Patterns of C1-Inhibitor/Plasma Serine Protease Complexes in Healthy Humans and in Hereditary Angioedema Patients.

C1-inhibitor (C1-INH) is an important regulator of the complement, coagulation, fibrinolytic and contact systems. The quantity of protease/C1-INH complexes in the blood is proportional to the level of the in vivo activation of these four cascade-like plasma enzyme systems. Parallel determination of C1-INH-containing activation complexes could be important to understand the regulatory role of C1-INH in diseases such as hereditary angioedema (HAE) due to C1-INH deficiency (C1-INH-HAE). We developed in-house ELISAs to measure the concentration of complexes of C1-INH formed with active proteases: C1r, C1s, MASP-1, MASP-2, plasma kallikrein, factor XIIa, factor XIa, and thrombin, as well as to determine total and functionally active C1-INH. We measured the concentration of the complexes in EDTA plasma from 6 healthy controls, from 5 with type I and 5 with type II C1-INH-HAE patients during symptom-free periods and from five patients during HAE attacks. We also assessed the concentration of these complexes in blood samples taken from one C1-INH-HAE patient during the kinetic follow-up of a HAE attack. The overall pattern of complexed C1-INH was similar in controls and C1-INH-HAE patients. C1-INH formed the highest concentration complexes with C1r and C1s. We observed higher plasma kallikrein/C1-INH complex concentration in both type I and type II C1-INH-HAE, and higher concentration of MASP-1/C1-INH, and MASP-2/C1-INH complexes in type II C1-INH-HAE patients compared to healthy controls and type I patients. Interestingly, none of the C1-INH complex concentrations changed significantly during HAE attacks. During the kinetic follow-up of an HAE attack, the concentration of plasma kallikrein/C1-INH complex was elevated at the onset of the attack. In parallel, C1r, FXIIa and FXIa complexes of C1-INH also tended to be elevated, and the changes in the concentrations of the complexes followed rather rapid kinetics. Our results suggest that the complement classical pathway plays a critical role in the metabolism of C1-INH, however, in C1-INH-HAE, contact system activation is the most significant in this respect. Due to the fast changes in the concentration of complexes, high resolution kinetic follow-up studies are needed to clarify the precise molecular background of C1-INH-HAE pathogenesis.

Hereditary angioedema attack: what happens to vasoactive mediators?

Hereditary angioedema is a disabling, life-threatening condition caused by deficiency (type I) or dysfunction (type II) of the C1 inhibitor protein (C1-INH-HAE) leading to bradykinin accumulation and recurrent episodes of edema attack. Vascular leakage is a complex process sustained by the coordinated production of several permeabilizing factors including vascular endothelial growth factors (VEGFs), angiopoietins (ANGPTs) and phospholipase A2 enzymes (PLA2). We previously reported that patients with C1-INH-HAE in remission have increased plasma levels of VEGFs, ANGPTs and secreted PLA2. In this study, we sought to analyze plasma levels of these mediators in 15 patients with C1-INH-HAE during the acute attack compared to remission. Plasma concentrations of VEGF-A, VEGF-C and VEGF-D were not altered during attack compared to remission. Moreover, VEGF-D concentrations were not altered also in remission phase compared to controls. Concentrations of ANGPT1, a vascular stabilizer, were increased during attacks compared to symptoms-free periods, whereas ANGPT2 levels were not altered. The ANGPT2/ANGPT1 ratio was decreased during angioedema attacks. Platelet activating factor acetylhydrolase activity was increased in patients with C1-INH-HAE in remission compared to controls and was decreased during angioedema attacks. Our results emphasize the complexity by which several vasoactive mediators are involved not only in the pathophysiology of C1-INH-HAE, but also during angioedema attacks and its resolution.

Increased fibrinolysis-induced bradykinin formation in hereditary angioedema confirmed using stored plasma and biotechnological inhibitors.

