Coagulation factor XI regulates endothelial cell permeability and barrier function in vitro and in vivo.

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.

Safety and tolerability of the protein C activator AB002 in end-stage renal disease patients on hemodialysis: a randomized phase 2 trial.

BACKGROUND: The protein C system regulates blood coagulation, inflammation, and vascular integrity. AB002 is an injectable protein C activating enzyme under investigation to safely prevent and treat thrombosis. In preclinical models, AB002 is antithrombotic, cytoprotective, and anti-inflammatory. Since prophylactic use of heparin is contraindicated during hemodialysis in some end-stage renal disease (ESRD) patients, we propose using AB002 as a short-acting alternative to safely limit blood loss due to clotting in the dialysis circuit. METHODS: This phase 2, randomized, double-blind, placebo-controlled, single-dose study evaluates the safety and tolerability of AB002 administered into the hemodialysis line of ESRD patients during hemodialysis at one study center in the United States (ClinicalTrials.gov: NCT03963895). In this study, 36 patients were sequentially enrolled into two cohorts and randomized to AB002 or placebo in a 2:1 ratio. In cohort 1, patients received 1.5 µg/kg AB002 (n = 12) or placebo (n = 6); in cohort 2, patients received 3 µg/kg AB002 (n = 12) or placebo (n = 6). Patients underwent five heparin-free hemodialysis sessions over 10 days and were dosed with AB002 or placebo during session four. RESULTS: Here we show that AB002 is safe and well-tolerated in ESRD patients, with no treatment-related adverse events. Clinically relevant bleeding did not occur in any patient, and the time to hemostasis at the vascular access sites is not affected by AB002. CONCLUSIONS: As far as we are aware, this proof-of-concept study is the first clinical trial assessing the therapeutic potential of protein C activation. The results herein support additional investigation of AB002 to safely prevent and treat thrombosis in at-risk populations.

Some people with kidney disease require hemodialysis, a process in which a machine filters the blood to remove waste products. The process of hemodialysis can trigger blood clotting in the hemodialysis circuit. Therefore, the blood-thinner heparin is commonly used to prevent blood from clotting. However, some patients cannot tolerate heparin. Here we describe a clinical trial in which we tested whether a drug called AB002 is safe and can reduce hemodialysis circuit clotting in people with permanent kidney disease (end-stage renal disease) undergoing hemodialysis. AB002 appears to be safe and well-tolerated, and we observed reduced clotting without any signs of increased bleeding. Further studies are required in more patients to determine whether AB002 can be used routinely during hemodialysis to safely prevent or treat blood clots.

Pharmacologic targeting of coagulation factors XII and XI by monoclonal antibodies reduces thrombosis in nitinol stents under flow.

BACKGROUND: Cardiovascular implantable devices, such as vascular stents, are critical for the treatment of cardiovascular diseases. However, their success is dependent on robust and often long-term antithrombotic therapies. Yet, the current standard-of-care therapies often pose significant bleeding risks to patients. Coagulation factor (F)XI and FXII have emerged as potentially safe and efficacious targets to safely reduce pathologic thrombin generation in medical devices. OBJECTIVES: To study the efficacy of monoclonal antibody-targeting FXII and FXI of the contact pathway in preventing vascular device-related thrombosis. METHODS: The effects of inhibition of FXII and FXI using function-blocking monoclonal antibodies were examined in a nonhuman primate model of nitinol stent-related thrombosis under arterial and venous flow conditions. RESULTS: We found that function-blocking antibodies of FXII and FXI reduced markers of stent-induced thrombosis in vitro and ex vivo. However, FXI inhibition resulted in more effective mitigation of thrombosis markers under varied flow conditions. CONCLUSION: This work provides further support for the translation of contact pathway of coagulation inhibitors for their adjunctive clinical use with cardiovascular devices.

Activation of coagulation FXI promotes endothelial inflammation and amplifies platelet activation in a nonhuman primate model of hyperlipidemia.

