Combined transcriptomics and TMT-proteomics reveal abnormal complement and coagulation cascades in cow's milk protein allergy.

Cow's milk protein allergy (CMPA) is primarily due to the inability of the intestinal mucosa to establish typical immunological tolerance to proteins found in cow's milk, and the specific molecular mechanism is still unclear. In order to investigate molecular alterations in intestinal tissues during CMPA occurrence, this study analyzed the jejunal tissue of ß-lactoglobulin (BLG)-sensitized mice through transcriptomics and quantitative tandem mass tag (TMT)-labeled proteomics. A total of 475 differentially expressed genes (256 up-regulated, 219 down-regulated) and 94 differentially expressed proteins (65 up-regulated, 29 down-regulated) were identified. Comparing the KEGG pathways of the two groups, it was found that both were markedly enriched in the signaling pathways of complement and coagulation cascade. Among these, kallikrein B1 (KLKB1) in this pathway is speculated to be pivotal in CMPA. It may potentially enhance the release of bradykinin by activating the kallikrein-kinin system, leading to pro-inflammatory effects and exacerbating intestinal mucosal damage. This study suggests that the pathways of complement and coagulation cascades could be significant in the context of intestinal immunity in CMPA, and KLKB1 may be its potential therapeutic target.

The Dysregulation of the Renin-Angiotensin System in COVID-19 Studied by Serum Proteomics: Angiotensinogen Increases with Disease Severity.

(1) Background: ACE and CPN serum activity correlated with disease severity in an earlier study of 45 hospitalized COVID-19 patients. The serum protein profile was investigated in the same cohort here to shed more light on the involvement of the renin-angiotensin system (RAS). (2) Methods: High-definition mass spectrometry-based protein expression analysis was performed, followed by multivariate statistical and network analyses. (3) Results: The protein profiles of hospitalized patients (HoP) differed significantly from those of convalescent and healthy probands. Surprisingly, HoP samples separated into six groups according to their protein profiles: group (G) 1 represented the youngest and the least afflicted patients, and G6 the oldest and critically ill patients. At least two major pathophysiological schemes were indicated based on differing involvement of the kallikrein-kinin system (KKS), the RAS and complement activation. The serum angiotensinogen concentration increased with disease severity. (4) Conclusions: The important role of the RAS in the response to COVID-19 infection was substantiated, but other pathways such as the KKS, plasminogen activation and complement activation influence the systemic response to the infection.

A review of oral kallikrein inhibitor berotralstat for hereditary angioedema.

Orladeyo, a once-daily oral formulation of berotralstat (formerly BCX-7353), is a novel oral small-molecule drug developed by BioCryst Pharmaceuticals for the prevention of hereditary angioedema (HAE) attacks. It was first approved by the U.S. Food and Drug Administration (FDA) in 2020, and in 2021 also gained approval for marketing in Japan and the European Union. Preclinical and phase I studies showed promising efficacy and safety, and several multicenter international Angioedema Prophylaxis (APeX) phase II and III trials have since been initiated to further evaluate berotralstat. The ongoing phase III APeX-2 trial showed a 67% reduction in HAE attacks at the standard 150-mg dosing. Mild to moderate gastrointestinal side effects are most commonly seen and minimal serious adverse effects have been reported. Other first-line therapies for HAE prophylaxis rely on burdensome subcutaneous or intravenous routes. Thus far berotralstat has shown to be effective and well tolerated for HAE prophylaxis with the convenience of once-daily oral dosing.

Serum KLKB1 as a Potential Prognostic Biomarker for Hepatocellular Carcinoma Based on Data-Independent Acquisition and Parallel Reaction Monitoring.

PURPOSE: With the advancement of minimally invasive surgery and catheters for hepatocellular carcinoma (HCC), it is becoming more and more inconvenient to get tissues or the tissues gained are insufficient for testing. Screening of blood-derived markers is of great significance for prognosis assessment. PATIENTS AND METHODS: Data-independent acquisition (DIA) and parallel reaction monitoring (PRM) were implemented to identify valuable prognostic HCC biomarkers in 48 patients with different prognosis. The potential candidate biomarkers were examined in 205 HCC patients using enzyme-linked immunosorbent assay (ELISA) and then validated in The Cancer Genome Atlas (TCGA) HCC cohort. RESULTS: DIA screened 86 significantly differentially regulated proteins between patients with poor prognosis and those with good prognosis. Eight proteins from the DIA proteomic analyses were quantified by PRM, and six of them (KLKB1, IGFBP3, SHBG, SAA1, C7, and CD44) presented consistent expression trends between DIA and PRM. Then, the results of ELISA indicated that KLKB1 was abnormally expressed in HCC patients, and the serum level of KLKB1 also exhibited significant changes before and after treatment (P = 0.016). Patients with higher KLKB1 serum levels had significantly superior overall survival (P = 0.035) and progression-free survival (P = 0.027) than those with lower KLKB1 expression. In the TCGA-HCC cohort, Cox regression analysis suggested that KLKB1 was an independent prognostic factor for overall survival (P = 0.032) of HCC patients. CONCLUSION: Aberrant expression of KLKB1 was strongly associated with the prognosis of HCC patients. KLKB1 may be used to evaluate the prognosis and guide the treatment for HCC.