OBJECTIVE: We recently investigated the pathways of immunoreactive bradykinin (iBK) formation in fresh blood of normal volunteers and of patients with hereditary angioedema due to C1-esterase inhibitor deficiency (HAE-1/-2). Herein, we adapted the techniques to small volumes (200 µl) of previously frozen citrated plasma and further analyzed the mechanisms of iBK formation with additional biotechnological inhibitors. RESULTS: Measurable iBK formation was observed under stimulation with tissue kallikrein (KLK-1, 10 nM), the particulate material Kontact-APTT (concentration reduced to 2% v/v) or recombinant tissue plasminogen activator (tPA, 169 nM), with little background in unstimulated plasma incubated for up to 2 h. Plasma samples from HAE-1/-2 patients responded earlier to tPA than those from controls, as previously reported with whole blood. Lanadelumab inhibited iBK formation induced by Kontact-APTT and tPA. A highly specific plasmin inhibitor, DX-1000, abolished tPA-induced iBK formation in plasma but had no effect against Kontact-APTT, confirming the role of fibrinolysis in tPA-induced kinin formation. The anti-lanadelumab neutralizing antibody M293-D02 reversed the inhibitory effects of lanadelumab. Frozen plasma is a suitable material for measuring iBK formation kinetics, with possible applications such as investigating the effect of rare disease states on the kallikrein-kinin system and monitoring the effect of HAE prophylactic treatments.

Hereditary angioedema with C1 inhibitor (C1-INH) deficit: the strength of recognition (51 cases).

Hereditary angioedema (HAE) is a rare autosomal dominant disease due to C1 esterase inhibitor deficiency (C1-INH). The disease is characterized by subcutaneous and submucosal edema in the absence of urticaria due to the accumulation of bradykinin. This descriptive study aimed to evaluate the clinical characteristics of patients with a confirmed diagnosis of HAE referred to our Outpatient Clinic between December 2009 and November 2017. Fifty-one patients (38 F, 13 M) with a mean age of 32 years (range: 7-70 y) were included. Family history of HAE was reported in 70% (36/51) of the cases; 33/46 patients became symptomatic by 18 years of age. The median time between onset of symptoms and diagnosis was 13 years (3 mo-50 y). The most frequent triggering factors for attacks were stress (74.4%), trauma (56.4%), and hormonal variations (56%). The main symptoms were subcutaneous edema in 93.5% (43/46) of patients, gastrointestinal symptoms in 84.8% (39/46), and obstruction in the upper airways in 34.8% (16/46). Hospitalization occurred in 65.2%, of whom 13.3% had to be transferred to the Intensive Care Unit. Prophylactic treatment was instituted in 87% (40/46) of patients, and 56.5% (26/46) required additional treatment to control attacks. Owing to our data collection over a period of 8 years, a significant number of patients were identified by this HAE reference center. Despite early recognition and prophylactic treatment, a high percentage of patients were hospitalized. HAE is still diagnosed late, reinforcing the need for more reference centers specialized in diagnosis and educational projects for health professionals.

Hereditary angioedema: the plasma contact system out of control.

The plasma contact system contributes to thrombosis in experimental models. Even though our standard blood coagulation tests are prolonged when plasma lacks contact factors, this enzyme system appears to have a minor (if any) role in hemostasis. In this review, we explore the clinical phenotype of C1 esterase inhibitor (C1-INH) deficiency. C1-INH is the key plasma inhibitor of the contact system enzymes, and its deficiency causes hereditary angioedema (HAE). This inflammatory disorder is characterized by recurrent aggressive attacks of tissue swelling that occur at unpredictable locations throughout the body. Bradykinin, which is considered to be a byproduct of the plasma contact system during in vitro coagulation, is the main disease mediator in HAE. Surprisingly, there is little evidence for thrombotic events in HAE patients, suggesting mechanistic uncoupling from the intrinsic pathway of coagulation. In addition, it is questionable whether a surface is responsible for contact system activation in HAE. In this review, we discuss the clinical phenotype, disease modifiers and diagnostic challenges of HAE. We subsequently describe the underlying biochemical mechanisms and contributing disease mediators. Furthermore, we review three types of HAE that are not caused by C1-INH inhibitor deficiency. Finally, we propose a central enzymatic axis that we hypothesize to be responsible for bradykinin production in health and disease.

