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Journal of Thrombosis and Haemostasis :... Jun 2024Factor XII (FXII) triggers contact activation by binding to foreign surfaces, with the EGF-1 domain being the primary binding site. Blocking FXII surface-binding might...
BACKGROUND
Factor XII (FXII) triggers contact activation by binding to foreign surfaces, with the EGF-1 domain being the primary binding site. Blocking FXII surface-binding might hold therapeutic value to prevent medical device-induced thrombosis.
OBJECTIVES
To unravel and prevent EGF-1 mediated FXII surface-binding with VH.
METHODS
FXII variants with glutamine substitutions of two positively-charged amino acid patches within the EGF-1 domain were created. Their role in FXII contact activation was assessed using kaolin pull-down experiments, amidolytic activity assays, and clotting assays. FXII EGF-1 domain specific VHHs were raised to inhibit EGF-1 mediated FXII contact activation while preserving quiescence.
RESULTS
Two unique, positively-charged patches in the EGF1 domain were identified (upstream: 73K74K76K78H81K82H; downstream: 87K113K). Neutralizing the charge of both patches led to a 99% reduction in FXII kaolin binding, subsequent decrease in auto-activation of 94% and prolongation of clot formation in aPTT assays from 36 (±2) to 223 (±13) seconds. Three FXII EGF-1 specific VHHs were developed, that are capable of inhibiting kaolin binding and subsequent contact system activation in plasma. The most effective V HH 'F2' binds the positively-charged patches and thereby dose-dependently extends aPTT clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence.
CONCLUSION
The two unique, positively-charged patches in FXII EGF-1 cooperatively mediate FXII surface-binding making both patches crucial for contact activation. Targeting these with FXII EGF-1 specific VHHs can exclusively decrease FXII surface-binding and subsequent contact activation, while preserving zymogen quiescence. These patches thus have potential as druggable target in preventing medical device-induced thrombosis.
PubMed: 38897387
DOI: 10.1016/j.jtha.2024.06.005 -
Journal of Neuroinflammation Jun 2024Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key...
Activation of the kallikrein-kinin system promotes vascular leakage, inflammation, and neurodegeneration in ischemic stroke. Inhibition of plasma kallikrein (PK) - a key component of the KKS - in the acute phase of ischemic stroke has been reported to reduce thrombosis, inflammation, and damage to the blood-brain barrier. However, the role of PK during the recovery phase after cerebral ischemia is unknown. To this end, we evaluated the effect of subacute PK inhibition starting from day 3 on the recovery process after transient middle artery occlusion (tMCAO). Our study demonstrated a protective effect of PK inhibition by reducing infarct volume and improving functional outcome at day 7 after tMCAO. In addition, we observed reduced thrombus formation in cerebral microvessels, fewer infiltrated immune cells, and an improvement in blood-brain barrier integrity. This protective effect was facilitated by promoting tight junction reintegration, reducing detrimental matrix metalloproteinases, and upregulating regenerative angiogenic markers. Our findings suggest that PK inhibition in the subacute phase might be a promising approach to accelerate the post-stroke recovery process.
Topics: Animals; Recovery of Function; Male; Plasma Kallikrein; Mice; Mice, Inbred C57BL; Infarction, Middle Cerebral Artery; Blood-Brain Barrier; Stroke; Thrombosis; Ischemic Stroke; Inflammation
PubMed: 38872149
DOI: 10.1186/s12974-024-03149-w -
Journal of Biotechnology Jun 2024Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat...
Ecallantide comprises Kunitz Domain 1 of Tissue Factor Pathway Inhibitor, mutated at seven amino acid positions to inhibit plasma kallikrein (PK). It is used to treat acute hereditary angioedema (HAE). We appended hexahistidine tags to the N- or C-terminus of recombinant Ecallantide (rEcall) and expressed and purified the resulting proteins, with or without fusion to human serum albumin (HSA), using Pichia pastoris. The inhibitory constant (K) of rEcall-H6 or H6-rEcall for PK was not increased by albumin fusion. When I-labelled rEcall proteins were injected intravenously into mice, the area under the clearance curve (AUC) was significantly increased, 3.4- and 3.6-fold, for fusion proteins H6-rEcall-HSA and HSA-rEcall-H6 versus their unfused counterparts but remained 2- to 3-fold less than that of HSA-H6. The terminal half-life of H6-rEcall-HSA and HSA-H6 did not differ, although that of HSA-rEcall-H6 was significantly shorter than either other protein. Receptor Associated Protein (RAP), a Low-density lipoprotein Receptor-related Protein (LRP1) antagonist, competed H6-rEcall-HSA clearance more effectively than intravenous immunoglobulin (IVIg), a neonatal Fc receptor (FcRn) antagonist. HSA fusion decreases rEcall clearance in vivo, but LRP1-mediated clearance remains more important than FcRn-mediated recycling for rEcall fusion proteins. The properties of H6-rEcall-HSA warrant investigation in a murine model of HAE.
