Analysis of coagulation factors in angioedema/urticaria: increased values of D-dimer and fibrinogen in isolated angioedema Maja Štrajtenberger1, Liborija Lugović-Mihić2,3 ✉, Asja Stipić-Marković1, Marinko Artuković1,4, Roman Mihić5, Lorena Dolački6, Nika-Barbara Pravica7, Ivica Lokner1,8 1Department of Pulmonology, Special Hospital for Pulmonary Diseases, Zagreb, Croatia. 2Department of Dermatology, University Hospital Center Sestre Milosrdnice, Zagreb, Croatia. 3School of Dental Medicine, University of Zagreb, Zagreb, Croatia. 4Osijek Faculty of Dental Medicine and Health, Osijek, Croatia. 5Department of Clinical Chemistry, University Hospital Center Sestre Milosrdnice, Zagreb, Croatia. 6Institute of Emergency Medicine of Zagreb County, Velika Gorica, Croatia. 7Department of Emergency Medicine, University Hospital Center Sestre Milosrdnice, Zagreb, Croatia. 8Department of Laboratory Diagnostics, Department of Pulmonology, Special Hospital for Pulmonary Diseases, Zagreb, Croatia. 63 2024;33:63-68 doi: 10.15570/actaapa.2024.13 Introduction Angioedema is a painless and limited swelling of the subcutane- ous or submucosal tissue, typically occurring in episodes affecting areas such as the face, lips, oral cavity, genitalia, or gastrointes- tinal regions (1–9). Attacks are unpredictable and asymmetrical. Angioedema can occur in isolation or in association with wheals (urticaria) in approximately 40% to 60% of patients (4–7). By definition, urticaria is a condition characterized by the develop- ment of wheals (hives), angioedema, or both (1, 2). Based on its duration, urticaria is classified as acute or chronic, and according to definite triggers it is classified as inducible or spontaneous (1). Chronic spontaneous urticaria (CSU) is defined by the presence of wheals, angioedema, or both for more than 6 weeks and can be characterized by angioedema, excruciatingly itchy recurrent hives, or both. Angioedema can be seen in mast cell–mediated disorders, bradykinin-mediated disorders, or other conditions (infections and some rare disorders), or it can appear as idiopathic angi- oedema (8). Angioedema is the result of fluid extravasation from increased vascular permeability, which is mediated by vasoactive mediators. There are two main types of angioedema: the rare he- reditary angioedema (HAE) and the more common non-heredi- tary angioedema (non-HAE) (9–11). Angioedema is also classified by mediators, the two key mediators involved in its pathogenesis being histamine and bradykinin. The variant mediated by brady- kinin can be hereditary or acquired; for example, angioedema caused by angiotensin-converting enzyme (ACE) inhibitor. Angi- oedema mediated by histamine is mostly an allergic type, but it can also be caused by direct mast cell / basophil activation and consequent release of inflammatory mediators or by disruption of the arachidonic acid pathway (9–19). The third type has a com- bination of mediators (e.g., angioedema caused by pseudoaller- gic reactions to non-steroidal anti-inflammatory drugs). There has been a recent increase in the incidence of all types of angi- oedema, including drug-mediated angioedema, such as those triggered by ACE inhibitors or vaccines, and allergic angioedema, which is in line with the general increase in prevalence of allergic diseases (7, 12, 19). Although there are clear categories of angi- oedema, the mechanisms responsible for the differences between isolated angioedema and angioedema with wheals remain to be fully elucidated. In diagnostics, the causes of angioedema are established based on clinical symptoms (including related autoimmune or in- fectious diseases), deficiency of C1 inhibitor (C1-INH), and factor XII mutation. Because there is no standard defined methodology for diagnosing angioedema, the recommendations for diagnosing and treating CSU should be followed. The blood tests for these patients include a differential blood count (DBC), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), total IgE anti- bodies, and anti-thyroid peroxidase (TPO) IgG antibodies, among others (1, 2, 20–22). Bloodwork shows that both isolated and an- gioedema with wheals are associated with higher inflammatory markers (CRP, ESR), and serum values for D-dimers and fibrinogen Abstract Introduction: Recent research has shown that blood coagulation and the extrinsic coagulation cascade are involved in the patho- genesis of chronic spontaneous urticaria (CSU), but little is known about the coagulation factors in angioedema. Methods: This study included 58 participants: 29 patients with chronic angioedema (14 with isolated angioedema and 15 with angioedema with wheals) and 29 healthy controls (HCs). We compared