The expression patterns of vascular endothelial (VE)-cadherin and other proteins of interest were examined by western blot or immunofluorescence. Endothelial cell permeability was analyzed by Transwell assay. We demonstrate that FXIa increases endothelial cell permeability by inducing cleavage of the VE-cadherin extracellular domain, releasing a soluble fragment. The activation of a disintegrin and metalloproteinase 10 (ADAM10) mediates the FXIa-dependent cleavage of VE-cadherin, because adding an ADAM10 inhibitor prevented the cleavage of VE-cadherin induced by FXIa. The binding of FXIa with plasminogen activator inhibitor 1 and very low-density lipoprotein receptor on HUVEC or HAEC surfaces activates vascular endothelial growth receptor factor 2 (VEGFR2). The activation of VEGFR2 triggers the mitogen-activated protein kinase (MAPK) signaling pathway and promotes the expression of active ADAM10 on the cell surface. In a pilot experiment using an established baboon model of sepsis, the inhibition of FXI activation significantly decreased the levels of soluble VE-cadherin to preserve barrier function. This study reveals a novel pathway by which FXIa regulates vascular permeability. The effect of FXIa on barrier function may be another way by which FXIa contributes to the development of inflammatory diseases.


Asunto(s)
Proteína ADAM10 , Antígenos CD , Cadherinas , Permeabilidad Capilar , Células Endoteliales de la Vena Umbilical Humana , Humanos , Animales , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Cadherinas/metabolismo , Antígenos CD/metabolismo , Proteína ADAM10/metabolismo , Factor XIa/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas ADAM/metabolismo , Células Endoteliales/metabolismo , Factor XI/metabolismo , Células Cultivadas , Papio
4.
Blood ; 141(15): 1871-1883, 2023 04 13.
Artículo en Inglés | MEDLINE | ID: mdl-36706361

RESUMEN

A hypercoagulable state, chronic inflammation, and increased risk of venous thrombosis and stroke are prominent features in patients with sickle cell disease (SCD). Coagulation factor XII (FXII) triggers activation of the contact system that is known to be involved in both thrombosis and inflammation, but not in physiological hemostasis. Therefore, we investigated whether FXII contributes to the prothrombotic and inflammatory complications associated with SCD. We found that when compared with healthy controls, patients with SCD exhibit increased circulating biomarkers of FXII activation that are associated with increased activation of the contact pathway. We also found that FXII, but not tissue factor, contributes to enhanced thrombin generation and systemic inflammation observed in sickle cell mice challenged with tumor necrosis factor α. In addition, FXII inhibition significantly reduced experimental venous thrombosis, congestion, and microvascular stasis in a mouse model of SCD. Moreover, inhibition of FXII attenuated brain damage and reduced neutrophil adhesion to the brain vasculature of sickle cell mice after ischemia/reperfusion induced by transient middle cerebral artery occlusion. Finally, we found higher FXII, urokinase plasminogen activator receptor, and αMß2 integrin expression in neutrophils of patients with SCD compared with healthy controls. Our data indicate that targeting FXII effectively reduces experimental thromboinflammation and vascular complications in a mouse model of SCD, suggesting that FXII inhibition may provide a safe approach for interference with inflammation, thrombotic complications, and vaso-occlusion in patients with SCD.


Asunto(s)
Anemia de Células Falciformes , Factor XII , Animales , Ratones , Anemia de Células Falciformes/complicaciones , Anemia de Células Falciformes/metabolismo , Factor XII/metabolismo , Inflamación , Accidente Cerebrovascular , Trombosis/metabolismo
5.
Arterioscler Thromb Vasc Biol ; 44(1): 290-299, 2024 01.
Artículo en Inglés | MEDLINE | ID: mdl-37970718

RESUMEN

BACKGROUND: Despite the ubiquitous utilization of central venous catheters in clinical practice, their use commonly provokes thromboembolism. No prophylactic strategy has shown sufficient efficacy to justify routine use. Coagulation factors FXI (factor XI) and FXII (factor XII) represent novel targets for device-associated thrombosis, which may mitigate bleeding risk. Our objective was to evaluate the safety and efficacy of an anti-FXI mAb (monoclonal antibody), gruticibart (AB023), in a prospective, single-arm study of patients with cancer receiving central line placement. METHODS: We enrolled ambulatory cancer patients undergoing central line placement to receive a single dose of gruticibart (2 mg/kg) administered through the venous catheter within 24 hours of placement and a follow-up surveillance ultrasound at day 14 for evaluation of catheter thrombosis. A parallel, noninterventional study was used as a comparator. RESULTS: In total, 22 subjects (n=11 per study) were enrolled. The overall incidence of catheter-associated thrombosis was 12.5% in the interventional study and 40.0% in the control study. The anti-FXI mAb, gruticibart, significantly prolonged the activated partial thromboplastin time in all subjects on day 14 compared with baseline (P<0.001). Gruticibart was well tolerated and without infusion reactions, drug-related adverse events, or clinically relevant bleeding. Platelet flow cytometry demonstrated no difference in platelet activation following administration of gruticibart. T (thrombin)-AT (antithrombin) and activated FXI-AT complexes increased following central line placement in the control study, which was not demonstrated in our intervention study. CRP (C-reactive protein) did not significantly increase on day 14 in those who received gruticibart, but it did significantly increase in the noninterventional study. CONCLUSIONS: FXI inhibition with gruticibart was well tolerated without any significant adverse or bleeding-related events and resulted in a lower incidence of catheter-associated thrombosis on surveillance ultrasound compared with the published literature and our internal control study. These findings suggest that targeting FXI could represent a safe intervention to prevent catheter thrombosis. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifier: NCT04465760.


