Lecanemab blocks the effects of the Aß/fibrinogen complex on blood clots and synapse toxicity in organotypic culture.

Proteinaceous brain inclusions, neuroinflammation, and vascular dysfunction are common pathologies in Alzheimer's disease (AD). Vascular deficits include a compromised blood-brain barrier, which can lead to extravasation of blood proteins like fibrinogen into the brain. Fibrinogen's interaction with the amyloid-beta (Aß) peptide is known to worsen thrombotic and cerebrovascular pathways in AD. Lecanemab, an FDA-approved antibody therapy for AD, clears Aß plaque from the brain and slows cognitive decline. Here, we show that lecanemab blocks fibrinogen's binding to Aß protofibrils, preventing Aß/fibrinogen-mediated delayed fibrinolysis and clot abnormalities in vitro and in human plasma. Additionally, we show that lecanemab dissociates the Aß/fibrinogen complex and prevents fibrinogen from exacerbating Aß-induced synaptotoxicity in mouse organotypic hippocampal cultures. These findings reveal a possible protective mechanism by which lecanemab may slow disease progression in AD.

Lecanemab Blocks the Effects of the Aß/Fibrinogen Complex on Blood Clots and Synapse Toxicity in Organotypic Culture.

Proteinaceous brain inclusions, neuroinflammation, and vascular dysfunction are common pathologies in Alzheimer's disease (AD). Vascular deficits include a compromised blood-brain barrier, which can lead to extravasation of blood proteins like fibrinogen into the brain. Fibrinogen's interaction with the amyloid-beta (Aß) peptide is known to worsen thrombotic and cerebrovascular pathways in AD. Lecanemab, an FDA-approved antibody therapy for AD, shows promising results in facilitating reduction of Aß from the brain and slowing cognitive decline. Here we show that lecanemab blocks fibrinogen's binding to Aß protofibrils, normalizing Aß/fibrinogen-mediated delayed fibrinolysis and clot abnormalities in vitro and in human plasma. Additionally, we show that lecanemab dissociates the Aß/fibrinogen complex and prevents fibrinogen from exacerbating Aß-induced synaptotoxicity in mouse organotypic hippocampal cultures. These findings reveal a possible protective mechanism by which lecanemab may slow disease progression in AD.

A possible mechanism for the enhanced toxicity of beta-amyloid protofibrils in Alzheimer's disease.

The amyloid-beta peptide (Aß) is a driver of Alzheimer's disease (AD). Aß monomers can aggregate and form larger soluble (oligomers/protofibrils) and insoluble (fibrils) forms. There is evidence that Aß protofibrils are the most toxic form, but the reasons are not known. Consistent with a critical role for this form of Aß in AD, a recently FDA-approved therapeutic antibody targeted against protofibrils, lecanemab, slows the progression of AD in patients. The plasma contact system, which can promote coagulation and inflammation, has been implicated in AD pathogenesis. This system is activated by Aß which could lead to vascular and inflammatory pathologies associated with AD. We show here that the contact system is preferentially activated by protofibrils of Aß. Aß protofibrils bind to coagulation factor XII and high molecular weight kininogen and accelerate the activation of the system. Furthermore, lecanemab blocks Aß protofibril activation of the contact system. This work provides a possible mechanism for Aß protofibril toxicity in AD and why lecanemab is therapeutically effective.

Fiberoptic hemodynamic spectroscopy reveals abnormal cerebrovascular reactivity in a freely moving mouse model of Alzheimer's disease.

Many Alzheimer's disease (AD) patients suffer from altered cerebral blood flow and damaged cerebral vasculature. Cerebrovascular dysfunction could play an important role in this disease. However, the mechanism underlying a vascular contribution in AD is still unclear. Cerebrovascular reactivity (CVR) is a critical mechanism that maintains cerebral blood flow and brain homeostasis. Most current methods to analyze CVR require anesthesia which is known to hamper the investigation of molecular mechanisms underlying CVR. We therefore combined spectroscopy, spectral analysis software, and an implantable device to measure cerebral blood volume fraction (CBVF) and oxygen saturation (SO2) in unanesthetized, freely-moving mice. Then, we analyzed basal CBVF and SO2, and CVR of 5-month-old C57BL/6 mice during hypercapnia as well as during basic behavior such as grooming, walking and running. Moreover, we analyzed the CVR of freely-moving AD mice and their wildtype (WT) littermates during hypercapnia and could find impaired CVR in AD mice compared to WT littermates. Our results suggest that this optomechanical approach to reproducibly getting light into the brain enabled us to successfully measure CVR in unanesthetized freely-moving mice and to find impaired CVR in a mouse model of AD.