Extremely Delayed Diagnosis of Type II Hereditary Angioedema: Case Report and Review of the Literature.

We present a case with extremely late diagnosis of type II hereditary angioedema (HAE). Given recent advances in HAE treatment, we want to bring physician awareness to this condition and aid in earlier detection. HAE is a disorder associated with episodes of angioedema of the face, larynx, lips, abdomen, or extremities. Late diagnosis of HAE can lead to significant morbidity and is severely impairing due to recurring attacks. The diagnosis of HAE is ordinarily made during childhood and adolescence. Delayed diagnoses in early and middle adulthood have been documented in the literature. Gastrointestinal symptoms are common features of HAE and can be misdiagnosed as disease of primary gastrointestinal pathology, such as irritable bowel syndrome, recurrent pancreatitis, or appendicitis. These attacks are characterized by recurrent attacks of subcutaneous and submucosal edema without the presence of urticaria.We present a case of an elderly veteran whose diagnoses was extremely delayed into the eighth decade of life subsequent to unexplained abdominal symptoms. After diagnosis, the patient's symptoms were well controlled with medication due to advances in HAE treatment. To prevent further atypically delayed diagnoses, physicians should consider HAE in patients with recurrent attacks of unexplained abdominal pain.

Hereditary angioedema with C1 inhibitor (C1-INH) deficit: the strength of recognition (51 cases)

Hereditary angioedema (HAE) is a rare autosomal dominant disease due to C1 esterase inhibitor deficiency (C1-INH). The disease is characterized by subcutaneous and submucosal edema in the absence of urticaria due to the accumulation of bradykinin. This descriptive study aimed to evaluate the clinical characteristics of patients with a confirmed diagnosis of HAE referred to our Outpatient Clinic between December 2009 and November 2017. Fifty-one patients (38 F, 13 M) with a mean age of 32 years (range: 7-70 y) were included. Family history of HAE was reported in 70% (36/51) of the cases; 33/46 patients became symptomatic by 18 years of age. The median time between onset of symptoms and diagnosis was 13 years (3 mo-50 y). The most frequent triggering factors for attacks were stress (74.4%), trauma (56.4%), and hormonal variations (56%). The main symptoms were subcutaneous edema in 93.5% (43/46) of patients, gastrointestinal symptoms in 84.8% (39/46), and obstruction in the upper airways in 34.8% (16/46). Hospitalization occurred in 65.2%, of whom 13.3% had to be transferred to the Intensive Care Unit. Prophylactic treatment was instituted in 87% (40/46) of patients, and 56.5% (26/46) required additional treatment to control attacks. Owing to our data collection over a period of 8 years, a significant number of patients were identified by this HAE reference center. Despite early recognition and prophylactic treatment, a high percentage of patients were hospitalized. HAE is still diagnosed late, reinforcing the need for more reference centers specialized in diagnosis and educational projects for health professionals.

Endocan: A Novel Marker of Endothelial Dysfunction in C1-Inhibitor-Deficient Hereditary Angioedema.

BACKGROUND: Hereditary angioedema (HAE) related to C1-inhibitor deficiency is a rare autosomal dominant disorder. Vascular cell adhesion molecules (VCAM) are known as endothelial activation markers. Endocan (also called ESM-1) is proposed as an endothelial dysfunction indicator. We aimed to investigate endothelial activation in attack-free periods in HAE patients by measuring their levels of endocan and VCAM-1. METHODS: Twenty-six HAE patients (22 female, mean age 40 ± 13 years) and 38 healthy control patients (13 female, mean age 36.9 ± 12 years) were included in the study. Peripheral blood samples were collected from HAE patients during symptom-free periods and control subjects. Endocan and VCAM-1 levels were measured using the enzyme-linked immunosorbent assay method. RESULTS: The median serum levels of endocan (647 ± 101 ng/mL) and VCAM-1 (500 ± 79 ng/mL) in the HAE patients were significantly higher than in the control patients (391 ± 41 and 325 ± 4; p < 0.001 for both). CONCLUSION: The increased endocan and VCAM-1 levels may reflect an endothelial activation even in attack-free periods in HAE patients.