Asunto(s)
Neoplasias , Trombosis , Humanos , Factor XI/metabolismo , Estudios Prospectivos , Trombosis/etiología , Trombosis/prevención & control , Trombosis/tratamiento farmacológico , Hemorragia/inducido químicamente , Catéteres/efectos adversos , Neoplasias/tratamiento farmacológico , Neoplasias/complicaciones
6.
N Engl J Med ; 385(23): 2161-2172, 2021 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-34780683

RESUMEN

BACKGROUND: Factor XIa inhibitors for the prevention and treatment of venous and arterial thromboembolism may be more effective and result in less bleeding than conventional anticoagulants. Additional data are needed regarding the efficacy and safety of milvexian, an oral factor XIa inhibitor. METHODS: In this parallel-group, phase 2 trial, we randomly assigned 1242 patients undergoing knee arthroplasty to receive one of seven postoperative regimens of milvexian (25 mg, 50 mg, 100 mg, or 200 mg twice daily or 25 mg, 50 mg, or 200 mg once daily) or enoxaparin (40 mg once daily). The primary efficacy outcome was venous thromboembolism (which was a composite of asymptomatic deep-vein thrombosis, confirmed symptomatic venous thromboembolism, or death from any cause). The principal safety outcome was bleeding. RESULTS: Among the patients receiving milvexian twice daily, venous thromboembolism developed in 27 of 129 (21%) taking 25 mg, in 14 of 124 (11%) taking 50 mg, in 12 of 134 (9%) taking 100 mg, and in 10 of 131 (8%) taking 200 mg. Among those receiving milvexian once daily, venous thromboembolism developed in 7 of 28 (25%) taking 25 mg, in 30 of 127 (24%) taking 50 mg, and in 8 of 123 (7%) taking 200 mg, as compared with 54 of 252 patients (21%) taking enoxaparin. The dose-response relationship with twice-daily milvexian was significant (one-sided P<0.001), and the 12% incidence of venous thromboembolism with twice-daily milvexian was significantly lower than the prespecified benchmark of 30% (one-sided P<0.001). Bleeding of any severity occurred in 38 of 923 patients (4%) taking milvexian and in 12 of 296 patients (4%) taking enoxaparin; major or clinically relevant nonmajor bleeding occurred in 1% and 2%, respectively; and serious adverse events were reported in 2% and 4%, respectively. CONCLUSIONS: Postoperative factor XIa inhibition with oral milvexian in patients undergoing knee arthroplasty was effective for the prevention of venous thromboembolism and was associated with a low risk of bleeding. (Funded by Bristol Myers Squibb and Janssen Research and Development; AXIOMATIC-TKR ClinicalTrials.gov number, NCT03891524.).


Asunto(s)
Anticoagulantes/uso terapéutico , Artroplastia de Reemplazo de Rodilla , Factor XIa/antagonistas & inhibidores , Complicaciones Posoperatorias/prevención & control , Pirimidinas/uso terapéutico , Triazoles/uso terapéutico , Tromboembolia Venosa/prevención & control , Administración Oral , Anciano , Anciano de 80 o más Años , Anticoagulantes/efectos adversos , Relación Dosis-Respuesta a Droga , Enoxaparina/efectos adversos , Enoxaparina/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Pirimidinas/administración & dosificación , Pirimidinas/efectos adversos , Triazoles/administración & dosificación , Triazoles/efectos adversos
7.
Blood ; 139(18): 2816-2829, 2022 05 05.
Artículo en Inglés | MEDLINE | ID: mdl-35100351

