Aprotinin (I): Understanding the Role of Host Proteases in COVID-19 and the Importance of Pharmacologically Regulating Their Function.

Proteases are produced and released in the mucosal cells of the respiratory tract and have important physiological functions, for example, maintaining airway humidification to allow proper gas exchange. The infectious mechanism of severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), takes advantage of host proteases in two ways: to change the spatial conformation of the spike (S) protein via endoproteolysis (e.g., transmembrane serine protease type 2 (TMPRSS2)) and as a target to anchor to epithelial cells (e.g., angiotensin-converting enzyme 2 (ACE2)). This infectious process leads to an imbalance in the mucosa between the release and action of proteases versus regulation by anti-proteases, which contributes to the exacerbation of the inflammatory and prothrombotic response in COVID-19. In this article, we describe the most important proteases that are affected in COVID-19, and how their overactivation affects the three main physiological systems in which they participate: the complement system and the kinin-kallikrein system (KKS), which both form part of the contact system of innate immunity, and the renin-angiotensin-aldosterone system (RAAS). We aim to elucidate the pathophysiological bases of COVID-19 in the context of the imbalance between the action of proteases and anti-proteases to understand the mechanism of aprotinin action (a panprotease inhibitor). In a second-part review, titled "Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions", we explain in depth the pharmacodynamics, pharmacokinetics, toxicity, and use of aprotinin as an antiviral drug.

Aprotinin (II): Inhalational Administration for the Treatment of COVID-19 and Other Viral Conditions.

Aprotinin is a broad-spectrum inhibitor of human proteases that has been approved for the treatment of bleeding in single coronary artery bypass surgery because of its potent antifibrinolytic actions. Following the outbreak of the COVID-19 pandemic, there was an urgent need to find new antiviral drugs. Aprotinin is a good candidate for therapeutic repositioning as a broad-spectrum antiviral drug and for treating the symptomatic processes that characterise viral respiratory diseases, including COVID-19. This is due to its strong pharmacological ability to inhibit a plethora of host proteases used by respiratory viruses in their infective mechanisms. The proteases allow the cleavage and conformational change of proteins that make up their viral capsid, and thus enable them to anchor themselves by recognition of their target in the epithelial cell. In addition, the activation of these proteases initiates the inflammatory process that triggers the infection. The attraction of the drug is not only its pharmacodynamic characteristics but also the possibility of administration by the inhalation route, avoiding unwanted systemic effects. This, together with the low cost of treatment (≈2 Euro/dose), makes it a good candidate to reach countries with lower economic means. In this article, we will discuss the pharmacodynamic, pharmacokinetic, and toxicological characteristics of aprotinin administered by the inhalation route; analyse the main advances in our knowledge of this medication; and the future directions that should be taken in research in order to reposition this medication in therapeutics.

Factor XII contact activation can be prevented by targeting 2 unique patches in its epidermal growth factor-like 1 domain with a nanobody.

BACKGROUND: Factor (F)XII triggers contact activation by binding to foreign surfaces, with the epidermal growth factor-like 1 (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 variable domains of a heavy chain-only antibody (VHH). METHODS: FXII variants with glutamine substitutions of 2 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 EGF-1 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 autoactivation of 94%, and prolongation of clot formation in activated partial thromboplastin time 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 VHH "F2" binds the positively charged patches and thereby dose-dependently extends activated partial thromboplastin time clotting times from 29 (±2) to 43 (±3) seconds without disrupting FXII quiescence. CONCLUSION: The 2 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 targets in preventing medical device-induced thrombosis.

Target-based discovery of antagonists of the tick (Rhipicephalus microplus) kinin receptor identifies small molecules that inhibit midgut contractions.