2D-LC-MS/MS to measure cleaved high-molecular-weight kininogen in human plasma as a biomarker for C1-INH-HAE.

AIM: C1-INH-HAE is caused by activation of plasma kallikrein which subsequently cleaves high-molecular-weight kininogen (HMWK) to generate bradykinin and cHMWK. MATERIALS & METHODS: A novel ion-pair 2D LC-MS/MS assay was developed to measure the 46 kDa cHMWK in plasma as a biomarker for C1-INH-HAE. The sample preparation included sodium dodecyl sulfate denaturation, methanol crash, chymotryptic digestion and peptide enrichment by solid phase extraction. RESULTS: The LLOQ was 200 ng/ml. The overall cHMWK recovery combining crash and digestion was 57.5%. The precision of the method was ≤12.7% and accuracy ≤-13.8%. CONCLUSION: A reagent-free LC-MS assay has been developed for the quantitation of 46 kDa cHMWK, which was shown to be elevated in plasma of C1-INH-HAE patients due to C1-INH deficiency relative to that of healthy subjects.

Glucocorticoid receptor gene polymorphisms in hereditary angioedema with C1-inhibitor deficiency.

BACKGROUND: Hereditary angioedema caused by C1-inhibitor deficiency (C1-INH-HAE) is a rare, autosomal dominant disorder. C1-INH-HAE is characterized by edema-formation, which may occur in response to stress. The individual's response to stress stimuli is partly genetically determined. Activation of the hypothalamic-pituitary-adrenal axis results in the release of cortisol. In turn, the secreted gluco- and mineralocorticoids affect the metabolism, as well as the cardiovascular and immune systems. We hypothesized that changes in serum cortisol level and polymorphisms of the glucocorticoid receptor (GR) modify the individual sensitivity to stressor stimuli of C1-INH-HAE patients. RESULTS: We compared the response to stress with Rahe's Brief Stress and Coping Inventory of 43 C1-INH-HAE patients, 18 angioedema patients and 13 healthy controls. 139 C1-INH-HAE patients and 160 healthy controls were genotyped for glucocorticoid receptor polymorphisms BclI, N363S and A3669G. Serum cortisol levels were determined during attacks and during symptom-free periods in 36 C1-INH-HAE patients. The relationships between clinical, laboratory data and GR SNPs (Single Nucleotide Polymorphisms) were assessed using ANOVA. C1-INH-HAE patients have decreased coping capabilities compared to healthy controls. Cortisol levels were significantly higher during attacks than in symptom-free periods (p = 0.004). The magnitude of the elevation of cortisol levels did not show a significant correlation with any clinical or laboratory data. Among the C1-INH-HAE patients, the carriers of the A3669G allele had significantly lower cortisol levels, and increased body mass index compared with non-carriers. CONCLUSIONS: The higher cortisol level observed during attacks may reflect the effect of a stressful situation (such as of the attack itself), on the patients' neuroendocrine system. In A3669G carriers, the lower cortisol levels might reflect altered feedback to the hypothalamic-pituitary-adrenal axis, due to decreased sensitivity to glucocorticoids.

Thyroid hormones and complement parameters in hereditary angioedema with C1-inhibitor deficiency.