RESUMEN

Patients with hereditary angioedema (HAE) experience episodes of bradykinin (BK)-induced swelling of skin and mucosal membranes. The most common cause is reduced plasma activity of C1 inhibitor, the main regulator of the proteases plasma kallikrein (PKa) and factor XIIa (FXIIa). Recently, patients with HAE were described with a Lys311 to glutamic acid substitution in plasminogen (Plg), the zymogen of the protease plasmin (Plm). Adding tissue plasminogen activator to plasma containing Plg-Glu311 vs plasma containing wild-type Plg (Plg-Lys311) results in greater BK generation. Similar results were obtained in plasma lacking prekallikrein or FXII (the zymogens of PKa and FXIIa) and in normal plasma treated with a PKa inhibitor, indicating Plg-Glu311 induces BK generation independently of PKa and FXIIa. Plm-Glu311 cleaves high and low molecular weight kininogens (HK and LK, respectively), releasing BK more efficiently than Plm-Lys311. Based on the plasma concentrations of HK and LK, the latter may be the source of most of the BK generated by Plm-Glu311. The lysine analog ε-aminocaproic acid blocks Plm-catalyzed BK generation. The Glu311 substitution introduces a lysine-binding site into the Plg kringle 3 domain, perhaps altering binding to kininogens. Plg residue 311 is glutamic acid in most mammals. Glu311 in patients with HAE, therefore, represents reversion to the ancestral condition. Substantial BK generation occurs during Plm-Glu311 cleavage of human HK, but not mouse HK. Furthermore, mouse Plm, which has Glu311, did not liberate BK from human kininogens more rapidly than human Plg-Lys311. This indicates Glu311 is pathogenic in the context of human Plm when human kininogens are the substrates.


Asunto(s)
Angioedemas Hereditarios , Angioedemas Hereditarios/genética , Angioedemas Hereditarios/patología , Animales , Bradiquinina/metabolismo , Factor XIIa/metabolismo , Fibrinolisina , Ácido Glutámico , Humanos , Quininógenos/metabolismo , Lisina , Mamíferos/metabolismo , Ratones , Calicreína Plasmática , Plasminógeno/genética , Plasminógeno/metabolismo , Activador de Tejido Plasminógeno
8.
Proc Natl Acad Sci U S A ; 118(3)2021 01 19.
Artículo en Inglés | MEDLINE | ID: mdl-33397811

RESUMEN

Kallikrein (PKa), generated by activation of its precursor prekallikrein (PK), plays a role in the contact activation phase of coagulation and functions in the kallikrein-kinin system to generate bradykinin. The general dogma has been that the contribution of PKa to the coagulation cascade is dependent on its action on FXII. Recently this dogma has been challenged by studies in human plasma showing thrombin generation due to PKa activity on FIX and also by murine studies showing formation of FIXa-antithrombin complexes in FXI deficient mice. In this study, we demonstrate high-affinity binding interactions between PK(a) and FIX(a) using surface plasmon resonance and show that these interactions are likely to occur under physiological conditions. Furthermore, we directly demonstrate dose- and time-dependent cleavage of FIX by PKa in a purified system by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and chromogenic assays. By using normal pooled plasma and a range of coagulation factor-deficient plasmas, we show that this action of PKa on FIX not only results in thrombin generation, but also promotes fibrin formation in the absence of FXII or FXI. Comparison of the kinetics of either FXIa- or PKa-induced activation of FIX suggest that PKa could be a significant physiological activator of FIX. Our data indicate that the coagulation cascade needs to be redefined to indicate that PKa can directly activate FIX. The circumstances that drive PKa substrate specificity remain to be determined.


Asunto(s)
Bradiquinina/metabolismo , Factor IX/metabolismo , Factor XII/metabolismo , Fibrina/metabolismo , Calicreínas/metabolismo , Trombina/metabolismo , Coagulación Sanguínea/fisiología , Bradiquinina/química , Calcio/química , Calcio/metabolismo , Cationes Bivalentes , Factor IX/química , Factor XI/química , Factor XI/metabolismo , Factor XII/química , Fibrina/química , Humanos , Calicreínas/química , Cinética , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Fosfatidiletanolaminas/química , Fosfatidiletanolaminas/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/metabolismo , Unión Proteica , Trombina/química
9.
J Biol Chem ; 298(2): 101567, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35007530