Background: Hyperlipidemia is associated with chronic inflammation and thromboinflammation. This is an underlying cause of several cardiovascular diseases, including atherosclerosis. In diseased blood vessels, rampant thrombin generation results in the initiation of the coagulation cascade, activation of platelets, and endothelial cell dysfunction. Coagulation factor (F) XI represents a promising therapeutic target to reduce thromboinflammation, as it is uniquely positioned at an intersection between inflammation and thrombin generation. Objectives: This study aimed to investigate the role of FXI in promoting platelet and endothelial cell activation in a model of hyperlipidemia. Methods: Nonhuman primates (NHPs) were fed a standard chow diet (lean, n = 6) or a high-fat diet (obese, n = 8) to establish a model of hyperlipidemia. Obese NHPs were intravenously administered a FXI blocking antibody (2 mg/kg) and studied at baseline and at 1, 7, 14, 21, and 28 days after drug administration. Platelet activation and inflammatory markers were measured using fluorescence-activated cell sorting or enzyme-linked immunosorbent assay. Molecular imaging was used to quantify vascular cell adhesion molecule 1 (VCAM-1) expression at the carotid bifurcation. Results: Obese NHPs demonstrated increased sensitivity for platelet P-selectin expression and phosphatidylserine exposure in response to platelet GPVI or PAR agonists compared with lean NHPs. Obese NHPs exhibited elevated levels of C-reactive protein, cathepsin D, and myeloperoxidase compared with lean NHPs. Following pharmacological inhibition of FIX activation by FXIa, platelet priming for activation by GPVI or PAR agonists, C-reactive protein levels, and endothelial VCAM-1 levels were reduced in obese NHPs. Conclusion: FXI activation promotes the proinflammatory phenotype of hyperlipidemia by priming platelet activation and inciting endothelial cell dysfunction.

Factor XI Inhibition for the Prevention of Catheter-Associated Thrombosis in Patients With Cancer Undergoing Central Line Placement: A Phase 2 Clinical Trial.

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.

Cytoprotective E-WE thrombin reduces disease severity in a murine model of relapsing-remitting multiple sclerosis.

The blood-brain barrier is composed of microvascular endothelial cells, immune cells, and astrocytes that work in concert with the coagulation cascade to control inflammation and immune cell infiltration into the central nervous system. Endothelial cell dysfunction leading to increased permeability and compromised barrier function are hallmarks of neuroinflammatory and autoimmune disorders, including multiple sclerosis (MS). Therapeutic strategies that improve or protect endothelial barrier function may be beneficial in the treatment or prevention of neuroinflammatory diseases. We therefore tested the hypothesis that biasing thrombin toward anticoagulant and cytoprotective activities would provide equivalent or even additive benefit compared with standard-of-care therapeutic strategies, including corticosteroids. In a mouse model of relapsing-remitting MS, treatment with the thrombin mutant, E-WE thrombin, an engineered thrombin mutant with cytoprotective activities that is biased toward anticoagulant and cytoprotective activity, reduced neuroinflammation and extracellular fibrin formation in SJL mice inoculated with proteolipid protein (PLP) peptide. When administered at the onset of detectable disease, E-WE thrombin significantly improved the disease severity of the initial attack as well as the relapse and delayed the onset of relapse to a similar extent as observed with methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment. These results provide rationale for considering engineered forms of thrombin biased toward anticoagulant and cytoprotective activity as a therapeutic strategy and perhaps an effective alternative to high-dose methylprednisolone for the management of acute relapsing MS attacks.NEW & NOTEWORTHY There are limited treatment options for mitigating acute relapsing attacks for patients with multiple sclerosis. We tested the hypothesis that harnessing the cytoprotective activity of the blood coagulation enzyme, thrombin, would provide benefit and protection against relapsing disease in a mouse model of MS. Our results provide rationale for considering engineered forms of thrombin biased toward cytoprotective activity as a therapeutic strategy and perhaps an alternative to steroids for the management of relapsing MS attacks.

Factor XI Inhibition With Heparin Reduces Clot Formation in Simulated Pediatric Extracorporeal Membrane Oxygenation.