Striatal fibrinogen extravasation and vascular degeneration correlate with motor dysfunction in an aging mouse model of Alzheimer's disease.

Introduction: Alzheimer's Disease (AD) patients exhibit signs of motor dysfunction, including gait, locomotion, and balance deficits. Changes in motor function often precede other symptoms of AD as well as correlate with increased severity and mortality. Despite the frequent occurrence of motor dysfunction in AD patients, little is known about the mechanisms by which this behavior is altered. Methods and Results: In the present study, we investigated the relationship between cerebrovascular impairment and motor dysfunction in a mouse model of AD (Tg6799). We found an age-dependent increase of extravasated fibrinogen deposits in the cortex and striatum of AD mice. Interestingly, there was significantly decreased cerebrovascular density in the striatum of the 15-month-old as compared to 7-month-old AD mice. We also found significant demyelination and axonal damage in the striatum of aged AD mice. We analyzed striatum-related motor function and anxiety levels of AD mice at both ages and found that aged AD mice exhibited significant impairment of motor function but not in the younger AD mice. Discussion: Our finding suggests an enticing correlation between extravasated fibrinogen, cerebrovascular damage of the striatum, and motor dysfunction in an AD mouse model, suggesting a possible mechanism underlying motor dysfunction in AD.

Anti-HK antibody inhibits the plasma contact system by blocking prekallikrein and factor XI activation in vivo.

A dysregulated plasma contact system is involved in various pathological conditions, such as hereditary angioedema, Alzheimer disease, and sepsis. We previously showed that the 3E8 anti-high molecular weight kininogen (anti-HK) antibody blocks HK cleavage and bradykinin generation in human plasma ex vivo. Here, we show that 3E8 prevented not only HK cleavage but also factor XI (FXI) and prekallikrein (PK) activation by blocking their binding to HK in mouse plasma in vivo. 3E8 also inhibited contact system-induced bradykinin generation in vivo. Interestingly, FXII activation was also inhibited, likely because of the ability of 3E8 to block the positive feedback activation of FXII by kallikrein (PKa). In human plasma, 3E8 also blocked PK and FXI binding to HK and inhibited both thrombotic (FXI activation) and inflammatory pathways (PK activation and HK cleavage) of the plasma contact system activation ex vivo. Moreover, 3E8 blocked PKa binding to HK and dose-dependently inhibited PKa cleavage of HK. Our results reveal a novel strategy to inhibit contact system activation in vivo, which may provide an effective method to treat human diseases involving contact system dysregulation.

Contact and intrinsic coagulation pathways are activated and associated with adverse clinical outcomes in COVID-19.

Coagulation activation is a prominent feature of severe acute respiratory syndrome coronavirus 2 (COVID-19) infection. Activation of the contact system and intrinsic pathway has increasingly been implicated in the prothrombotic state observed in both sterile and infectious inflammatory conditions. We therefore sought to assess activation of the contact system and intrinsic pathway in individuals with COVID-19 infection. Baseline plasma levels of protease:serpin complexes indicative of activation of the contact and intrinsic pathways were measured in samples from inpatients with COVID-19 and healthy individuals. Cleaved kininogen, a surrogate for bradykinin release, was measured by enzyme-linked immunosorbent assay, and extrinsic pathway activation was assessed by microvesicle tissue factor-mediated factor Xa (FXa; MVTF) generation. Samples were collected within 24 hours of COVID-19 diagnosis. Thirty patients with COVID-19 and 30 age- and sex-matched controls were enrolled. Contact system and intrinsic pathway activation in COVID-19 was demonstrated by increased plasma levels of FXIIa:C1 esterase inhibitor (C1), kallikrein:C1, FXIa:C1, FXIa:α1-antitrypsin, and FIXa:antithrombin (AT). MVTF levels were also increased in patients with COVID-19. Because FIXa:AT levels were associated with both contact/intrinsic pathway complexes and MVTF, activation of FIX likely occurs through both contact/intrinsic and extrinsic pathways. Among the protease:serpin complexes measured, FIXa:AT complexes were uniquely associated with clinical indices of disease severity, specifically total length of hospitalization, length of intensive care unit stay, and extent of lung computed tomography changes. We conclude that the contact/intrinsic pathway may contribute to the pathogenesis of the prothrombotic state in COVID-19. Larger prospective studies are required to confirm whether FIXa:AT complexes are a clinically useful biomarker of adverse clinical outcomes.

Anti-HK antibody reveals critical roles of a 20-residue HK region for Aß-induced plasma contact system activation.