BACKGROUND: A GPCR (G protein-coupled receptor) target-based approach was applied to identify antagonists of the arthropod-specific tick kinin receptor. These small molecules were expected to reproduce the detrimental phenotypic effects that had been observed in Rhipicephalus microplus females when the kinin receptor was silenced by RNA interference. Rhipicephalus microplus, the southern cattle tick, cattle fever tick, or Asian blue tick, is the vector of pathogenic microorganisms causing the deadly bovine babesiosis and anaplasmosis. The widespread resistance to acaricides in tick populations worldwide emphasizes that exploring novel targets for effective tick control is imperative. RESULTS: Fifty-three structural analogs of previously identified tick kinin antagonists were screened in a 'dual-addition' calcium fluorescence assay using a CHO-K1 cell line expressing the tick kinin receptor. Seven molecules were validated as non-cytotoxic antagonists, four of which were partial (SACC-0428764, SACC-0428780, SACC-0428800, and SACC-0428803), and three were full antagonists (SACC-0428799, SACC-0428801, and SACC-0428815). Four of these antagonists (SACC-0428764, SACC-0428780, SACC-0428799, and SACC-0428815) also inhibited the tick midgut contractions induced by the myotropic kinin agonist analog 1728, verifying their antagonistic bioactivity. The small molecules were tested on recombinant human neurokinin (NK) receptors, the one most similar to the invertebrate kinin receptors. Most molecules were inhibitors of the NK1 receptor, except SACC-0412066, a previously identified tick kinin receptor antagonist, which inhibited the NK1 receptor only at the highest concentration tested (25 µm). None of the molecules inhibited the NK3 human receptor. CONCLUSION: Molecules identified through this approach could be useful probes for studying the tick kinin signaling system and midgut physiology. © 2024 The Author(s). Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Delayed plasma kallikrein inhibition fosters post-stroke recovery by reducing thrombo-inflammation.

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.

Increased Prolylcarboxypeptidase Expression Can Serve as a Biomarker of Senescence in Culture.

Prolylcarboxypeptidase (PRCP, PCP, Lysosomal Pro-X-carboxypeptidase, Angiotensinase C) controls angiotensin- and kinin-induced cell signaling. Elevation of PRCP appears to be activated in chronic inflammatory diseases [cardiovascular disease (CVD), diabetes] in proportion to severity. Vascular endothelial cell senescence and mitochondrial dysfunction have consistently been shown in models of CVD in aging. Cellular senescence, a driver of age-related dysfunction, can differentially alter the expression of lysosomal enzymes due to lysosomal membrane permeability. There is a lack of data demonstrating the effect of age-related dysfunction on the expression and function of PRCP. To explore the changes in PRCP, the PRCP-dependent prekallikrein (PK) pathway was characterized in early- and late-passage human pulmonary artery endothelial cells (HPAECs). Detailed kinetic analysis of cells treated with high molecular weight kininogen (HK), a precursor of bradykinin (BK), and PK revealed a mechanism by which senescent HPAECs activate the generation of kallikrein upon the assembly of the HK-PK complex on HPAECs in parallel with an upregulation of PRCP and endothelial nitric oxide (NO) synthase (eNOS) and NO formation. The NO production and expression of both PRCP and eNOS increased in early-passage HPAECs and decreased in late-passage HPAECs. Low activity of PRCP in late-passage HPAECs was associated with rapid decreased telomerase reverse transcriptase mRNA levels. We also found that, with an increase in the passage number of HPAECs, reduced PRCP altered the respiration rate. These results indicated that aging dysregulates PRCP protein expression, and further studies will shed light into the complexity of the PRCP-dependent signaling pathway in aging.

A quantitative systems pharmacology model of plasma kallikrein-kinin system dysregulation in hereditary angioedema.

Hereditary angioedema (HAE) due to C1-inhibitor deficiency is a rare, debilitating, genetic disorder characterized by recurrent, unpredictable, attacks of edema. The clinical symptoms of HAE arise from excess bradykinin generation due to dysregulation of the plasma kallikrein-kinin system (KKS). A quantitative systems pharmacology (QSP) model that mechanistically describes the KKS and its role in HAE pathophysiology was developed based on HAE attacks being triggered by autoactivation of factor XII (FXII) to activated FXII (FXIIa), resulting in kallikrein production from prekallikrein. A base pharmacodynamic model was constructed and parameterized from literature data and ex vivo assays measuring inhibition of kallikrein activity in plasma of HAE patients or healthy volunteers who received lanadelumab. HAE attacks were simulated using a virtual patient population, with attacks recorded when systemic bradykinin levels exceeded 20 pM. The model was validated by comparing the simulations to observations from lanadelumab and plasma-derived C1-inhibitor clinical trials. The model was then applied to analyze the impact of nonadherence to a daily oral preventive therapy; simulations showed a correlation between the number of missed doses per month and reduced drug effectiveness. The impact of reducing lanadelumab dosing frequency from 300 mg every 2 weeks (Q2W) to every 4 weeks (Q4W) was also examined and showed that while attack rates with Q4W dosing were substantially reduced, the extent of reduction was greater with Q2W dosing. Overall, the QSP model showed good agreement with clinical data and could be used for hypothesis testing and outcome predictions.