RESUMEN

Skeletal muscle myosin (SkM) has been shown to possess procoagulant activity; however, the mechanisms of this coagulation-enhancing activity involving plasma coagulation pathways and factors are incompletely understood. Here, we discovered direct interactions between immobilized SkM and coagulation factor XI (FXI) using biolayer interferometry (Kd = 0.2 nM). In contrast, we show that prekallikrein, a FXI homolog, did not bind to SkM, reflecting the specificity of SkM for FXI binding. We also found that the anti-FXI monoclonal antibody, mAb 1A6, which recognizes the Apple (A) 3 domain of FXI, potently inhibited binding of FXI to immobilized SkM, implying that SkM binds FXI A3 domain. In addition, we show that SkM enhanced FXI activation by thrombin in a concentration-dependent manner. We further used recombinant FXI chimeric proteins in which each of the four A domains of the heavy chain (designated A1 through A4) was individually replaced with the corresponding A domain from prekallikrein to investigate SkM-mediated enhancement of thrombin-induced FXI activation. These results indicated that activation of two FXI chimeras with substitutions of either the A3 domains or A4 domains was not enhanced by SkM, whereas substitution of the A2 domain did not reduce the thrombin-induced activation compared with wildtype FXI. These data strongly suggest that functional interaction sites on FXI for SkM involve the A3 and A4 domains. Thus, this study is the first to reveal and support the novel intrinsic blood coagulation pathway concept that the procoagulant mechanisms of SkM include FXI binding and enhancement of FXI activation by thrombin.


Asunto(s)
Coagulación Sanguínea , Factor XI , Miosinas del Músculo Esquelético , Trombina , Anticuerpos Monoclonales/química , Sitios de Unión , Factor XI/química , Factor XI/genética , Factor XI/metabolismo , Precalicreína/química , Precalicreína/metabolismo , Dominios Proteicos , Proteínas Recombinantes de Fusión/química , Miosinas del Músculo Esquelético/metabolismo , Trombina/metabolismo
10.
Semin Thromb Hemost ; 2023 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-37276883

RESUMEN

Factor XII (FXII), the zymogen of the protease FXIIa, contributes to pathologic processes such as bradykinin-dependent angioedema and thrombosis through its capacity to convert the homologs prekallikrein and factor XI to the proteases plasma kallikrein and factor XIa. FXII activation and FXIIa activity are enhanced when the protein binds to a surface. Here, we review recent work on the structure and enzymology of FXII with an emphasis on how they relate to pathology. FXII is a homolog of pro-hepatocyte growth factor activator (pro-HGFA). We prepared a panel of FXII molecules in which individual domains were replaced with corresponding pro-HGFA domains and tested them in FXII activation and activity assays. When in fluid phase (not surface bound), FXII and prekallikrein undergo reciprocal activation. The FXII heavy chain restricts reciprocal activation, setting limits on the rate of this process. Pro-HGFA replacements for the FXII fibronectin type 2 or kringle domains markedly accelerate reciprocal activation, indicating disruption of the normal regulatory function of the heavy chain. Surface binding also enhances FXII activation and activity. This effect is lost if the FXII first epidermal growth factor (EGF1) domain is replaced with pro-HGFA EGF1. These results suggest that FXII circulates in blood in a "closed" form that is resistant to activation. Intramolecular interactions involving the fibronectin type 2 and kringle domains maintain the closed form. FXII binding to a surface through the EGF1 domain disrupts these interactions, resulting in an open conformation that facilitates FXII activation. These observations have implications for understanding FXII contributions to diseases such as hereditary angioedema and surface-triggered thrombosis, and for developing treatments for thrombo-inflammatory disorders.

11.
Blood ; 138(22): 2173-2184, 2021 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-34086880

RESUMEN

End-stage renal disease (ESRD) patients on chronic hemodialysis have repeated blood exposure to artificial surfaces that can trigger clot formation within the hemodialysis circuit. Dialyzer clotting can lead to anemia despite erythropoietin and iron supplementation. Unfractionated heparin prevents clotting during hemodialysis, but it is not tolerated by all patients. Although heparin-free dialysis is performed, intradialytic blood entrapment can be problematic. To address this issue, we performed a randomized, double-blind, phase 2 study comparing AB023, a unique antibody that binds factor XI (FXI) and blocks its activation by activated FXII, but not by thrombin, to placebo in 24 patients with ESRD undergoing heparin-free hemodialysis. Patients were randomized to receive a single predialysis dose of AB023 (0.25 or 0.5 mg/kg) or placebo in a 2:1 ratio, and safety and preliminary efficacy were compared with placebo and observations made prior to dosing within each treatment arm. AB023 administration was not associated with impaired hemostasis or other drug-related adverse events. Occlusive events requiring hemodialysis circuit exchange were less frequent and levels of thrombin-antithrombin complexes and C-reactive protein were lower after AB023 administration compared with data collected prior to dosing. AB023 also reduced potassium and iron entrapment in the dialyzers, consistent with less blood accumulation within the dialyzers. We conclude that despite the small sample size, inhibition of contact activation-induced coagulation with AB023 was well tolerated and reduced clotting within the dialyzer. This trial was registered at www.clinicaltrials.gov as #NCT03612856.


Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Antitrombinas/uso terapéutico , Fallo Renal Crónico/terapia , Diálisis Renal/métodos , Adulto , Anticuerpos Monoclonales Humanizados/efectos adversos , Antitrombinas/efectos adversos , Método Doble Ciego , Factor XI/antagonistas & inhibidores , Femenino , Hemostasis/efectos de los fármacos , Humanos , Masculino , Persona de Mediana Edad , Efecto Placebo , Diálisis Renal/efectos adversos , Trombosis/etiología , Trombosis/prevención & control
12.
Blood ; 138(2): 178-189, 2021 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-33598692

RESUMEN

Activation of coagulation factor (F) XI promotes multiorgan failure in rodent models of sepsis and in a baboon model of lethal systemic inflammation induced by infusion of heat-inactivated Staphylococcus aureus. Here we used the anticoagulant FXII-neutralizing antibody 5C12 to verify the mechanistic role of FXII in this baboon model. Compared with untreated control animals, repeated 5C12 administration before and at 8 and 24 hours after bacterial challenge prevented the dramatic increase in circulating complexes of contact system enzymes FXIIa, FXIa, and kallikrein with antithrombin or C1 inhibitor, and prevented cleavage and consumption of high-molecular-weight kininogen. Activation of several coagulation factors and fibrinolytic enzymes was also prevented. D-dimer levels exhibited a profound increase in the untreated animals but not in the treated animals. The antibody also blocked the increase in plasma biomarkers of inflammation and cell damage, including tumor necrosis factor, interleukin (IL)-1ß, IL-6, IL-8, IL-10, granulocyte-macrophage colony-stimulating factor, nucleosomes, and myeloperoxidase. Based on clinical presentation and circulating biomarkers, inhibition of FXII prevented fever, terminal hypotension, respiratory distress, and multiorgan failure. All animals receiving 5C12 had milder and transient clinical symptoms and were asymptomatic at day 7, whereas untreated control animals suffered irreversible multiorgan failure and had to be euthanized within 2 days after the bacterial challenge. This study confirms and extends our previous finding that at least 2 enzymes of the contact activation complex, FXIa and FXIIa, play critical roles in the development of an acute and terminal inflammatory response in baboons challenged with heat-inactivated S aureus.


Asunto(s)
Factor XII/metabolismo , Insuficiencia Multiorgánica/metabolismo , Insuficiencia Multiorgánica/microbiología , Staphylococcus aureus/fisiología , Animales , Anticuerpos/uso terapéutico , Trastornos de la Coagulación Sanguínea/complicaciones , Trastornos de la Coagulación Sanguínea/inmunología , Trastornos de la Coagulación Sanguínea/microbiología , Plaquetas/metabolismo , Microambiente Celular , Activación de Complemento , Factor XII/inmunología , Femenino , Fibrinógeno/metabolismo , Calor , Inflamación/complicaciones , Inflamación/patología , Masculino , Insuficiencia Multiorgánica/inmunología , Papio , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/inmunología , Infecciones Estafilocócicas/microbiología , Infecciones Estafilocócicas/patología , Análisis de Supervivencia
13.
J Immunol ; 206(8): 1784-1792, 2021 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-33811105

RESUMEN

Complement factor H (CFH) is the major inhibitor of the alternative pathway of the complement system and is structurally related to beta2-glycoprotein I, which itself is known to bind to ligands, including coagulation factor XI (FXI). We observed reduced complement activation when FXI activation was inhibited in a baboon model of lethal systemic inflammation, suggesting cross-talk between FXI and the complement cascade. It is unknown whether FXI or its activated form, activated FXI (FXIa), directly interacts with the complement system. We explored whether FXI could interact with and inhibit the activity of CFH. We found that FXIa neutralized CFH by cleavage of the R341/R342 bonds. FXIa reduced the capacity of CFH to enhance the cleavage of C3b by factor I and the decay of C3bBb. The binding of CFH to human endothelial cells was also reduced after incubating CFH with FXIa. The addition of either short- or long-chain polyphosphate enhanced the capacity of FXIa to cleave CFH. FXIa also cleaved CFH that was present on endothelial cells and in the secretome from blood platelets. The generation of FXIa in plasma induced the cleavage of CFH. Moreover, FXIa reduced the cleavage of C3b by factor I in serum. Conversely, we observed that CFH inhibited FXI activation by either thrombin or FXIIa. Our study provides, to our knowledge, a novel molecular link between the contact pathway of coagulation and the complement system. These results suggest that FXIa generation enhances the activity of the complement system and thus may potentiate the immune response.