BACKGROUND: Thyroid hormones control and up-regulate the synthesis of many plasma proteins. OBJECTIVE: To explore possible associations between thyroid hormone and complement levels in patients with hereditary angioedema resulting from the deficiency of the C1-inhibitor (C1-INH-HAE). METHODS: In this case-control study, serum thyrotropin, free triiodothyronine (FT3), and free thyroxine (FT4) levels, anti-thyroid peroxidase and antithyroglobulin antibody titers, and C1-INH concentrations were measured in 117 euthyroid patients with C1-INH-HAE and compared with their clinical properties. The control group comprised 150 healthy, age- and sex-matched, euthyroid individuals. RESULTS: The thyrotropin and antithyroglobulin levels were similar between the patients and the controls. Significantly lower FT3 (P < .001) and FT4 (P = .002) levels, as well as higher anti-thyroid peroxidase titers (P < .001), were seen in the patients with C1-INH-HAE. The proportion of patients with reduced C1-INH activity was greater among those with below-median FT4 levels than among those with above-median values (P = .02). Patients who experienced more edematous attacks per year had lower FT4 levels (within the normal range) than those afflicted by fewer episodes (P = .01). The FT3 and FT4 levels were significantly higher in patients undergoing long-term danazol therapy than in those who did not receive this drug (P = .01 and P = .02, respectively). The proportion of patients with FT4 levels in the below-median range was higher in the subset with increased d-dimer concentration (P = .009). CONCLUSION: Minor variations of the thyroid hormone levels (within the reference range) can influence the function of C1-INH in C1-INH-HAE. Our findings suggest a role for the endocrine system in the pathophysiology of C1-INH-HAE.

Elevated plasma levels of vascular permeability factors in C1 inhibitor-deficient hereditary angioedema.

BACKGROUND: Hereditary angioedema with C1 inhibitor deficiency (C1-INH-HAE) is a rare inherited genetic disease characterized by recurrent swelling episodes of the skin, gastrointestinal tract, and upper airways. Angioedema attacks result from increased vascular permeability due to the release of bradykinin from high molecular weight kininogen. Currently, there are no biomarkers predicting the frequency of angioedema attacks. Vascular permeability is modulated by several factors, including vascular endothelial growth factors (VEGFs) and angiopoietins (Angs). As increased circulating levels of VEGFs and Angs have been observed in diseases associated with higher vascular permeability (e.g., systemic capillary leak syndrome and sepsis), we sought to analyze plasma concentrations of VEGFs and Angs in patients with C1-INH-HAE. METHODS: Sixty-eight healthy controls and 128 patients with C1-INH-HAE were studied. Concentrations of angiogenic (VEGF-A, Ang1, Ang2), anti-angiogenic (VEGF-A165b ) and lymphangiogenic (VEGF-C) factors were evaluated by ELISA. C1-INH functional activity was assessed by EIA. RESULTS: Plasma concentrations of VEGF-A, VEGF-C, Ang1, and Ang2 were higher in patients with C1-INH-HAE in remission than in healthy controls. Concentration of VEGF-A was further increased in patients with lower C1-INH functional activity. Patients with C1-INH-HAE experiencing more than 12 angioedema attacks per year were characterized by higher plasma levels of VEGF-A, VEGF-C, and Ang2 compared with the other patients. CONCLUSIONS: We hypothesize that VEGFs and Angs induce a state of 'vascular preconditioning' that may predispose to angioedema attacks. In addition, the identification of increased plasma levels of VEGFs and Angs in patients with C1-INH-HAE may prompt the investigation of VEGFs and Angs as biomarkers of C1-INH-HAE severity.

Elevated D-dimers in attacks of hereditary angioedema are not associated with increased thrombotic risk.