A fluorescently-tagged tick kinin neuropeptide triggers peristalsis and labels tick midgut muscles.

Ticks are blood-feeding arthropods that require heme for their successful reproduction. During feeding they also acquire pathogens that are subsequently transmitted to humans, wildlife and/or livestock. Understanding the regulation of tick midgut is important for blood meal digestion, heme and nutrient absorption processes and for aspects of pathogen biology in the host. We previously demonstrated the activity of tick kinins on the cognate G protein-coupled receptor. Herein we uncovered the physiological role of the kinin receptor in the tick midgut. A fluorescently-labeled kinin peptide with the endogenous kinin 8 sequence (TMR-RK8), identical in the ticks Rhipicephalus microplus and R. sanguineus, activated and labeled the recombinant R. microplus receptor expressed in CHO-K1 cells. When applied to the live midgut the TMR-RK8 labeled the kinin receptor in muscles while the labeled peptide with the scrambled-sequence of kinin 8 (TMR-Scrambled) did not. The unlabeled kinin 8 peptide competed TMR-RK8, decreasing confocal microscopy signal intensity, indicating TMR-RK8 specificity to muscles. TMR-RK8 was active, inducing significant midgut peristalsis that was video-recorded and evaluated with video tracking software. The TMR-Scrambled peptide used as a negative control did not elicit peristalsis. The myotropic function of kinins in eliciting tick midgut peristalsis was established.

Bradykinin protects nucleus pulposus cells from tert-butyl hydroperoxide-induced damage and delays intervertebral disc degeneration.

Intervertebral disc degeneration (IVDD) is a leading cause of degenerative spinal disorders, involving complex biological processes. This study investigates the role of the kallikrein-kinin system (KKS) in IVDD, focusing on the protective effects of bradykinin (BK) on nucleus pulposus cells (NPCs) under oxidative stress. Clinical specimens were collected, and experiments were conducted using human and rat primary NPCs to elucidate BK's impact on tert-butyl hydroperoxide (TBHP)-induced oxidative stress and damage. The results demonstrate that BK significantly inhibits TBHP-induced NPC apoptosis and restores mitochondrial function. Further analysis reveals that this protective effect is mediated through the BK receptor 2 (B2R) and its downstream PI3K/AKT pathway. Additionally, BK/PLGA sustained-release microspheres were developed and validated in a rat model, highlighting their potential therapeutic efficacy for IVDD. Overall, this study sheds light on the crucial role of the KKS in IVDD pathogenesis and suggests targeting the B2R as a promising therapeutic strategy to delay IVDD progression and promote disc regeneration.

Recent advances in the discovery and development of drugs targeting the kallikrein-kinin system.

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.

Role of kinin receptors in skin pigmentation.

Previous studies have shown that all kinin system is constitutively expressed in the normal and inflamed skin, with a potential role in both physiological and pathological processes. However, the understanding regarding the involvement of the kinin system in skin pigmentation and pigmentation disorders remains incomplete. In this context, the present study was designed to determine the role of kinins in the Monobenzone (MBZ)-induced vitiligo-like model. Our findings showed that MBZ induces higher local skin depigmentation in kinin receptors knockout mice (KOB1R, KOB2R and KOB1B2R) than in wild type (WT). Remarkably, lower levels of melanin content and reduced ROS generation were detected in KOB1R and KOB2R mice treated with MBZ. In addition, both KOB1R and KOB2R show increased dermal cell infiltrate in vitiligo-like skin, when compared to WT-MBZ. Additionally, lack of B1R was associated with greater skin accumulation of IL-4, IL-6, and IL-17 by MBZ, while KOB1B2R presented lower levels of TNF and IL-1. Of note, the absence of both kinin B1 and B2 receptors demonstrates a protective effect by preventing the increase in polymorphonuclear and mononuclear cell infiltrations, as well as inflammatory cytokine levels induced by MBZ. In addition, in vitro assays confirm that B1R and B2R agonists increase intracellular melanin synthesis, while bradykinin significantly enhanced extracellular melanin levels and proliferation of B16F10 cells. Our findings highlight that the lack of kinin receptors caused more severe depigmentation in the skin, as well as genetic deletion of both B1/B2 receptors seems to be linked with changes in levels of constitutive melanin levels, suggesting the involvement of kinin system in crucial skin pigmentation pathways.