Extracorporeal membrane oxygenation (ECMO) supplies circulatory support and gas exchange to critically ill patients. Despite the use of systemic anticoagulation, blood exposure to ECMO surfaces causes thromboembolism complications. Inhibition of biomaterial surface-mediated activation of coagulation factor XI (FXI) may prevent device-associated thrombosis. Blood was collected from healthy volunteers (n = 13) following the U.S. Army Institute of Surgical Research standard operating procedure for testing in an ex vivo ECMO circuit. A roller-pump circuit circulated either 0.5 U/ml of unfractionated heparin alone or in combination with the anti-FXI immunoglobulin G (IgG) (AB023) for 6 hours or until clot formation caused device failure. Coagulation factor activity, platelet counts, time to thrombin generation, peak thrombin, and endogenous thrombin potential were quantified. AB023 in addition to heparin sustained circuit patency in all tested circuits (5/5) after 6 hours, while 60% of circuits treated with heparin alone occluded (3/8), log-rank p < 0.03. AB023 significantly prolonged the time to clot formation as compared to heparin alone (15.5 vs . 3.3 minutes; p < 0.01) at the 3-hour time point. AB023 plus heparin significantly reduced peak thrombin compared to heparin alone (123 vs . 217 nM; p < 0.01). Inhibition of contact pathway activation of FXI may be an effective adjunct to anticoagulation in extracorporeal life support.

Pharmacological targeting of coagulation factor XI attenuates experimental autoimmune encephalomyelitis in mice.

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.

E-WE thrombin, a protein C activator, reduces disease severity and spinal cord inflammation in relapsing-remitting murine experimental autoimmune encephalomyelitis.

Objective: Relapses in patients with relapsing-remitting multiple sclerosis (RRMS) are typically treated with high-dose corticosteroids including methylprednisolone. However, high-dose corticosteroids are associated with significant adverse effects, can increase the risk for other morbidities, and often do not impact disease course. Multiple mechanisms are proposed to contribute to acute relapses in RRMS patients, including neuroinflammation, fibrin formation and compromised blood vessel barrier function. The protein C activator, E-WE thrombin is a recombinant therapeutic in clinical development for its antithrombotic and cytoprotective properties, including protection of endothelial cell barrier function. In mice, treatment with E-WE thrombin reduced neuroinflammation and extracellular fibrin formation in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). We therefore tested the hypothesis that E-WE thrombin could reduce disease severity in a relapsing-remitting model of EAE. Methods: Female SJL mice were inoculated with proteolipid protein (PLP) peptide and treated with E-WE thrombin (25 µg/kg; iv) or vehicle at onset of detectable disease. In other experiments, E-WE thrombin was compared to methylprednisolone (100 mg/kg; iv) or the combination of both. Results: Compared to vehicle, administration of E-WE thrombin significantly improved disease severity of the initial attack and relapse and delayed onset of relapse as effectively as methylprednisolone. Both methylprednisolone and E-WE thrombin reduced demyelination and immune cell recruitment, and the combination of both treatments had an additive effect. Conclusion: The data presented herein demonstrate that E-WE thrombin is protective in mice with relapsing-remitting EAE, a widely used model of MS. Our data indicate that E-WE thrombin is as effective as high-dose methylprednisolone in improving disease score and may exert additional benefit when administered in combination. Taken together, these data suggest that E-WE thrombin may be an effective alternative to high-dose methylprednisolone for managing acute MS attacks.

Thrombogenic potential of picomolar coagulation factor XIa is mediated by thrombin wave propagation.

Inhibitors of coagulation factor XIa (FXIa) are currently being investigated as potential anticoagulant therapies. We hypothesize that circulating FXIa could be a potential target for these therapies. Using previous analyses of FXIa impurities in immune globulin products involved in thrombotic adverse events, we estimated that picomolar levels of FXIa can be thrombogenic. In an in vitro clot-growth assay, 0.1-3 pM of FXIa did not, by itself, activate clotting but increased the size of growing clots. Spatio-temporal reconstruction of thrombin activity inside the clot revealed that FXIa's effect was limited to the clot-plasma interface, in which FXIa produced a taller than standard wave of thrombin. Factor-depleted plasma and a panel of selective anti-FXIa antibodies showed that exogenous FXIa effects are (1) blocked by anti-FXIa antibodies, (2) independent of FXI activation inside the clot, and (3) larger than the contribution of in situ FXIa. In a thrombin generation (TG) assay, picomolar FXIa did not initiate TG but rather promoted TG triggered by tissue factor or thrombin, suggesting that the effect of FXIa on the thrombin wave is mediated by the elevation of thrombin-triggered TG. In circulating bovine blood, low doses of human FXIa did not initiate clotting but increased the size of stenosis-triggered thrombi. FXIa injection in mice enhanced TG in plasma for at least 6 hours ex vivo, confirming the persistence of circulating FXIa. Our findings suggest that picomolar levels of circulating FXIa may not be able to initiate thrombosis but can facilitate thrombus growth through the facilitation of TG inside the clot.