BACKGROUND: Recommended management of attacks of hereditary angioedema (HAE) due to C1 esterase inhibitor (C1-INH) deficiency (C1-INH-HAE) includes therapy with exogenous C1INH. Thrombotic/thromboembolic events (TEE) have been reported with plasma-derived C1INH, but so far none with recombinant human C1INH (rhC1INH). This phase III, randomized, placebo (saline)-controlled study evaluated the safety of rhC1INH 50 IU/kg for the treatment of acute attacks in 74 patients with C1-INH-HAE. METHODS: Monitoring for TEE and assessment of risk of deep vein thrombosis (DVT) by the Wells prediction rule were performed, and levels of fibrin degradation products (plasma D-dimers) were assessed before study drug administration (baseline), 2 h, and 7 days posttreatment. RESULTS: Plasma D-dimer levels were elevated in 80% of the patients (median [25th-75th percentiles]: 2149 [480-5105] µg/l; normal ≤250 µg/l) and were higher in patients with submucosal (abdominal, oropharyngeal-laryngeal) attacks (3095 [890-10000] µg/l; n = 29) compared with subcutaneous (peripheral, facial) attacks (960 [450-4060] µg/l; n = 35). Median plasma D-dimer levels were comparable across treatment groups at baseline (1874 [475-4568] µg/l rhC1INH; 2259 [586-7533] µg/l saline) and 2 h postinfusion (2389 [760-4974] µg/l rhC1INH; 2550 [310-8410] µg/l saline); median plasma D-dimer levels were decreased by Day 7 in both groups (425 [232-3240] µg/l rhC1INH; 418 [246-2318] µg/l saline). No increased risk of DVT was identified, nor any TEE reported in rhC1INH treated or controls. CONCLUSION: Elevated plasma D-dimer levels were associated with acute C1-INH-HAE attacks, particularly with submucosal involvement. However, rhC1INH therapy was not associated with thrombotic events.

Enzymatic assays for the diagnosis of bradykinin-dependent angioedema.

BACKGROUND: The kinins (primarily bradykinin, BK) represent the mediators responsible for local increase of vascular permeability in hereditary angioedema (HAE), HAE I-II associated with alterations of the SERPING1 gene and HAE with normal C1-Inhibitor function (HAE-nC1INH). Besides C1-Inhibitor function and concentration, no biological assay of kinin metabolism is actually available to help physicians for the diagnosis of angioedema (AE). We describe enzymatic tests on the plasma for diagnosis of BK-dependent AE. METHODS: The plasma amidase assays are performed using the Pro-Phe-Arg-p-nitroanilide peptide substrate to evaluate the spontaneous amidase activity and the proenzyme activation. We analyzed data of 872 patients presenting with BK-dependent AE or BK-unrelated diseases, compared to 303 controls. Anti-high MW kininogen (HK) immunoblot was achieved to confirm HK cleavage in exemplary samples. Reproducibility, repeatability, limit of blank, limit of detection, precision, linearity and receiver operating characteristics (ROC) were used to calculate the diagnostic performance of the assays. RESULTS: Spontaneous amidase activity was significantly increased in all BK-dependent AE, associated with the acute phase of disease in HAE-nC1INH, but preserved in BK-unrelated disorders. The increase of the amidase activity was associated to HK proteolysis, indicating its relevance to identify kininogenase activity. The oestrogens, known for precipitating AE episodes, were found as triggers of enzymatic activity. Calculations from ROC curves gave the optimum diagnostic cut-off for women (9.3 nmol⋅min(-1)⋅mL(-1), area under curve [AUC] 92.1%, sensitivity 80.0%, and specificity 90.1%) and for men (6.6 nmol·min(-1)⋅mL(-1), AUC 91.0%, sensitivity 87.0% and specificity 81.2%). CONCLUSION: The amidase assay represents a diagnostic tool to help physicians in the decision to distinguish between BK-related and -unrelated AE.

Population pharmacokinetics of recombinant human C1 inhibitor in patients with hereditary angioedema.