PubMed: 38844246
DOI: 10.1016/j.jbiotec.2024.06.002 -
Journal of Translational Medicine Apr 2024The kallikrein-kinin system is a key regulatory cascade involved in blood pressure maintenance, hemostasis, inflammation and renal function. Currently, approved drugs... (Review)
Review
BACKGROUND
The kallikrein-kinin system is a key regulatory cascade involved in blood pressure maintenance, hemostasis, inflammation and renal function. Currently, approved drugs remain limited to the rare disease hereditary angioedema. However, growing interest in this system is indicated by an increasing number of promising drug candidates for further indications.
METHODS
To provide an overview of current drug development, a two-stage literature search was conducted between March and December 2023 to identify drug candidates with targets in the kallikrein-kinin system. First, drug candidates were identified using PubMed and Clinicaltrials.gov. Second, the latest publications/results for these compounds were searched in PubMed, Clinicaltrials.gov and Google Scholar. The findings were categorized by target, stage of development, and intended indication.
RESULTS
The search identified 68 drugs, of which 10 are approved, 25 are in clinical development, and 33 in preclinical development. The three most studied indications included diabetic retinopathy, thromboprophylaxis and hereditary angioedema. The latter is still an indication for most of the drug candidates close to regulatory approval (3 out of 4). For the emerging indications, promising new drug candidates in clinical development are ixodes ricinus-contact phase inhibitor for thromboprophylaxis and RZ402 and THR-149 for the treatment of diabetic macular edema (all phase 2).
CONCLUSION
The therapeutic impact of targeting the kallikrein-kinin system is no longer limited to the treatment of hereditary angioedema. Ongoing research on other diseases demonstrates the potential of therapeutic interventions targeting the kallikrein-kinin system and will provide further treatment options for patients in the future.
Topics: Humans; Kallikrein-Kinin System; Drug Discovery; Drug Development; Animals
PubMed: 38671481
DOI: 10.1186/s12967-024-05216-5 -
The Journal of Allergy and Clinical... Apr 2024Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity....
BACKGROUND
Hereditary angioedema (HAE) is a genetic disorder that manifests as recurrent angioedema attacks, most frequently due to absent or reduced C1 inhibitor (C1INH) activity. C1INH is a crucial regulator of enzymatic cascades in the complement, fibrinolytic, and contact systems. Inter-α-trypsin inhibitor heavy chain 4 (ITIH4) is an abundant plasma protease inhibitor that can inhibit enzymes in the proteolytic pathways associated with HAE. Nothing is known about its role in HAE.
OBJECTIVE
We investigated ITIH4 activation in HAE, establishing it as a potential biomarker, and explored its involvement in HAE-associated proteolytic pathways.
METHODS
Specific immunoassays for noncleaved ITIH4 (intact ITIH4) and an assay detecting both intact and cleaved ITIH4 (total ITIH4) were developed. We initially tested serum samples from HAE patients (n = 20), angiotensin-converting enzyme inhibitor-induced edema patients (ACEI) (n = 20), and patients with HAE of unknown cause (HAE-UNK) (n = 20). Validation involved an extended cohort of 80 HAE patients (60 with HAE-C1INH type 1, 20 with HAE-C1INH type 2), including samples taken during attack and quiescent disease periods, as well as samples from 100 healthy controls.
RESULTS
In 63% of HAE patients, intact ITIH4 assay showed lower signals than total ITIH4 assay. This difference was not observed in ACEI and HAE-UNK patients. Western blot analysis confirmed cleaved ITIH4 with low intact ITIH4 samples. In serum samples lacking intact endogenous ITIH4, we observed immediate cleavage of added recombinant ITIH4, suggesting continuous enzymatic activity in the serum. Confirmatory HAE cohort analysis revealed significantly lower intact ITIH4 levels in both type 1 and type 2 HAE patients compared to controls, with consistently low intact/total ITIH4 ratios during clinical HAE attacks.
CONCLUSION
The disease-specific low intact ITIH4 levels highlight its unique nature in HAE. ITIH4 may exhibit compensatory mechanisms in HAE, suggesting its utility as a diagnostic and prognostic biomarker. The variations during quiescent and active disease periods raise intriguing questions about the dynamics of proteolytic pathways in HAE.