Alzheimer's disease (AD) is a neurodegenerative disorder and the leading cause of dementia. Vascular abnormalities and neuroinflammation play roles in AD pathogenesis. Plasma contact activation, which leads to fibrin clot formation and bradykinin release, is elevated in many AD patients, likely due to the ability of AD's pathogenic peptide ß-amyloid (Aß) to induce its activation. Since overactivation of this system may be deleterious to AD patients, the development of inhibitors could be beneficial. Here, we show that 3E8, an antibody against a 20-amino acid region in domain 6 of high molecular weight kininogen (HK), inhibits Aß-induced intrinsic coagulation. Mechanistically, 3E8 inhibits contact system activation by blocking the binding of prekallikrein (PK) and factor XI (FXI) to HK, thereby preventing their activation and the continued activation of factor XII (FXII). The 3E8 antibody can also disassemble HK/PK and HK/FXI complexes in normal human plasma in the absence of a contact system activator due to its strong binding affinity for HK, indicating its prophylactic ability. Furthermore, the binding of Aß to both FXII and HK is critical for Aß-mediated contact system activation. These results suggest that a 20-amino acid region in domain 6 of HK plays a critical role in Aß-induced contact system activation, and this region may provide an effective strategy to inhibit or prevent contact system activation in related disorders.

Vascular endothelial growth factor associated dissimilar cerebrovascular phenotypes in two different mouse models of Alzheimer's Disease.

Vascular perturbations and cerebral hypometabolism are emerging as important components of Alzheimer's disease (AD). While various in vivo imaging modalities have been designed to detect changes of cerebral perfusion and metabolism in AD patients and animal models, study results were often heterogenous with respect to imaging techniques and animal models. We therefore evaluated cerebral perfusion and glucose metabolism of two popular transgenic AD mouse strains, TgCRND8 and 5xFAD, at 7 and 12 months-of-age under identical conditions and analyzed possible molecular mechanisms underlying heterogeneous cerebrovascular phenotypes. Results revealed disparate findings in these two strains, displaying important aspects of AD progression. TgCRND8 mice showed significantly decreased cerebral blood flow and glucose metabolism with unchanged cerebral blood volume (CBV) at 12 months-of-age whereas 5xFAD mice showed unaltered glucose metabolism with significant increase in CBV at 12 months-of-age and a biphasic pattern of early hypoperfusion followed by a rebound to normal cerebral blood flow in late disease. Finally, immunoblotting assays suggested that VEGF dependent vascular tone change may restore normoperfusion and increase CBV in 5xFAD.

The contact activation system and vascular factors as alternative targets for Alzheimer's disease therapy.

Alzheimer's disease (AD) is the most common neurodegenerative disease, affecting millions of people worldwide. Extracellular beta-amyloid (Aß) plaques and neurofibrillary tau tangles are classical hallmarks of AD pathology and thus are the prime targets for AD therapeutics. However, approaches to slow or stop AD progression and dementia by reducing Aß production, neutralizing toxic Aß aggregates, or inhibiting tau aggregation have been largely unsuccessful in clinical trials. The contribution of dysregulated vascular components and inflammation is evident in AD pathology. Vascular changes are detectable early in AD progression, so treatment of vascular defects along with anti-Aß/tau therapy could be a successful combination therapeutic strategy for this disease. Here, we explain how vascular dysfunction mechanistically contributes to thrombosis as well as inflammation and neurodegeneration in AD pathogenesis. This review provides evidence that addressing vascular dysfunction in people with AD could be a promising therapeutic strategy.

Plasmin-mediated cleavage of high-molecular-weight kininogen contributes to acetaminophen-induced acute liver failure.

Acetaminophen (APAP)-induced liver injury is associated with activation of coagulation and fibrinolysis. In mice, both tissue factor-dependent thrombin generation and plasmin activity have been shown to promote liver injury after APAP overdose. However, the contribution of the contact and intrinsic coagulation pathways has not been investigated in this model. Mice deficient in individual factors of the contact (factor XII [FXII] and prekallikrein) or intrinsic coagulation (FXI) pathway were administered a hepatotoxic dose of 400 mg/kg of APAP. Neither FXII, FXI, nor prekallikrein deficiency mitigated coagulation activation or hepatocellular injury. Interestingly, despite the lack of significant changes to APAP-induced coagulation activation, markers of liver injury and inflammation were significantly reduced in APAP-challenged high-molecular-weight kininogen-deficient (HK-/-) mice. Protective effects of HK deficiency were not reproduced by inhibition of bradykinin-mediated signaling, whereas reconstitution of circulating levels of HK in HK-/- mice restored hepatotoxicity. Fibrinolysis activation was observed in mice after APAP administration. Western blotting, enzyme-linked immunosorbent assay, and mass spectrometry analysis showed that plasmin efficiently cleaves HK into multiple fragments in buffer or plasma. Importantly, plasminogen deficiency attenuated APAP-induced liver injury and prevented HK cleavage in the injured liver. Finally, enhanced plasmin generation and HK cleavage, in the absence of contact pathway activation, were observed in plasma of patients with acute liver failure due to APAP overdose. In summary, extrinsic but not intrinsic pathway activation drives the thromboinflammatory pathology associated with APAP-induced liver injury in mice. Furthermore, plasmin-mediated cleavage of HK contributes to hepatotoxicity in APAP-challenged mice independently of thrombin generation or bradykinin signaling.