the values of coagulation factors in patients with isolated angioedema to those with wheals. Plasma levels of D-dimer, fibrinogen, and factor VII were measured by enzyme-linked immuno- sorbent assay (ELISA) for all participants. Results: Significantly higher D-dimer (p = 0.016; ε² = 0.381) and fibrinogen (p = 0.044; ε² = 0.331) levels were recorded in patients with angioedema (both groups) than in the HCs, with higher levels for angioedema with wheals. Factor VII and fibrinogen levels did not differ significantly between the groups with angioedema, but coagulation factors were more often elevated in both angi- oedema groups than in HCs. Conclusions: One characteristic of angioedema is an elevated blood coagulation potential, which may help produce fibrin and may be important in controlling angioedema attacks. Keywords: angioedema, urticaria, D-dimer, fibrinogen, factor VII Acta Dermatovenerologica Alpina, Pannonica et Adriatica Acta Dermatovenerol APA Received: 30 January 2024 | Returned for modification: 10 March 2024 | Accepted: 17 April 2024 ✉ Corresponding author: liborija@sfzg.hr 64 Acta Dermatovenerol APA | 2024;33:63-68M. Štrajtenberger et al. are often used as additional biomarkers of angioedema severity (21–25). Concerning therapy, histaminergic angioedema is treated with antihistamines, corticosteroids, and epinephrine, or even anti-IgE receptor omalizumab (for idiopathic angioedema). Treatment for non-histamine-mediated angioedema involves blocking bradykinin release or activity. No licensed treatments for the remaining types of isolated angioedema exist (1, 26–28). According to research data on chronic urticaria, the blood coagulation cascade plays an active role in the disease; specifically, inflammation and coagulation activate each other (21, 22, 25, 29–35). In both urticaria and angioedema, the activation of skin mast cells takes place through the external coagulation cascade and can be explained by a combination of infection (e.g., Gram-negative bacteria) and histamine and other tissue factor (TF) inducers including tumor necrosis factor alpha (TNF-α), interleukins (IL)-1β and IL-33, and vascular endothelial growth factor (VEGF), leading to changes in vascular endothelial cells. Thus, CSU pathogenesis involves the extrinsic coagulation cascade triggered by TF and complement (C) factors (such as C3a and C5a), although it is unclear how the TF-triggered coagulation pathway and complement factors induce the activation of skin mast cells and peripheral basophils in CSU patients (29–31). It seems that TF, expressed on vascular endothelial cells, eosinophils, or monocytes, then activates the extrinsic coagulation cascade, which leads to the production of the active forms of coagulation factors, such as factor (F) VIIa, FXa, and FIIa. Thus, FXa and FIIa increase vascular permeability via protease-activated receptor 1 (PAR-1), expressed on vascular endothelial cells, and induce leakage of plasma containing active coagulation factor forms and complement components. Active coagulation factor forms, such as FXa and FIIa, may convert serum C5 to C5a in intravascular and leaked plasma, which then induces mast cells / basophils to release histamine via C5aR. In addition, a fibrinolysis factor, plasmin, converts C5 to C5a, resulting in mast cell activation (29). In response to C5a and/or autoantibodies, the activated dermal mast cells / basophils release a large amount of histamine, which results in long-lasting formation of edema and wheals, through H1 receptors expressed on vascular endothelial cells. Meanwhile, activated eosinophils migrate outside of blood vessels and release stored inflammatory mediators, which may also induce or enhance the degranulation of skin mast cells via Mas-related G protein- coupled receptor X2 (MrgX2). However, in patients with CSU (possibly also angioedema), elevated levels of several molecules (including coagulation factors) have been found: D-dimers, fibrin, prothrombin fragments 1 and 2, CRP, proinflammatory cytokines TNF-α, transforming growth factor-β (TGF-β), and IL-1, IL-6, IL-17, IL-31, and IL-33 (20–22, 35). So far, there is no evidence of elevated levels of these molecules in isolated recurrent angioedema. Thus, because recent research has indicated the involvement of the extrinsic coagulation cascade in CSU pathogenesis, and because little is known about the coagulation pathway’s connection with angioedema, we wanted to examine and compare the values of key coagulation factors in patients with isolated angioedema and patients with angioedema with wheals. Methods Participants and collection of blood samples This study was conducted in accordance with the Declaration of Helsinki, and