Kinin-kallikrein system: New perspectives in heart failure.

Heart failure (HF) is a pervasive clinical challenge characterized by compromised cardiac function and reduced quality of life. The kinin-kallikrein system (KSS), a multifaceted peptide cascade, has garnered substantial attention due to its potential role in HF. Through activation of B1 and/or B2 receptors and downstream signaling, kinins modulate various physiological processes, including inflammation, coagulation, pain, blood pressure control, and vascular permeability. Notably, aberrations in KKS components have been linked to HF risk. The elevation of vasodilatory bradykinin (BK) due to kallikrein activity reduces preload and afterload, while concurrently fostering sodium reabsorption inhibition. However, kallikrein's conversion of prorenin to renin leads to angiotensinsII upregulation, resulting in vasoconstriction and fluid retention, alongside increased immune cell activity that fuels inflammation and cardiac remodeling. Importantly, prolonged KKS activation resulting from volume overload and tissue stretch contributes to cardiac collagen loss. The conventional renin-angiotensin-aldosterone system (RAAS) inhibitors used in HF management may inadvertently intensify KKS activity, exacerbating collagen depletion and cardiac remodeling. It is crucial to balance the KKS's role in acute cardiac damage, which may temporarily enhance function and metabolic parameters against its detrimental long-term effects. Thus, KKS blockade emerges as a promising strategy to impede HF progression. By attenuating the link between immune system function and tissue damage, KKS inhibition can potentially reduce cardiac remodeling and alleviate HF symptoms. However, the nuanced roles of BK in various acute conditions necessitate further investigation into the sustained benefits of kallikrein inhibitors in patients with chronic HF.

Kinin Receptors and Kinin-Related Gene Expression in Astrocytic Brain Tumors.

Kinins are a set of peptides present in tissues that are involved in the inflammatory response and cancer progression. However, studies showing the expression of kinin receptors in human glioma samples are still incomplete and contradictory. The aim of the present study was to ascertain the expression of BDKRB1 and BDKRB2 genes, as well as the level of B1R and B2R proteins in human gliomas, depending on the degree of malignancy. Additionally, representative kinin-dependent genes with altered expression were indicated. The expression profile of kinin-dependent genes was determined using oligonucleotide microarray technique. In addition, RT-qPCR was used to assess the expression level of selected differentiating genes. The location of kinin receptors in brain gliomas was assessed using immunohistochemical methods. The oligonucleotide microarray method was used to identify 12 mRNA IDs of kinin-related genes whose expression was upregulated or downregulated in gliomas of different grades. In immunohistochemically stained samples, the concentrations of BR1 and BR2 proteins, measured by optical density, were statistically significantly higher in grade G3 vs. G2 and G4 vs. G3. Increased expression of kinin receptors BDKRB1 and BDKRB2 in brain gliomas, depending on the degree of malignancy, suggests the involvement of kinins and their receptors in the disease's pathogenesis. Quantitative assessment of mRNA BDKRB1, PRKAR1A, MAP2K, and EGFR in patients with brain tumors may hold diagnostic and therapeutic significance.

Bradykinin produced during Plasmodium falciparum erythrocytic cycle drives monocyte adhesion to human brain microvascular endothelial cells.

Cerebral malaria (CM) pathogenesis is described as a multistep mechanism. In this context, monocytes have been implicated in CM pathogenesis by increasing the sequestration of infected red blood cells to the brain microvasculature. In disease, endothelial activation is followed by reduced monocyte rolling and increased adhesion. Nowadays, an important challenge is to identify potential pro-inflammatory stimuli that can modulate monocytes behavior. Our group have demonstrated that bradykinin (BK), a pro-inflammatory peptide involved in CM, is generated during the erythrocytic cycle of P. falciparum and is detected in culture supernatant (conditioned medium). Herein we investigated the role of BK in the adhesion of monocytes to endothelial cells of blood brain barrier (BBB). To address this issue human monocytic cell line (THP-1) and human brain microvascular endothelial cells (hBMECs) were used. It was observed that 20% conditioned medium from P. falciparum infected erythrocytes (Pf-iRBC sup) increased the adhesion of THP-1 cells to hBMECs. This effect was mediated by BK through the activation of B2 and B1 receptors and involves the increase in ICAM-1 expression in THP-1 cells. Additionally, it was observed that angiotensin-converting enzyme (ACE) inhibitor, captopril, enhanced the effect of both BK and Pf-iRBC sup on THP-1 adhesion. Together these data show that BK, generated during the erythrocytic cycle of P. falciparum, could play an important role in adhesion of monocytes in endothelial cells lining the BBB.