AIMS: To characterize the pharmacokinetics (PK) of recombinant human C1 inhibitor (rhC1INH) in healthy volunteers and hereditary angioedema (HAE) patients. METHODS: Plasma levels of C1INH following 294 administrations of rhC1INH in 133 subjects were fitted using nonlinear mixed-effects modelling. The model was used to simulate maximal C1INH levels for the proposed dosing scheme. RESULTS: A one-compartment model with Michaelis-Menten elimination kinetics described the data. Baseline C1INH levels were 0.901 [95% confidence interval (CI): 0.839-0.968] and 0.176 U ml(-1) (95% CI: 0.154-0.200) in healthy volunteers and HAE patients, respectively. The volume of distribution of rhC1INH was 2.86 l (95% CI: 2.68-3.03). The maximal rate of elimination and the concentration corresponding to half this maximal rate were 1.63 U ml(-1) h(-1) (95% CI: 1.41-1.88) and 1.60 U ml(-1) (95% CI: 1.14-2.24), respectively, for healthy volunteers and symptomatic HAE patients. The maximal elimination rate was 36% lower in asymptomatic HAE patients. Peak C1INH levels did not change upon repeated administration of rhC1INH. Bodyweight was found to be an important predictor of the volume of distribution. Simulations of the proposed dosing scheme predicted peak C1INH concentrations above the lower level of the normal range (0.7 U ml(-1)) for at least 94% of all patients. CONCLUSIONS: The population PK model for C1INH supports a dosing scheme on a 50 U kg(-1) basis up to 84 kg, with a fixed dose of 4200 U above 84 kg. The PK of rhC1INH following repeat administration are consistent with the PK following the first administration.

Hereditary angioedema nationwide study in Slovenia reveals four novel mutations in SERPING1 gene.

Hereditary angioedema (HAE) is a rare autosomal dominant disease characterized by swelling of the face, lips, tongue, larynx, genitalia, or extremities, with abdominal pain caused by intra-abdominal edema. HAE is caused by mutations affecting the C1 inhibitor gene, SERPING1, resulting in low levels of C1 inhibitor (Type I HAE) or normal levels of ineffective C1 inhibitor (Type II HAE). A nationwide survey identified nine unrelated families with HAE in Slovenia, among whom 17 individuals from eight families were recruited for genetic analyses. A diagnosis of HAE was established in the presence of clinical and laboratory criteria (low C1 inhibitor antigenic levels and/or function), followed up by a positive family history. Genetic studies were carried out using PCR and sequencing to detect SERPING1 mutations in promoter, noncoding exon 1, the 7 coding exons, and exon-intron boundaries. Multiplex ligation-dependent probe amplification was performed in order to search for large deletions/duplications in SERPING1 gene. A mutation responsible for HAE was identified in patients from seven families with the disease. In HAE type I families, one previously reported substitution (Gln67Stop, c.265C>T) and four novel mutations were identified. The new mutations included two missense substitutions, Ser128Phe (c.449C>T), and Glu429Lys (c.1351G>A), together with two frameshift mutations, indel (c.49delGinsTT) and deletion (c.593_594delCT). Both families with HAE type II harbored the two well-known substitutions affecting the arginyl residue at the reactive center in exon 8, Arg444Cys (c.1396C>T) and Arg444His (c.1397G>A), respectively. In one patient only the homozygous variant g.566T>C (c.-21T>C) was identified. Our study identified four novel mutations in the Slovenian HAE population, highlighting the heterogeneity of mutations in the SERPING1 gene causing C1 inhibitor deficiency and HAE. In a single patient with HAE a homozygous variant g.566T>C (c.-21T>C) might be responsible for the disease.

The role of ficolins and MASPs in hereditary angioedema due to C1-inhibitor deficiency.