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.

Increased Contact System Activation in Mild Cognitive Impairment Patients with Impaired Short-Term Memory.

An activated plasma contact system is an abnormality observed in many Alzheimer's disease (AD) patients. Since mild cognitive impairment (MCI) patients often develop AD, we analyzed the status of contact system activation in MCI patients. We found that kallikrein activity, high molecular weight kininogen cleavage, and bradykinin levels- measures of contact system activation- were significantly elevated in MCI patient plasma compared to plasma from age- and education-matched healthy individuals. Changes were more pronounced in MCI patients with impaired short-term recall memory, indicating the possible role of the contact system in early cognitive changes.

Cerebral amyloid angiopathy-linked ß-amyloid mutations promote cerebral fibrin deposits via increased binding affinity for fibrinogen.

Cerebral amyloid angiopathy (CAA), where beta-amyloid (Aß) deposits around cerebral blood vessels, is a major contributor of vascular dysfunction in Alzheimer's disease (AD) patients. However, the molecular mechanism underlying CAA formation and CAA-induced cerebrovascular pathology is unclear. Hereditary cerebral amyloid angiopathy (HCAA) is a rare familial form of CAA in which mutations within the (Aß) peptide cause an increase in vascular deposits. Since the interaction between Aß and fibrinogen increases CAA and plays an important role in cerebrovascular damage in AD, we investigated the role of the Aß-fibrinogen interaction in HCAA pathology. Our work revealed the most common forms of HCAA-linked mutations, Dutch (E22Q) and Iowa (D23N), resulted in up to a 50-fold stronger binding affinity of Aß for fibrinogen. In addition, the stronger interaction between fibrinogen and mutant Aßs led to a dramatic perturbation of clot structure and delayed fibrinolysis. Immunofluorescence analysis of the occipital cortex showed an increase of fibrin(ogen)/Aß codeposition, as well as fibrin deposits in HCAA patients, compared to early-onset AD patients and nondemented individuals. Our results suggest the HCAA-type Dutch and Iowa mutations increase the interaction between fibrinogen and Aß, which might be central to cerebrovascular pathologies observed in HCAA.

High molecular weight kininogen contributes to early mortality and kidney dysfunction in a mouse model of sickle cell disease.

BACKGROUND: Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusive crises, chronic inflammation, and activation of coagulation. The clinical complications such as painful crisis, stroke, pulmonary hypertension, nephropathy and venous thromboembolism lead to cumulative organ damage and premature death. High molecular weight kininogen (HK) is a central cofactor for the kallikrein-kinin and intrinsic coagulation pathways, which contributes to both coagulation and inflammation. OBJECTIVE: We hypothesize that HK contributes to the hypercoagulable and pro-inflammatory state that causes end-organ damage and early mortality in sickle mice. METHODS: We evaluated the role of HK in the Townes mouse model of SCD. RESULTS/CONCLUSIONS: We found elevated plasma levels of cleaved HK in sickle patients compared to healthy controls, suggesting ongoing HK activation in SCD. We used bone marrow transplantation to generate wild type and sickle cell mice on a HK-deficient background. We found that short-term HK deficiency attenuated thrombin generation and inflammation in sickle mice at steady state, which was independent of bradykinin signaling. Moreover, long-term HK deficiency attenuates kidney injury, reduces chronic inflammation, and ultimately improves survival of sickle mice.

A critical role for plasminogen in inflammation.

Plasminogen and its active form, plasmin, have diverse functions related to the inflammatory response in mammals. Due to these roles in inflammation, plasminogen has been implicated in the progression of a wide range of diseases with an inflammatory component. In this review, we discuss the functions of plasminogen in inflammatory regulation and how this system plays a role in the pathogenesis of diseases spanning organ systems throughout the body.