Severe plasma prekallikrein deficiency: Clinical characteristics, novel KLKB1 mutations, and estimated prevalence.

BACKGROUND: Severe plasma prekallikrein (PK) deficiency is an autosomal-recessive defect characterized by isolated activated partial thromboplastin time prolongation. To date, no comprehensive methodologically firm analysis has investigated the diagnostic, clinical, and genetic characteristics of PK deficiency, and its prevalence remains unknown. PATIENTS/METHODS: We described new families with PK deficiency, retrieved clinical and laboratory information of cases systematically searched in the (gray) literature, and collected blood of these cases for complementary analyses. The Genome Aggregation Database (gnomAD) and the population-based Gutenberg Health Study served to study the prevalence of mutations and relevant genetic variants. RESULTS: We assembled a cohort of 111 cases from 89 families and performed new genetic analyses in eight families (three unpublished). We identified new KLKB1 mutations, excluded the pathogenicity of some of the previously described ones, and estimated a prevalence of severe PK deficiency of 1/155 668 overall and 1/4725 among Africans. One individual reported with PK deficiency had, in fact, congenital kininogen deficiency associated with decreased PK activity. One quarter of individuals had factor XII clotting activity below the reference range. Four major bleeding events were described in 96 individuals, of which 3 were provoked, for a prevalence of 4% and an annualized rate of 0.1%. The prevalence of cardiovascular events was 15% (6% <40 years; 21% 40-65 years; 33% >65 years) for an annualized rate of 0.4%. CONCLUSIONS: We characterized the genetic background of severe PK deficiency, critically appraised mutations, and provided prevalence estimates. Our data on laboratory characteristics and clinical course of severe PK deficiency may have clinical implications.

Design and development of a series of borocycles as selective, covalent kallikrein 5 inhibitors.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases, particularly Kallikrein 5 (KLK5) has been well established and it is expected that a KLK5 inhibitor would improve the dermal barrier and also reduce the pain and itch that afflict Netherton syndrome patients. One of the challenges of covalent protease inhibitors has been achieving selectivity over closely related targets. In this paper we describe the use of structural insight to design and develop a selective and highly potent reversibly covalent KLK5 inhibitor from an initial weakly binding fragment.

Structure guided drug design to develop kallikrein 5 inhibitors to treat Netherton syndrome.

The connection between Netherton syndrome and overactivation of epidermal/dermal proteases particularly KLK5 has been well established. To treat sufferers of this severe condition we wished to develop a topical KLK5 inhibitor in order to normalise epidermal shedding and reduce the associated inflammation and itching. In this paper we describe structure-based optimisation of a series of brightly coloured weak KLK5 inhibitors into colourless, non-irritant molecules with good KLK5 activity and selectivity over a range of serine proteases.

Genetic Determinants of C1 Inhibitor Deficiency Angioedema Age of Onset.

BACKGROUND: In view of the large heterogeneity in the clinical presentation of hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE), great efforts are being made towards detecting measurable biological determinants of disease severity that can help to improve the management of the disease. Considering the central role that plasma kallikrein plays in bradykinin production, we investigated the contribution of the functional polymorphism KLKB1-428G/A to the disease phenotype. METHODS: We studied 249 C1-INH-HAE patients from 114 European families, and we explored possible associations of C1-INH-HAE clinical features with carriage of KLKB1-428G/A, combined or not with that of the functional F12-46C/T polymorphism. RESULTS: Carriers of the G allele of the KLKB1-428G/A polymorphism exhibited a significantly delayed disease onset (i.e., by 4.1 years [p < 0.001], depending on the zygocity status), while carriers of both the KLKB1-428G/A and the F12-46C/T polymorphism displayed an 8.8-year delay in disease onset (p < 0.001) and a 64% lower probability of needing long-term prophylactic treatment (p = 0.019). CONCLUSIONS: These findings support our initial hypothesis that functional alterations in genes of proteins involved in bradykinin metabolism and function affect the clinical phenotype and possibly contribute to the pathogenesis of C1-INH-HAE. Given that an earlier onset of symptoms is inversely correlated with the subsequent course of the disease and, eventually, the need for long-term prophylaxis, these polymorphisms may be helpful prognostic biomarkers of disease severity.

Genetic predisposition to bevacizumab-induced hypertension.