Kinin B1 receptor modulates glucose homeostasis and physical exercise capacity by altering adrenal catecholamine synthesis and secretion.

Our group has shown in several papers that kinin B1 receptor (B1R) is involved in metabolic adaptations, mediating glucose homeostasis and interfering in leptin and insulin signaling. Since catecholamines are involved with metabolism management, we sought to evaluate B1R role in catecholamine synthesis/secretion. Using B1R global knockout mice, we observed increased basal epinephrine content, accompanied by decreased hepatic glycogen content and increased glucosuria. When these mice were challenged with maximal intensity exercise, they showed decreased epinephrine and norepinephrine response, accompanied by disturbed glycemic responses to effort and poor performance. This phenotype was related to alterations in adrenal catecholamine synthesis: increased basal epinephrine concentration and reduced norepinephrine content in response to exercise, as well decreased gene expression and protein content of tyrosine hydroxylase and decreased gene expression of dopamine beta hydroxylase and kinin B2 receptor. We conclude that the global absence of B1R impairs catecholamine synthesis, interfering with glucose metabolism at rest and during maximal exercise.

Functional characterization of the kinin receptor in the Chagas disease vector Rhodnius prolixus; activity of native kinins and potent biostable Aib-containing insect kinin analogs.

The causative agent for Chagas disease, Trypanosoma cruzi, is transmitted to a human host in the urine/feces of the kissing bug, Rhodnius prolixus, following blood feeding. Kinins are important chemical messengers in the overall control of blood feeding physiology in R. prolixus, including hindgut contractions and excretion. Thus, disruption in kinin signaling would have damaging consequences to the insect but also interfere with the transmission of Chagas Disease. Here, a heterologous functional receptor assay was used to confirm the validity of the previously cloned putative kinin G-protein-coupled receptor, RhoprKR, in Rhodnius prolixus. Three native R. prolixus kinins were chosen for analysis; two possessing the typical kinin WGamide C-terminal motif and one that possesses an atypical C-terminal WAamide. All three are potent (EC50 values in the nM range), with high efficacy, on CHO-K1-aeq cells expressing the RhoprKR, thereby confirming ligand binding. Members of three other R. prolixus peptide families, which are also myotropins (tachykinins, pyrokinins and sulfakinins) elicited little or no response. In addition, this heterologous receptor assay was used to test characteristics of kinin mimetics previously tested on tick and mosquito kinin receptors. Five α-aminoisobutyric acid (Aib) containing analogs were tested, and four found to have considerably higher potencies than the native kinins, with EC50 values in the pM range. Interestingly, adding Aib to the atypical WAamide kinin improves its EC50 value from 2 nM to 39 pM. Biostable kinin analogs may prove useful leads for novel pest control strategies. Since T. cruzi is transmitted to a human host in the urine/feces after blood feeding, disruption in kinin signaling would also interfere with the transmission of Chagas Disease.

Recessive SERPING1 Variant Leads to Kinin-Kallikrein System Control Failure in a Consanguineous Brazilian Family with Hereditary Angioedema.

Background: Hereditary angioedema (HAE) is a severe and potentially life-threatening disease. The most common forms are caused by variants in SERPING1, resulting in C1-inhibitor (C1-INH) deficiency (HAE-C1-INH). C1-INH is a serine protease inhibitor (SERPIN) that regulates multiple proteases pathways, including the kallikrein-kinin system (KKS) and its complement. In HAE-C1-INH patients, C1-INH deficiencies affect KKS control, resulting in the development of kallikrein activity in plasma and the subsequent release of bradykinin (BK). While the overwhelming majority of disease-causing SERPING1 variants are dominant, very few recessive variants have been described. We present a large Brazilian HAE-C1-INH family with a recessive form of HAE-C1-INH. Methods: Blood samples of family members were investigated for protein levels of C1-INH, C4, C1q, and C1-INH function. The SERPING1 gene was sequenced. Results: In two severely affected sisters, we identified a homozygous missense variant in SERPING1 (NM_000062.3:c.964G>A;p.Val322Met). Fourteen family members were asymptomatic heterozygous carriers of the variant. Data regarding C1-INH function in the plasma showed that homozygous p.Val322Met strongly impacts C1-INH function to inhibit C1s and kallikrein (PKa). When heterozygously expressed, it affects the C1-INH control of C1s more than that of PKa. Conclusions: These studies of the variant's effects on the structure-function relationship reinforce prior observations suggesting that C1-INH deficiency is a conformational disease.