PubMed: 38657796
DOI: 10.1016/j.jaci.2024.03.028 -
ACS Medicinal Chemistry Letters Apr 2024Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin...
Hereditary angioedema (HAE), a rare genetic disorder, is associated with uncontrolled plasma kallikrein (PKa) enzyme activity leading to the generation of bradykinin swelling in subcutaneous and submucosal membranes in various locations of the body. Herein, we describe a series of potent α-amidobenzylboronates as potential covalent inhibitors of PKa. These compounds exhibited time-dependent inhibition of PKa (compound IC 66 nM at 1 min, 70 pM at 24 h). Further compound dissociation studies demonstrated that showed no apparent reversibility comparable to d-Phe-Pro-Arg-chloromethylketone (PPACK) (), a known nonselective covalent PKa inhibitor.
PubMed: 38628785
DOI: 10.1021/acsmedchemlett.3c00572 -
Molecular Vision 2024Diabetic macular edema (DME) is a sight-threatening complication of diabetes. Consequently, studying the proteome of DME may provide novel insights into underlying...
PURPOSE
Diabetic macular edema (DME) is a sight-threatening complication of diabetes. Consequently, studying the proteome of DME may provide novel insights into underlying molecular mechanisms.
METHODS
In this study, aqueous humor samples from eyes with treatment-naïve clinically significant DME (n = 13) and age-matched controls (n = 11) were compared with label-free liquid chromatography-tandem mass spectrometry. Additional aqueous humor samples from eyes with treatment-naïve DME (n = 15) and controls (n = 8) were obtained for validation by enzyme-linked immunosorbent assay (ELISA). Best-corrected visual acuity (BCVA) was evaluated, and the severity of DME was measured as central subfield thickness (CST) employing optical coherence tomography. Control samples were obtained before cataract surgery. Significantly changed proteins were identified using a permutation-based calculation, with a false discovery rate of 0.05. A human donor eye with DME and a control eye were used for immunofluorescence.
RESULTS
A total of 101 proteins were differentially expressed in the DME. Regulated proteins were involved in complement activation, glycolysis, extracellular matrix interaction, and cholesterol metabolism. The highest-fold change was observed for the fibrinogen alpha chain (fold change = 17.8). Complement components C2, C5, and C8, fibronectin, and hepatocyte growth factor-like protein were increased in DME and correlated with best-corrected visual acuity (BCVA). Ceruloplasmin and complement component C8 correlated with central subfield thickness (CST). Hemopexin, plasma kallikrein, monocyte differentiation antigen CD14 (CD14), and lipopolysaccharide-binding protein (LBP) were upregulated in the DME. LBP was correlated with vascular endothelial growth factor. The increased level of LBP in DME was confirmed using ELISA. The proteins involved in desmosomal integrity, including desmocollin-1 and desmoglein-1, were downregulated in DME and correlated negatively with CST. Immunofluorescence confirmed the extravasation of fibrinogen at the retinal level in the DME.
CONCLUSION
Elevated levels of pro-inflammatory proteins, including the complement components LBP and CD14, were observed in DME. DME was associated with the loss of basal membrane proteins, compromised desmosomal integrity, and perturbation of glycolysis.
Topics: Humans; Macular Edema; Diabetic Retinopathy; Proteome; Vascular Endothelial Growth Factor A; Aqueous Humor; Tomography, Optical Coherence; Fibrinogen; Intravitreal Injections; Angiogenesis Inhibitors; Diabetes Mellitus
PubMed: 38586604
DOI: No ID Found -
IBRO Neuroscience Reports Jun 2024Biomarkers are measured to evaluate physiological and pathological processes as well as responses to a therapeutic intervention. Biomarkers can be classified as... (Review)
Review
Biomarkers are measured to evaluate physiological and pathological processes as well as responses to a therapeutic intervention. Biomarkers can be classified as diagnostic, prognostic, predictor, clinical, and therapeutic. In Alzheimer's disease (AD), multiple biomarkers have been reported so far. Nevertheless, finding a specific biomarker in AD remains a major challenge. Three databases, including PubMed, Web of Science, and Scopus were selected with the keywords of Alzheimer's disease, neuroimaging, biomarker, and blood. The results were finalized with 49 potential CSF/blood and 35 neuroimaging biomarkers. To distinguish normal from AD patients, amyloid-beta (Aβ), plasma glial fibrillary acidic protein (GFAP), and neurofilament light (NFL) as potential biomarkers in cerebrospinal fluid (CSF) as well as the serum could be detected. Nevertheless, most of the biomarkers fairly change in the CSF during AD, listed as kallikrein 6, virus-like particles (VLP-1), galectin-3 (Gal-3), and synaptotagmin-1 (Syt-1). From the neuroimaging aspect, atrophy is an accepted biomarker for the neuropathologic progression of AD. In addition, Magnetic resonance spectroscopy (MRS), diffusion weighted imaging (DWI), diffusion tensor imaging (DTI), tractography (DTT), positron emission tomography (PET), and functional magnetic resonance imaging (fMRI), can be used to detect AD. Using neuroimaging and CSF/blood biomarkers, in combination with artificial intelligence, it is possible to obtain information on prognosis and follow-up on the different stages of AD. Hence physicians could select the suitable therapy to attenuate disease symptoms and follow up on the efficiency of the prescribed drug.