BACKGROUND AND OBJECTIVE: Hereditary angioedema due to C1-inhibitor deficiency (HAE-C1-INH) causes disturbances in the complement system. However, the influence of HAE-C1-INH on the lectin pathway of complement is unresolved. Thus, we studied the main initiator molecules, enzymes and regulators in the lectin pathway in patients with HAE-C1-INH. METHODS: The serum concentrations of ficolin-2, ficolin-3, MBL, MASP-2, MASP-3, and MAP-1 were measured during symptom-free periods in 91 patients with HAE-C1-INH, and in 100 healthy controls using sandwich ELISAs. RESULTS: Compared with controls, the levels of ficolin-2 (p<0.0001) and MASP-2 (p=0.0238) were reduced, while the levels of MBL and MASP-3 were elevated (p=0.0028 and p<0.0001, respectively) in HAE-C1-INH patients. Ficolin-3 and MAP-1 levels did not differ significantly between the two groups. Ficolin-2 correlated with MASP-3 in patients (r=0.3443, p=0.0008), while these parameters showed an opposite relationship in controls (r=-0.4625, p<0.0001). In the patients, ficolin-3 correlated with MASP-2 (r=0.3698, p=0.001). Ficolin-2, -3, and MAP-1 correlated negatively with the annual requirement of plasma derived C1-INH concentrate (r=-0.2863, p=0.0059; r=-0.2654, p=0.0110 and r=-0.2501, p=0.0168, respectively). Ficolin-3 showed a negative correlation with the annual number of attacks (r=-0.2478, p=0.0179). CONCLUSIONS: We found significant differences between patients and controls in the levels of some of the molecules belonging to the lectin complement pathway. Low concentrations of particularly ficolin-2 and -3 were inversely correlated with the severity of HAE-C1-INH, while this was not observed for MBL. This suggests a previously unrecognized involvement of the ficolin-dependent lectin complement pathway in the pathophysiology of HAE-C1-INH.

Hereditary and acquired complement component 1 esterase inhibitor deficiency: a review for the hematologist.

Hereditary angioedema (HAE), a rare autosomal dominant disorder, was first described in the late 19th century. The disease remained poorly understood and without therapeutic options until the latter half of the 20th century. Advances in the understanding of immunologic and hematologic pathways have shed light on HAE, a disease characterized by painful and unpredictable recurrent attacks of nonpitting edema without urticaria. Recognition that a deficiency of complement component 1 (C1) esterase inhibitor leads to overproduction of vasoactive kinins that cause angioedema paved the way for the development of early treatments. Increased understanding of the role of bradykinin in hereditary and acquired forms of C1 esterase inhibitor deficiency has led to the development of more targeted treatments for this painful, debilitating and potentially life-threatening disease.

Alterations of coagulation and fibrinolysis in patients with angioedema due to C1-inhibitor deficiency.

Patients with functional deficiency of C1-inhibitor (C1-INH) suffer from recurrent acute attacks (AA) of localized oedema associated with activation of the contact system, complement and fibrinolysis. To unravel further the role of coagulation and fibrinolysis in the pathophysiology of C1-INH deficiency, we performed simultaneous thrombin and plasmin generation measurements in plasma from patients with hereditary angioedema (HAE) due to C1-INH deficiency during AA (n = 23), in remission (R) (n = 20) and in controls (n = 20). During AA thrombin generation after in-vitro activation of plasma was higher than in controls, as demonstrated by shorter thrombin peak-time (P < 0·05), higher thrombin peak-height (P < 0·001) and increased area under the curve (AUC) (P < 0·05). Additionally, elevated levels of prothrombin fragment 1+2 (P < 0·0001) were observed in non-activated plasma from the same patients. In contrast, in activated plasma from patients during AA plasmin generation estimated as plasmin peak-height (P < 0·05) and plasmin potential (P < 0·05) was reduced, but non-activated plasma of the same patients showed elevated plasmin-anti-plasmin (PAP) complexes (P < 0·001). This apparent discrepancy can be reconciled by elevated soluble thrombomodulin (sTM) (P < 0·01) and thrombin activatable fibrinolysis inhibitor (TAFI) in patients during AA providing possible evidence for a regulatory effect on fibrinolysis. Plasminogen activator inhibitor-1 (PAI-1) was reduced in patients during AA indicating, together with the observed reduction of plasmin generation, the consumption of fibrinolytic factors. In conclusion, our results support the involvement of coagulation and fibrinolysis in the pathophysiology of HAE and show the possible application of simultaneous measurement of thrombin and plasmin generation to evaluate different clinical conditions in HAE patients.