Asunto(s)
Plaquetas/metabolismo , Factor H de Complemento/metabolismo , Células Endoteliales/metabolismo , Factor XIa/metabolismo , Inflamación/metabolismo , Animales , Coagulación Sanguínea , Complemento C3b/metabolismo , Vía Alternativa del Complemento , Fibrinógeno/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Papio , Unión Proteica , Receptor Cross-Talk
14.
Metab Brain Dis ; 38(7): 2383-2391, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37341855

RESUMEN

Multiple sclerosis (MS) is the most common causes of non-traumatic disability in young adults worldwide. MS pathophysiologies include the formation of inflammatory lesions, axonal damage and demyelination, and blood brain barrier (BBB) disruption. Coagulation proteins, including factor (F)XII, can serve as important mediators of the adaptive immune response during neuroinflammation. Indeed, plasma FXII levels are increased during relapse in relapsing-remitting MS patients, and previous studies showed that reducing FXII levels was protective in a murine model of MS, experimental autoimmune encephalomyelitis (EAE). Our objective was to determine if pharmacological targeting of FXI, a major substrate of activated FXII (FXIIa), improves neurological function and attenuates CNS damage in the setting of EAE. EAE was induced in male mice using murine myelin oligodendrocyte glycoprotein peptides combined with heat-inactivated Mycobacterium tuberculosis and pertussis toxin. Upon onset of symptoms, mice were treated every other day intravenously with anti-FXI antibody, 14E11, or saline. Disease scores were recorded daily until euthanasia for ex vivo analyses of inflammation. Compared to the vehicle control, 14E11 treatment reduced the clinical severity of EAE and total mononuclear cells, including CD11b+CD45high macrophage/microglia and CD4+ T cell numbers in brain. Following pharmacological targeting of FXI, BBB disruption was reduced, as measured by decreased axonal damage and fibrin(ogen) accumulation in the spinal cord. These data demonstrate that pharmacological inhibition of FXI reduces disease severity, immune cell migration, axonal damage, and BBB disruption in mice with EAE. Thus, therapeutic agents targeting FXI and FXII may provide a useful approach for treating autoimmune and neurologic disorders.


Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Animales , Masculino , Ratones , Encéfalo/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Factor XI/antagonistas & inhibidores , Factor XI/metabolismo , Ratones Endogámicos C57BL , Esclerosis Múltiple/patología , Glicoproteína Mielina-Oligodendrócito , Médula Espinal/metabolismo
15.
Curr Opin Hematol ; 29(5): 233-243, 2022 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-35916558

RESUMEN

PURPOSE OF REVIEW: Factor XII (FXII), the precursor of the protease FXIIa, contributes to pathologic processes including angioedema and thrombosis. Here, we review recent work on structure-function relationships for FXII based on studies using recombinant FXII variants. RECENT FINDINGS: FXII is a homolog of pro-hepatocyte growth factor activator (Pro-HGFA). We prepared FXII in which domains are replaced by corresponding parts of Pro-HGA, and tested them in FXII activation and activity assays. In solution, FXII and prekallikrein undergo reciprocal activation to FXIIa and kallikrein. The rate of this process is restricted by the FXII fibronectin type-2 and kringle domains. Pro-HGA replacements for these domains accelerate FXII and prekallikrein activation. When FXII and prekallikrein bind to negatively charged surfaces, reciprocal activation is enhanced. The FXII EGF1 domain is required for surface binding. SUMMARY: We propose a model in which FXII is normally maintained in a closed conformation resistant to activation by intramolecular interactions involving the fibronectin type-2 and kringle domains. These interactions are disrupted when FXII binds to a surface through EGF1, enhancing FXII activation and prekallikrein activation by FXIIa. These observations have important implications for understanding the contributions of FXII to disease, and for developing therapies to treat thrombo-inflammatory disorders.


Asunto(s)
Factor XII , Precalicreína , Coagulación Sanguínea , Factor XII/metabolismo , Fibronectinas , Humanos , Calicreínas , Precalicreína/metabolismo
16.
Blood ; 135(8): 558-567, 2020 02 20.
Artículo en Inglés | MEDLINE | ID: mdl-31800958

RESUMEN

Prekallikrein (PK) is the precursor of the trypsin-like plasma protease kallikrein (PKa), which cleaves kininogens to release bradykinin and converts the protease precursor factor XII (FXII) to the enzyme FXIIa. PK and FXII undergo reciprocal conversion to their active forms (PKa and FXIIa) by a process that is accelerated by a variety of biological and artificial surfaces. The surface-mediated process is referred to as contact activation. Previously, we showed that FXII expresses a low level of proteolytic activity (independently of FXIIa) that may initiate reciprocal activation with PK. The current study was undertaken to determine whether PK expresses similar activity. Recombinant PK that cannot be converted to PKa was prepared by replacing Arg371 with alanine at the activation cleavage site (PK-R371A, or single-chain PK). Despite being constrained to the single-chain precursor form, PK-R371A cleaves high-molecular-weight kininogen (HK) to release bradykinin with a catalytic efficiency ∼1500-fold lower than that of kallikrein cleavage of HK. In the presence of a surface, PK-R371A converts FXII to FXIIa with a specific activity ∼4 orders of magnitude lower than for PKa cleavage of FXII. These results support the notion that activity intrinsic to PK and FXII can initiate reciprocal activation of FXII and PK in solution or on a surface. The findings are consistent with the hypothesis that the putative zymogens of many trypsin-like proteases are actually active proteases, explaining their capacity to undergo processes such as autoactivation and to initiate enzyme cascades.