Titanium is a potent inducer of contact activation: implications for intravascular devices.

BACKGROUND: Titanium (Ti) and its alloys are widely used in manufacturing medical devices because of their strength and resistance to corrosion. Although Ti compounds are considered compatible with blood, they appear to support plasma contact activation and may be thrombogenic. OBJECTIVES: The objective of this study was to compare Ti and titanium nitride (TiN) with known activators of contact activation (kaolin and silica) in plasma-clotting assays and to assess binding and activation of factor XII, (FXII), factor XI (FXI), prekallikrein, and high-molecular-weight kininogen (HK) with Ti/TiN. METHODS: Ti-based nanospheres and foils were compared with kaolin, silica, and aluminum in plasma-clotting assays. Binding and activation of FXII, prekallikrein, HK, and FXI to surfaces was assessed with western blots and chromogenic assays. RESULTS: Using equivalent surface amounts, Ti and TiN were comparable with kaolin and superior to silica, for inducing coagulation and FXII autoactivation. Similar to many inducers of contact activation, Ti and TiN are negatively charged; however, their effects on FXII are not neutralized by the polycation polybrene. Antibodies to FXII, prekallikrein, or FXI or coating Ti with poly-L-arginine blocked Ti-induced coagulation. An antibody to FXII reduced FXII and PK binding to Ti, kallikrein generation, and HK cleavage. CONCLUSION: Titanium compounds induce contact activation with a potency comparable with that of kaolin. Binding of FXII with Ti shares some features with FXII binding to soluble polyanions but may have unique features. Inhibitors targeting FXII or FXI may be useful in mitigating Ti-induced contact activation in patients with titanium-based implants that are exposed to blood.

Pharmacological reduction of coagulation factor XI reduces macrophage accumulation and accelerates deep vein thrombosis resolution in a mouse model of venous thrombosis.

BACKGROUND: Deep vein thrombosis (DVT) and post-thrombotic syndrome (PTS) remain highly prevalent despite modern medical therapy. Contact activation is a promising target for safe antithrombotic anticoagulation. The anti-factor XI (FXI) monoclonal antibody 14E11 reduces circulating levels of FXI without compromising hemostasis. The human recombinant analog, AB023, is in clinical development. The role of FXI in mediation of inflammation during DVT resolution is unknown. OBJECTIVES: Investigate the effects of pharmacological targeting of FXI with 14E11 in an experimental model of venous thrombosis. METHODS: Adult wild-type CD1 mice were treated with subcutaneous anti-FXI antibody (14E11, 5 mg/kg) versus saline prior to undergoing surgical constriction of the inferior vena cava (IVC). Mice were evaluated at various time points to assess thrombus weight and volume, as well as histology analysis, ferumoxytol enhanced magnetic resonance imaging (Fe-MRI), and whole blood flow cytometry. RESULTS: 14E11-treated mice had reduced thrombus weights and volumes after IVC constriction on day 7 compared to saline-treated mice. 14E11 treatment reduced circulating monocytes by flow cytometry and macrophage content within thrombi as evaluated by histologic staining and Fe-MRI. Collagen deposition was increased at day 3 while CD31 and smooth muscle cell actin expression was increased at day 7 in the thrombi of 14E11-treated mice compared to saline-treated mice. CONCLUSION: Pharmacologic targeting of FXI enhances the early stages of experimental venous thrombus resolution in wild-type CD1 mice, and may be of interest for future clinical evaluation of the antibody in DVT and PTS.

Revised model of the tissue factor pathway of thrombin generation: Role of the feedback activation of FXI.

BACKGROUND: Biochemical reaction networks are self-regulated in part due to feedback activation mechanisms. The tissue factor (TF) pathway of blood coagulation is a complex reaction network controlled by multiple feedback loops that coalesce around the serine protease thrombin. OBJECTIVES: Our goal was to evaluate the relative contribution of the feedback activation of coagulation factor XI (FXI) in TF-mediated thrombin generation using a comprehensive systems-based analysis. MATERIALS AND METHODS: We developed a systems biology model that improves the existing Hockin-Mann (HM) model through an integrative approach of mathematical modeling and in vitro experiments. Thrombin generation measured using in vitro assays revealed that the feedback activation of FXI contributes to the propagation of thrombin generation based on the initial concentrations of TF or activated coagulation factor X (FXa). We utilized experimental data to improve the robustness of the HM model to capture thrombin generation kinetics without a role for FXI before including the feedback activation of FXI by thrombin to construct the extended (ext.) HM model. RESULTS AND CONCLUSIONS: Using the ext.HM model, we predicted that the contribution of positive feedback of FXI activation by thrombin can be abolished by selectively eliminating the inhibitory function of tissue factor pathway inhibitor (TFPI), a serine protease inhibitor of FXa and TF-activated factor VII (FVIIa) complex. This prediction from the ext.HM model was experimentally validated using thrombin generation assays with function blocking antibodies against TFPI and plasmas depleted of FXI. Together, our results demonstrate the applications of combining experimental and modeling techniques in predicting complex biochemical reaction systems.