PubMed: 38497046
DOI: 10.1016/j.ibneur.2024.02.007 -
Research Square Mar 2024Fluoride exposure during pregnancy has been associated with various effects on offspring, including changes in behavior and IQ. To provide clues to possible mechanisms...
BACKGROUND
Fluoride exposure during pregnancy has been associated with various effects on offspring, including changes in behavior and IQ. To provide clues to possible mechanisms by which fluoride affects human fetal development, we completed proteomic analyses of cord blood serum collected from second-trimester pregnant women residing in Northern California with either high or low fluoride exposure, as identified by maternal serum fluoride concentrations.
OBJECTIVE
To identify changes in cord blood proteins associated with maternal serum fluoride concentration in pregnant women living in Northern California.
METHODS
The proteomes of 19 archived second-trimester cord blood samples representing highest and lowest serum fluoride concentrations from a cohort of 48 women living in Northern California, previously analyzed for serum, urine and amniotic fluoride concentrations, were characterized by mass spectrometry. Proteins highly correlated to maternal serum fluoride concentrations were identified, and further compared in a group of samples from women with the highest serum fluoride to the group with the lowest maternal serum fluoride concentrations.
RESULTS
Nine cord blood proteins were significantly correlated with maternal serum fluoride concentrations. Six of these proteins, including apolipoprotein B-100, delta homolog 1, coagulation factor X, mimecan, plasma kallikrein, and vasorin, were significantly decreased in the cord blood from women with the highest serum fluoride levels.
CONCLUSION
Changes in the relative amounts of second trimester cord blood proteins included proteins associated with the development of the fetal hematopoetic system.
PubMed: 38464284
DOI: 10.21203/rs.3.rs-3995767/v1 -
The World Allergy Organization Journal Mar 2024Berotralstat is a once-daily oral inhibitor of plasma kallikrein for the prophylaxis of hereditary angioedema (HAE) in patients ≥12 years. APeX-J aimed to evaluate the...
BACKGROUND
Berotralstat is a once-daily oral inhibitor of plasma kallikrein for the prophylaxis of hereditary angioedema (HAE) in patients ≥12 years. APeX-J aimed to evaluate the efficacy and safety of berotralstat in Japan.
METHODS
APeX-J was a Phase III trial comprising 3 parts (NCT03873116). Part 1 was a randomized, placebo-controlled evaluation of berotralstat 150 or 110 mg over 24 weeks. Part 2 was a 28-week dose-blinded phase in which berotralstat-treated patients continued the same dose and placebo patients were re-randomized to berotralstat 150 or 110 mg. In Part 3, all patients remaining on study received berotralstat 150 mg in an open-label manner for up to an additional 52 weeks. The primary endpoint of Parts 2 and 3 was long-term safety and tolerability, and secondary endpoints examined effectiveness.
RESULTS
Seventeen patients entered Part 2, and 11 continued into Part 3. Treatment-emergent adverse events (TEAEs) were reported by 14/17 patients (82.4%) in Parts 2 or 3; the most common were nasopharyngitis, abdominal pain, cystitis, influenza, and vertigo. One patient (5.9%) experienced a drug-related TEAE (Grade 4 increased hepatic enzyme). No drug-related serious TEAEs were reported. For patients who completed 26 months of treatment with berotralstat 150 mg (n = 5), mean (standard error of the mean) monthly HAE attack rates and on-demand medication use decreased from baseline by 1.15 (0.09) attacks/month and 2.8 (0.64) doses/month, respectively. Sustained improvements were also observed in patient quality of life and treatment satisfaction.
CONCLUSIONS
Long-term prophylaxis with berotralstat raised no new safety signals and was effective at reducing attacks and improving patient-reported outcomes.
TRIAL REGISTRATION
ClinicalTrials.gov NCT03873116. Registered March 13, 2019. Retrospectively registered.
PubMed: 38445295
DOI: 10.1016/j.waojou.2024.100882