Asunto(s)
Coagulación Sanguínea , Bradiquinina/metabolismo , Precalicreína/metabolismo , Sustitución de Aminoácidos , Animales , Factor XII/metabolismo , Células HEK293 , Humanos , Quininógeno de Alto Peso Molecular/metabolismo , Ratones Endogámicos C57BL , Precalicreína/química , Precalicreína/genética , Proteolisis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
17.
J Stroke Cerebrovasc Dis ; 31(10): 106742, 2022 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-36037679

RESUMEN

BACKGROUND: Individuals with ischemic stroke or transient ischemic attack (TIA) have a high early risk of ischemic stroke despite dual antiplatelet therapy. The risk of ischemic stroke, and associated disability, represents a significant unmet clinical need. Genetic variants resulting in reduced factor XI levels are associated with reduced risk for ischemic stroke but are not associated with increased intracranial bleeding. Milvexian is an oral small-molecule inhibitor of FXIa that binds activated factor XI with high affinity and selectivity and may reduce the risk of stroke when added to antiplatelet drugs without significant bleeding. We aimed to evaluate the dose-response relationship of milvexian in participants treated with dual antiplatelets. METHODS: We began a phase II, double-blinded, randomized, placebo-controlled trial at 367 sites in 2019. Participants (N = 2366) with ischemic stroke (National Institutes of Health Stroke Scale score ≤7) or high-risk TIA (ABCD2 score ≥6) were randomized to 1 of 5 doses of milvexian or placebo for 90 days. Participants also received clopidogrel 75 mg daily for the first 21 days and aspirin 100 mg for 90 days. The efficacy endpoint was the composite of ischemic stroke or incident infarct on magnetic resonance imaging. Major bleeding, defined as type 3 or 5 bleeding according to the Bleeding Academic Research Consortium, was the safety endpoint. Participant follow-up will end in 2022. CONCLUSION: The AXIOMATIC-SSP trial will evaluate the dose-response of milvexian for ischemic stroke occurrence in participants with ischemic stroke or TIA.


Asunto(s)
Ataque Isquémico Transitorio , Accidente Cerebrovascular Isquémico , Accidente Cerebrovascular , Tromboembolia , Aspirina/efectos adversos , Clopidogrel/uso terapéutico , Método Doble Ciego , Quimioterapia Combinada , Factor XIa , Fibrinolíticos/efectos adversos , Hemorragia , Humanos , Ataque Isquémico Transitorio/diagnóstico por imagen , Ataque Isquémico Transitorio/tratamiento farmacológico , Inhibidores de Agregación Plaquetaria/uso terapéutico , Prevención Secundaria , Accidente Cerebrovascular/diagnóstico por imagen , Accidente Cerebrovascular/tratamiento farmacológico , Tromboembolia/tratamiento farmacológico , Resultado del Tratamiento
18.
Am J Physiol Cell Physiol ; 320(3): C365-C374, 2021 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-33471623

RESUMEN

Factor XI (FXI) has been shown to bind platelets, but the functional significance of this observation remains unknown. Platelets are essential for hemostasis and play a critical role in thrombosis, whereas FXI is not essential for hemostasis but promotes thrombosis. An apparent functional contradiction, platelets are known to support thrombin generation, yet platelet granules release protease inhibitors, including those of activated FXI (FXIa). We aim to investigate the secretory and binding mechanisms by which platelets could support or inhibit FXIa activity. The presence of platelets enhanced FXIa activity in a purified system and increased coagulation Factor IX (FIX) activation by FXIa and fibrin generation in human plasma. In contrast, platelets reduced the activation of FXI by activated coagulation factor XII (FXIIa) and the activation of FXII by kallikrein (PKa). Incubation of FXIa with the platelet secretome, which contains FXIa inhibitors, such as protease nexin-II, abolished FXIa activity, yet in the presence of activated platelets, the secretome was not able to block the activity of FXIa. FXIa variants lacking the anion-binding sites did not alter the effect of platelets on FXIa activity or interaction. Western blot analysis of bound FXIa [by FXIa-platelet membrane immunoprecipitation] showed that the interaction with platelets is zinc dependent and, unlike FXI binding to platelets, not dependent on glycoprotein Ib. FXIa binding to the platelet membrane increases its capacity to activate FIX in plasma likely by protecting it from inhibition by inhibitors secreted by activated platelets. Our findings suggest that an interaction of FXIa with the platelet surface may induce an allosteric modulation of FXIa.