The contact activation inhibitor AB023 in heparin-free hemodialysis: results of a randomized phase 2 clinical trial.

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.

Cross-Talk between the Complement Pathway and the Contact Activation System of Coagulation: Activated Factor XI Neutralizes Complement Factor H.

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.

Factor XII plays a pathogenic role in organ failure and death in baboons challenged with Staphylococcus aureus.

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.

Role of platelets in regulating activated coagulation factor XI activity.

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.

Pharmacological targeting of coagulation factor XI mitigates the development of experimental atherosclerosis in low-density lipoprotein receptor-deficient mice.

BACKGROUND: Human coagulation factor (F) XI deficiency, a defect of the contact activation system, protects against venous thrombosis, stroke, and heart attack, whereas FXII, plasma prekallikrein, or kininogen deficiencies are asymptomatic. FXI deficiency, inhibition of FXI production, activated FXI (FXIa) inhibitors, and antibodies to FXI that interfere with FXI/FXII interactions reduce experimental thrombosis and inflammation. FXI inhibitors are antithrombotic in patients, and FXI and FXII deficiencies are atheroprotective in apolipoprotein E-deficient mice. OBJECTIVES: Investigate the effects of pharmacological targeting of FXI in experimental models of atherogenesis and established atherosclerosis. METHODS AND RESULTS: Low-density lipoprotein receptor-knockout (Ldlr-/- ) mice were administered high-fat diet (HFD) for 8 weeks; concomitantly, FXI was targeted with anti-FXI antibody (14E11) or FXI antisense oligonucleotide (ASO). 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas when compared with controls, and 14E11 also reduced aortic sinus lesions. In an established disease model, in which therapy was given after atherosclerosis had developed, Ldlr-/- mice were fed HFD for 8 weeks and then administered 14E11 or FXI-ASO weekly until 16 weeks on HFD. In this established disease model, 14E11 and FXI-ASO reduced atherosclerotic lesion area in proximal aortas, but not in aortic sinus. In cultures of human endothelium, FXIa exposure disrupted VE-Cadherin expression and increased endothelial lipoprotein permeability. Strikingly, we found that 14E11 prevented the disruption of VE-Cadherin expression in aortic sinus lesions observed in the atherogenesis mouse model. CONCLUSION: Pharmacological targeting of FXI reduced atherogenesis in Ldlr-/- mice. Interference with the contact activation system may safely reduce development or progression of atherosclerosis.

The contact activation system as a potential therapeutic target in patients with COVID-19.

Coronavirus disease 2019 (COVID-19) is predicted to overwhelm health care capacity in the United States and worldwide, and, as such, interventions that could prevent clinical decompensation and respiratory compromise in infected patients are desperately needed. Excessive cytokine release and activation of coagulation appear to be key drivers of COVID-19 pneumonia and associated mortality. Contact activation has been linked to pathologic upregulation of both inflammatory mediators and coagulation, and accumulating preclinical and clinical data suggest it to be a rational therapeutic target in patients with COVID-19. Pharmacologic inhibition of the interaction between coagulation factors XI and XII has been shown to prevent consumptive coagulopathy, pathologic systemic inflammatory response, and mortality in at least 2 types of experimental sepsis. Importantly, inhibition of contact activation also prevented death from Staphylococcus aureus-induced lethal systemic inflammatory response syndrome in nonhuman primates. The contact system is likely dispensable for hemostasis and may not be needed for host immunity, suggesting it to be a reasonably safe target that will not result in immunosuppression or bleeding. As a few drugs targeting contact activation are already in clinical development, immediate clinical trials for their use in patients with COVID-19 are potentially feasible for the prevention or treatment of respiratory distress.