it was approved by the Ethics Committee of Uni- versity Hospital Center Sestre Milosrdnice in Zagreb, Croatia, in December 2022 (protocol no. 251-29-11-21-08) for studies involving humans. Participants were gathered at the Department of Immunology and Rheumatology at the Special Hospital for Pulmonary Dis- eases in Zagreb. We collected patients with chronic angioedema based on the criteria for chronic urticaria, which is defined as the occurrence of wheals, angioedema, or both for more than 6 weeks (1). We excluded patients with hereditary angioedema, acute urti- caria, inducible (physical and non-physical) urticaria, or any form of urticaria other than spontaneous urticaria (1, 9). With adher- ence to these inclusion and exclusion criteria, our study encom- passed 29 patients diagnosed with chronic angioedema, span- ning both those with isolated angioedema and those with chronic angioedema accompanied by wheals. In total, our participant pool comprised three distinct groups: 14 patients with isolated angioedema, 15 patients with chronic angioedema accompanied by wheals, and 29 healthy controls (HCs). Therefore, the cumula- tive sample size amounted to 58 participants. Coagulation assays First, participants’ blood was drawn into evacuated anticoagulant tubes, nine volumes of blood to one volume of 3.8% (weight/vol- ume) trisodium citrate solution. After centrifugation for 15 min- utes at 1,500 g, the platelet-poor plasma was stored at −80 °C and thawed at 37 °C immediately prior to performing the assays pre- sented in Table 1 (factor VII, fibrinogen, and D-dimer). D-dimer was measured on the Cobas Integra 400 plus analyzer (Roche Diagnostics, Vienna, Austria) using original manufacturer reagents and protocols. It was determined by latex-immunoturbi- dimetry using the original D-dimer reagent cassette D-DI2 and the D-dimer gen2 Calibrator Set. This method has been standardized against the Asserachrom D-dimer method. Factor VII and fibrino- gen were measured on the BCS XP analyzer (Siemens Health- ineers, Marburg, Germany) using the manufacturer’s reagents and protocols. Fibrinogen concentration was determined by the Clauss clotting method with the Multifibren U reagent (Siemens Healthineers, Marburg, Germany). Finally, the activity of factor VII was measured with the coagulometric assay using factor VII– deficient plasma (Siemens Healthineers, Marburg, Germany). A mixture of factor VII–deficient plasma and patient plasma were tested with the Thromborel S (Siemens Healthineers, Marburg, Germany), and the results were interpreted as a percentage ac- cording to a calibration curve made by using dilutions of Stand- ard Human Plasma (Siemens Healthineers, Marburg, Germany). Standard Human Plasma is traceable to the International Stand- ard WHO 09/172. Table 1 | Sample description and examined factors/characteristics. Variable Mean SD Minimum Maximum Median IQR Age (years) 47.0 13.9 21 79 45 35.8–58.3 Factor VII (% of activity) 102.8 24.0 11 151 104 86.5–119.3 Fibrinogen (g/L) 3.6 0.7 2.3 5.2 3.5 3.2–4.1 D-dimer (FEU/L) 0.37 0.37 0.13 2.11 0.29 0.19–0.37 SD = standard deviation, IQR = interquartile range. 65 Acta Dermatovenerol APA | 2024;33:63-68 Coagulation factors in chronic angioedema and urticaria Statistical analysis Age and coagulation factor values were compared across groups using the Kruskal–Wallis test and post-hoc Mann–Whitney test with Bonferroni adjustment for multiple comparisons. Effect size was calculated with the formula ε² = H / [(n² − 1) / (n + 1)] for the Kruskal–Wallis test, and r = z / √N for the Mann–Whitney test. Co- hen’s criteria were used for interpretation—for the Mann–Whitney test, a small effect size (r) was 0.25–0.30, 0.30–0.50 was moderate, 0.50–0.70 was large, and > 0.70 was a very large effect size. The squared values of Cohen’s criterion were used for the interpreta- tion of ε² in the Kruskal–Wallis test. To compare frequency, the χ² test was utilized, and Cramer’s V was used to calculate the effect’s size. The aforementioned Cohen’s criteria were applied as limit- ers for the assessment of the effect size. Spearman’s correlation and Cohen’s interpretational standards were used to analyze rela- tionships between variables. Two-way analysis of covariance was used to analyze whether there was an interaction between group and sex with the control of age at the level of coagulation factors, and the effect size was quantified using η² and interpreted with the help of the squared values of Cohen’s criteria. Results Our study involved 58 participants, comprising individuals diag- nosed with chronic angioedema/urticaria and HCs. The age range of the participants was from 21 to 79 years, with a median