Asunto(s)
Plaquetas/metabolismo , Factor XIa/metabolismo , Adolescente , Precursor de Proteína beta-Amiloide/metabolismo , Sitios de Unión/fisiología , Coagulación Sanguínea/fisiología , Femenino , Hemostasis/fisiología , Humanos , Masculino , Trombina/metabolismo , Trombosis/metabolismo
19.
RNA ; 25(8): 921-934, 2019 08.
Artículo en Inglés | MEDLINE | ID: mdl-31053653

RESUMEN

Biological roles for extracellular RNA (eRNA) have become apparent. For example, eRNA can induce contact activation in blood via activation of the plasma proteases factor XII (FXII) and factor XI (FXI). We sought to reveal the biological role of the secretory enzyme ribonuclease 1 (RNase 1) in an organismal context by generating and analyzing RNase 1 knockout (Rnase1-/-) mice. We found that these mice are viable, healthy, and fertile, though larger than Rnase1+/+ mice. Rnase1-/- plasma contains more RNA than does the plasma of Rnase1+/+ mice. Moreover, the plasma of Rnase1-/- mice clots more rapidly than does wild-type plasma. This phenotype appeared to be due to increased levels of the active form of FXII (FXIIa) in the plasma of Rnase1-/- mice compared to Rnase1+/+ mice, and is consistent with the known effects of eRNA on FXII activation. The apparent activity of FXI in the plasma of Rnase1-/- mice was 1000-fold higher when measured in an assay triggered by a low concentration of tissue factor than in assays based on recalcification, consistent with eRNA enhancing FXI activation by thrombin. These findings suggest that one of the physiological functions of RNase 1 is to degrade eRNA in blood plasma. Loss of this function facilitates FXII and FXI activation, which could have effects on inflammation and blood coagulation. We anticipate that Rnase1-/- mice will be a useful tool for evaluating other hypotheses about the functions of RNase 1 and of eRNA in vivo.


Asunto(s)
Neurotoxina Derivada del Eosinófilo/deficiencia , Factor XII/metabolismo , ARN/química , Animales , Coagulación Sanguínea , Tamaño Corporal , Neurotoxina Derivada del Eosinófilo/genética , Factor XI/metabolismo , Femenino , Fertilidad , Técnicas de Inactivación de Genes , Masculino , Ratones , Modelos Animales , Fenotipo , ARN/sangre , Estabilidad del ARN , Regulación hacia Arriba
20.
Blood ; 133(10): 1152-1163, 2019 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-30591525

RESUMEN

The plasma proteins factor XII (FXII) and prekallikrein (PK) undergo reciprocal activation to the proteases FXIIa and kallikrein by a process that is enhanced by surfaces (contact activation) and regulated by the serpin C1 inhibitor. Kallikrein cleaves high-molecular-weight kininogen (HK), releasing the vasoactive peptide bradykinin. Patients with hereditary angioedema (HAE) experience episodes of soft tissue swelling as a consequence of unregulated kallikrein activity or increased prekallikrein activation. Although most HAE cases are caused by reduced plasma C1-inhibitor activity, HAE has been linked to lysine/arginine substitutions for Thr309 in FXII (FXII-Lys/Arg309). Here, we show that FXII-Lys/Arg309 is susceptible to cleavage after residue 309 by coagulation proteases (thrombin and FXIa), resulting in generation of a truncated form of FXII (δFXII). The catalytic efficiency of δFXII activation by kallikrein is 15-fold greater than for full-length FXII. The enhanced rate of reciprocal activation of PK and δFXII in human plasma and in mice appears to overwhelm the normal inhibitory function of C1 inhibitor, leading to increased HK cleavage. In mice given human FXII-Lys/Arg309, induction of thrombin generation by infusion of tissue factor results in enhanced HK cleavage as a consequence of δFXII formation. The effects of δFXII in vitro and in vivo are reproduced when wild-type FXII is bound by an antibody to the FXII heavy chain (HC; 15H8). The results contribute to our understanding of the predisposition of patients carrying FXII-Lys/Arg309 to angioedema after trauma, and reveal a regulatory function for the FXII HC that normally limits PK activation in plasma.


Asunto(s)
Factor XII/química , Factor XIa/química , Angioedema Hereditario Tipo III/sangre , Angioedema Hereditario Tipo III/genética , Angioedemas Hereditarios , Animales , Arginina/química , Coagulación Sanguínea , Bradiquinina/sangre , Catálisis , Proteína Inhibidora del Complemento C1/química , Factor XIIa/química , Células HEK293 , Humanos , Quininógenos/sangre , Lisina/química , Ratones , Ratones Endogámicos C57BL , Calicreína Plasmática/química , Precalicreína/química , Unión Proteica , Proteínas Recombinantes/química , Propiedades de Superficie , Trombina/genética
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