Increased plasma bradykinin level is associated with cognitive impairment in Alzheimer's patients.

Alzheimer's disease (AD) is characterized by the presence of proteinaceous brain deposits, brain atrophy, vascular dysfunction, and chronic inflammation. Along with cerebral inflammation, peripheral inflammation is also evident in many AD patients. Bradykinin, a proinflammatory plasma peptide, is also linked to AD pathology. For example, bradykinin infusion into the hippocampus causes learning and memory deficits in rats, and blockade of the bradykinin receptor lessens cognitive impairment in AD mouse models. Even though it has been hypothesized that plasma bradykinin could contribute to inflammation in AD, the level of plasma bradykinin and its association with beta-amyloid (Aß) pathology in AD patients had not been explored. Here, we assessed plasma bradykinin levels in AD patients and age-matched non-demented (ND) control individuals. We found significantly elevated plasma bradykinin levels in AD patients compared to ND subjects. Additionally, changes in plasma bradykinin levels were more profound in many AD patients with severe cognitive impairment, suggesting that peripheral bradykinin could play a role in dementia most likely via inflammation. Bradykinin levels in the cerebrospinal fluid (CSF) were reduced in AD patients and exhibited an inverse correlation with the CSF Aß40/Aß42 ratio. We also report that bradykinin interacts with the fibrillar form of Aß and co-localizes with Aß plaques in the post-mortem human AD brain. These findings connect the peripheral inflammatory pathway to cerebral abnormalities and identify a novel mechanism of inflammatory pathology in AD.

An antibody against HK blocks Alzheimer's disease peptide ß-amyloid-induced bradykinin release in human plasma.

Bradykinin is a proinflammatory factor that mediates angioedema and inflammation in many diseases. It is a key player in some types of hereditary angioedema and is involved in septic shock, traumatic injury, Alzheimer's disease (AD), and stroke, among others. Activation of the plasma contact system leads to elevated levels of plasma kallikrein, which cleaves high molecular weight kininogen (HK) to release bradykinin. Drug development for bradykinin-meditated pathologies has focused on designing inhibitors to the enzymes that cleave HK (to prevent bradykinin release) or antagonists of endothelial bradykinin receptors (to prevent downstream bradykinin action). Here we show a strategy to block bradykinin generation by using an HK antibody that binds to HK, preventing its cleavage and subsequent bradykinin release. We show that this antibody blocks dextran sodium sulfate-induced HK cleavage and bradykinin production. Moreover, while the pathogenic AD peptide ß-amyloid (Aß)42 cleaves HK and induces a dramatic increase in bradykinin production, our HK antibody blocked these events from occurring. These results may provide strategies for developing treatments for bradykinin-driven pathologies.

Long-Term Dabigatran Treatment Delays Alzheimer's Disease Pathogenesis in the TgCRND8 Mouse Model.

BACKGROUND: Alzheimer's disease (AD) is a multifactorial neurodegenerative disorder with important vascular and hemostatic alterations that should be taken into account during diagnosis and treatment. OBJECTIVES: This study evaluates whether anticoagulation with dabigatran, a clinically approved oral direct thrombin inhibitor with a low risk of intracerebral hemorrhage, ameliorates AD pathogenesis in a transgenic mouse model of AD. METHODS: TgCRND8 AD mice and their wild-type littermates were treated for 1 year with dabigatran etexilate or placebo. Cognition was evaluated using the Barnes maze, and cerebral perfusion was examined by arterial spin labeling. At the molecular level, Western blot and histochemical analyses were performed to analyze fibrin content, amyloid burden, neuroinflammatory activity, and blood-brain barrier (BBB) integrity. RESULTS: Anticoagulation with dabigatran prevented memory decline, cerebral hypoperfusion, and toxic fibrin deposition in the AD mouse brain. In addition, long-term dabigatran treatment significantly reduced the extent of amyloid plaques, oligomers, phagocytic microglia, and infiltrated T cells by 23.7%, 51.8%, 31.3%, and 32.2%, respectively. Dabigatran anticoagulation also prevented AD-related astrogliosis and pericyte alterations, and maintained expression of the water channel aquaporin-4 at astrocytic perivascular endfeet of the BBB. CONCLUSIONS: Long-term anticoagulation with dabigatran inhibited thrombin and the formation of occlusive thrombi in AD; preserved cognition, cerebral perfusion, and BBB function; and ameliorated neuroinflammation and amyloid deposition in AD mice. Our results open a field for future investigation on whether the use of direct oral anticoagulants might be of therapeutic value in AD.