Recombinant C1 inhibitor in the prevention of severe COVID-19: a randomized, open-label, multi-center phase IIa trial.

Background: Conestat alfa (ConA), a recombinant human C1 inhibitor, may prevent thromboinflammation. Methods: We conducted a randomized, open-label, multi-national clinical trial in which hospitalized adults at risk for progression to severe COVID-19 were assigned in a 2:1 ratio to receive either 3 days of ConA plus standard of care (SOC) or SOC alone. Primary and secondary endpoints were day 7 disease severity on the WHO Ordinal Scale, time to clinical improvement within 14 days, and safety, respectively. Results: The trial was prematurely terminated because of futility after randomization of 84 patients, 56 in the ConA and 28 in the control arm. At baseline, higher WHO Ordinal Scale scores were more frequently observed in the ConA than in the control arm. On day 7, no relevant differences in the primary outcome were noted between the two arms (p = 0.11). The median time to defervescence was 3 days, and the median time to clinical improvement was 7 days in both arms (p = 0.22 and 0.56, respectively). Activation of plasma cascades and endothelial cells over time was similar in both groups. The incidence of adverse events (AEs) was higher in the intervention arm (any AE, 30% with ConA vs. 19% with SOC alone; serious AE, 27% vs. 15%; death, 11% vs. 0%). None of these were judged as being related to the study drug. Conclusion: The study results do not support the use of ConA to prevent COVID-19 progression. Clinical trial registration: https://clinicaltrials.gov, identifier NCT04414631.

Pathophysiology of bradykinin and histamine mediated angioedema.

Angioedema is characterized by swelling localized to the subcutaneous and submucosal tissues. This review provides an overview of angioedema, including the different types, triggers, and underlying pathophysiologic mechanisms. Hereditary and acquired angioedema are caused by dysregulation of the complement and kinin pathways. In contrast, drug-induced and allergic angioedema involve the activation of the immune system and release of vasoactive mediators. Recent advances in the understanding of the pathophysiology of angioedema have led to the development of targeted therapies, such as monoclonal antibodies, bradykinin receptor antagonists, and complement inhibitors, which promise to improve clinical outcomes in patients with this challenging condition. To accurately diagnose and manage angioedema, an understanding of this condition's complex and varied pathophysiology is both necessary and critical.

Prevention of Inflammation, Neovascularization, and Retinal Dysfunction by Kinin B1 Receptor Antagonism in a Mouse Model of Age-Related Macular Degeneration.

The kallikrein-kinin system (KKS) contributes to vascular inflammation and neovascularization in age-related macular degeneration (AMD), particularly via the kinin B1 receptor (B1R). The aim of the present study was to determine the protective effects of the topical administration of the B1R antagonist (R-954) on inflammation, neovascularization, and retinal dysfunction in a murine model of neovascular AMD. Choroidal neovascularization (CNV) was induced in C57BL6 mice using an argon laser. A treatment with ocular drops of R-954 (100 µg/15 µL, twice daily in both eyes), or vehicle, was started immediately on day 0, for 7, 14, or 21 days. CNV, invasive microglia, and B1R immunoreactive glial cells, as well as electroretinography alterations, were observed within the retina and choroid of the CNV group but not in the control group. The staining of B1R was abolished by R-954 treatment as well as the proliferation of microglia. R-954 treatment prevented the CNV development (volume: 20 ± 2 vs. 152 ± 5 × 104 µm3 in R-954 vs. saline treatment). R-954 also significantly decreased photoreceptor and bipolar cell dysfunction (a-wave amplitude: -47 ± 20 vs. -34 ± 14 µV and b-wave amplitude: 101 ± 27 vs. 64 ± 17 µV in R-954 vs. saline treatment, day 7) as well as angiogenesis tufts in the retina. These results suggest that self-administration of R-954 by eye-drop treatment could be a promising therapy in AMD to preserve retinal health and vision.