OBJECTIVE: Bevacizumab, a monoclonal antibody to VEGF, has shown efficacy in ovarian, cervical and endometrial cancer in addition to several other solid tumors. Serious side effects include hypertension, proteinuria, bowel perforation, and thrombosis. We tested the hypothesis that genetic variation in hypertension-associated genes is associated with bevacizumab-induced hypertension (BIH). METHODS: Patients with solid tumors treated with bevacizumab in combination with other therapy were identified from six clinical trials. Haplotype-tagging (ht) SNPs for 10 candidate genes associated with hypertension were identified through the International Hapmap Project. Germline DNA was genotyped for 103 htSNPs using mass spectrometry. Bevacizumab toxicities were identified from clinical trial reports. Haplotypes were reconstructed from diploid genotyping data and frequencies were compared using standard two-sided statistical tests. RESULTS: The study included 114 patients with breast, lung, ovarian, or other cancers, of whom 38 developed BIH. WNK1, KLKB1, and GRK4 were found to contain single loci associated with BIH. Haplotype analysis of WNK1, KLKB1, and GRK4 identified risk haplotypes in each gene associated with grade 3/4 BIH. A composite risk model was created based on these haplotypes. Patients with the highest risk score were the most likely to develop grade 3/4 BIH (OR=6.45; P=0.005; 95%CI, 1.86-22.39). CONCLUSIONS: We concluded that genetic variation in WNK1, KLKB1, and GRK4 may be associated with BIH. These genes are biologically plausible mediators due to their role in blood pressure control, regulating sodium homeostasis and vascular tone. This preliminary risk model performed better than population-based risk models and when further validated may help risk-stratify patients for BIH prior to initiating therapy.

A Cross-sectional Study of KLKB1 and PRCP Polymorphisms in Patient Samples with Cardiovascular Disease.

Plasma kallikrein formed from prekallikrein (PK) produces bradykinin from kininogens and activates factor XII. Plasma PK is activated by factors αXIIa, ßXIIa, or prolylcarboxypeptidase (PRCP). A cross-sectional investigation determined if there is an association of PRCP and KLKB1 polymorphisms with cardiovascular disease (CVD). DNA was obtained from 2243 individuals from the Prevention of Events with Angiotensin Converting Enzyme trial. Two PRCP SNPs, rs7104980 and rs2298668, and two KLKB1 SNPs, rs3733402 and rs3087505, were genotyped. Logistic regression models were performed for history of diabetes, myocardial infarction, stroke, angina, angiographic coronary disease, CABG, intermittent claudication, percutaneous transluminal coronary angioplasty (PTCA), and transient ischemic attack. The PRCP SNP rs7104980 increased the odds of having a history of PTCA by 21% [odds ratio (OR) = 1.211; 95% confidence intervals (CI) = (1.008, 1.454)]; P = 0.041, but was non-significant after Bonferroni correction. Alternatively, having the G allele for rs3733402 (KLKB1 gene) decreased the odds of having a history of angiographic coronary disease by 24% [OR = 0.759; 95% CI = (0.622, 0.927)]; P = 0.007 that was statistically significant (P < 0.01) after Bonferroni correction for multiple hypothesis testing. When the best-fit model based on the Akaike information criterion controlled for age, weight, gender, hypertension, and history of angina, the G allele of KLKB1 rs3733402 that is associated with less plasma kallikrein activity correlated with reduced history of CVD.

Polymorphisms at the F12 and KLKB1 loci have significant trait association with activation of the renin-angiotensin system.

BACKGROUND: Plasma coagulation Factor XIIa (Hageman factor; encoded by F12) and kallikrein (KAL or Fletcher factor; encoded by KLKB1) are proteases of the kallikerin-kinin system involved in converting the inactive circulating prorenin to renin. Renin is a key enzyme in the formation of angiotensin II, which regulates blood pressure, fluid and electrolyte balance and is a biomarker for cardiovascular, metabolic and renal function. The renin-angiotensin system is implicated in extinction learning in posttraumatic stress disorder. METHODS & RESULTS: Active plasma renin was measured from two independent cohorts- civilian twins and siblings, as well as U.S. Marines, for a total of 1,180 subjects. Genotyping these subjects revealed that the carriers of the minor alleles at the two loci- F12 and KLKB1 had a significant association with reduced levels of active plasma renin. Meta-analyses confirmed the association across cohorts. In vitro studies verified digestion of human recombinant pro-renin by kallikrein (KAL) to generate active renin. Subsequently, the active renin was able to digest the synthetic substrate angiotensinogen to angiotensin-I. Examination of mouse juxtaglomerular cell line and mouse kidney sections showed co-localization of KAL with renin. Expression of either REN or KLKB1 was regulated in cell line and rodent models of hypertension in response to oxidative stress, interleukin or arterial blood pressure changes. CONCLUSIONS: The functional variants of KLKB1 (rs3733402) and F12 (rs1801020) disrupted the cascade of enzymatic events, resulting in diminished formation of active renin. Using genetic, cellular and molecular approaches we found that conversion of zymogen prorenin to renin was influenced by these polymorphisms. The study suggests that the variant version of protease factor XIIa due to the amino acid substitution had reduced ability to activate prekallikrein to KAL. As a result KAL has reduced efficacy in converting prorenin to renin and this step of the pathway leading to activation of renin affords a potential therapeutic target.