age of 45 years. The interquartile range was from 35.8 to 58.3 years. Among the participants, 79% were female (Table 1). The groups differed by age with a large effect size (p = 0.001; ε² = 0.532). The angioede- ma group was significantly older than HCs, with a large effect size (p = 0.001; r = −0.611; Fig. 1). Women were significantly younger than men, with a moderate effect size (median 42 vs. 59 years; p = 0.017; r = −0.314). There was also no significant difference in sex distribution between the groups (Fig. 1). Based on the results for coagulation factor levels for the three groups, the groups differed in fibrinogen levels, with a moder- ate effect size (p = 0.044; ε² = 0.331), and D-dimer (p = 0.016; ε² = 0.381), but not in factor VII levels (Figs. 2 and 3). When compar- ing parameter levels, differences between the three groups were significant only for D-dimer, for which patients with angioedema with wheals had higher D-dimer levels than HCs, with a moder- ate effect size (p = 0.004; r = −0.430). There were no statistically significant differences in the proportions of participants with el- evated coagulation factors; nonetheless, coagulation factors were more often elevated in patients with isolated angioedema and those with angioedema with wheals than in HCs (Table 2). Our results also show that coagulation factor values corre- lated with age, and that these correlations were weak to moder- ate, linear, and positive. With increasing age, factor VII increased (r = 0.270; p = 0.041), as did D-dimer levels (r = 0.308; p = 0.019). Fibrinogen levels were primarily dependent on age (r = 0.392; p = 0.002). Two-way analysis of covariance revealed that, when control- ling for age, there was no interaction between sex and study groups regarding the levels of coagulation factors. Furthermore, D-dimer values mostly depended on the subject’s group affiliation (p = 0.035; η² = 0.123), whereas fibrinogen levels mainly depended on age (p = 0.009; η² = 0.126). Additional statistics indicate that, when controlling data for age, sex, and data on specific angioede- ma groups, D-dimer primarily depends on the specific angioede- ma groups rather than age. There were no significant differences in the levels of coagula- tion factors or the prevalence of elevated coagulation factors be- tween men and women. Two-way analysis of covariance showed that, when controlling for age, there was no interaction between sex and study groups on the level of coagulation factors. Moreo- ver, D-dimer values mostly depended on the subject’s group affili- ation (p = 0.035; η² = 0.123), and fibrinogen mainly depended on age (p = 0.009; η² = 0.126). Figure 2 | Comparison of age (A) and D-dimer values (B) among the three study groups. Asterisks show extreme values. Groups connected by a horizontal line significantly differ statistically, accounting for Bonferroni correction (p ≤ 0.017). HCs = healthy controls, CSU = chronic spontaneous urticaria, AE = angioedema. Figure 1 | Data on participants’ sex in the three study groups. HCs = healthy controls, CSU = chronic spontaneous urticaria, AE = angioede- ma. 66 Acta Dermatovenerol APA | 2024;33:63-68M. Štrajtenberger et al. Discussion This study is the first attempt to analyze values of coagulation fac- tors in patients with isolated angioedema and compare them to values of coagulation factors in patients with angioedema with wheals and HCs. By conducting coagulation assays for the levels of coagulation factors and markers of activated coagulations, we were able to show that greater coagulation potential is also highly related to isolated angioedema, not only to angioedema accom- panied with wheals. Using traditional coagulation assays, we as- sessed hemostatic characteristics, for which both patients with isolated angioedema and patients with angioedema with wheals had greater levels of fibrinogen and D-dimer than HCs. The in- crease of factor VII levels, however, was not significant when compared with HCs. Although we did not find statistically signifi- cant differences in the proportions of participants with elevated coagulation factors, coagulation factors were more often elevated in patients with chronic angioedema (isolated angioedema and angioedema with wheals) than in HCs. It indicates that one of the characteristics of angioedema is an elevated blood coagulation potential, which may help produce fibrin and may be important in controlling angioedema attacks. The etiology of newly formed isolated angioedema is challeng- ing to differentiate, which may be important, particularly in emer- gency or clinical practice (7). Given that the angioedema group encompasses three main subgroups (histamine-mediated, brad- ykinin-mediated, and idiopathic angioedema types), the absence of a biological diagnostic marker for these angioedemas is why bradykinin-mediated angioedema is sometimes treated with an- tiallergic therapy and why clinicians rarely administer fast-acting bradykinin antagonists. However, over the last decade, significant progress has been made in understanding the pathophysiology of angioedema, especially HAE. Thus, in addition to the gene for C1- INH, several new mutations for proteins involved in blood enzyme cascades have been discovered (FXII, plasminogen, and angiopoi- etin) (7). Consequently, the deficiency of these particular enzymes disrupts the balance between blood enzymatic systems, such as the kinin-kallikrein system, the renin-angiotensin system, the contact coagulation pathway, fibrinolysis, and the complement pathway. This disruption leads to an excess of bradykinin forma- tion and increased vascular permeability. On the other hand, the development of histamine-induced angioedema differs, arising from an immune reaction (whether allergic or autoimmune), re- sulting in histamine release, vasodilation, and increased vascu- lar permeability. The role of coagulation in the pathogenesis of chronic urticaria, with or without angioedema, has been known for a long time. In addition, in HAE (bradykinin-mediated), there is clinical experience supporting the beneficial effect of coagula- tion blockage by tranexamic acid. However, it remains unclear how coagulation factors activate skin mast cell and peripheral ba- sophils in chronic angioedema or chronic urticaria. Furthermore, the mechanisms responsible for isolated angioedema versus chronic angioedema are not yet clearly understood. In addition, although the pathogenesis of chronic urticaria and angioedema has not been fully elucidated, according to cur- rent knowledge, the autoantibodies IgE and IgG predispose mast cell / basophil activation, with the involvement of coagulation and complement cascades. In their pathogenesis, the activation of skin mast cells takes place through the external coagulation cascade and may be associated with various microorganisms, cy- tokines, immune factors, and other blood factors (6, 30, 36–40). Concerning autoimmunity in CSU, according to several reports, around 30% to 50% of CSU patients have IgG autoantibodies against IgE or the high-affinity IgE receptors (FcεRIs) expressed on mast cells / basophils, which induce this cell activation, and subsequent release of stored mediators. Moreover, in certain CSU patients, IgE autoantibodies against several molecules such as double stranded DNA, TF, IL-24, and TPO have been detected. A role of IgG autoantibodies against IgE or IgE autoantibodies against self-molecules in CSU has been supported by the ineffec- tiveness of omalizumab (anti-IgE monoclonal antibody) in these patients. Figure 3 | Comparison of fibrinogen and coagulation factor VII between the three study groups. Circles show outliers; stars show extreme values. HCs = healthy controls, CSU = chronic spontaneous urticaria, AE = angioedema. HCs = healthy controls, CSU = chronic spontaneous urticaria, AE = angioedema, V = effect size (Cramer’s V), p = significance level according to χ² test. Table 2 | Comparison of the prevalence of elevated coagulation factors between the three study groups. Level HCs(n = 29) CSU ± AE (n = 15) AE (n = 14) Sum p V Factor VII, n (%) Normal 9 (31.0%) 4 (26.7%) 1 (7.1%) 14 (24.1%) Increased 20 (69.0%) 11 (73.3%) 13 (92.9%) 44 (75.9%) 0.222 0.228 Fibrinogen, n (%) Normal 27 (93.1%) 13 (86.7%) 12 (85.7%) 52 (89.7%) Increased 2 (6.9%) 2 (13.3%) 2 (14.3%) 6 (10.3%) 0.687 0.114 D-dimer, n (%) Normal 28 (96.6%) 11 (73.2%) 13 (92.9%) 52 (89.7%) Increased 1 (3.4%) 4 (26.7%) 1 (7.1%) 6 (10.3%) 0.051 0.320 67 Acta Dermatovenerol APA | 2024;33:63-68 Coagulation factors in chronic angioedema and urticaria References 1. Zuberbier T, Abdul Latiff AH, Abuzakouk M, Aquilina S, Asero R, Baker D, et al. The international EAACI/GA²LEN/EuroGuiDerm/APAAACI guideline for the defi- nition, classification, diagnosis, and management of urticaria. Allergy. 2022;77: 734–66. 2. Zuberbier T, Bernstein JA, Maurer M. Chronic spontaneous urticaria guidelines: what is new? J Allergy Clin Immunol. 2022;150:1249–55. 3. Azmy V, Brooks JP, Hsu FI. 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Isolated angioedema: an overview of clinical features and etiology. Exp Ther Med. 2019;17:1068–72. According to previous studies, the severity of angioedema with wheals positively correlated with D-dimer levels in plasma (which may indicate this possibility in isolated angioedema as well) (24, 25). Elevated plasma levels of D-dimer and fibrinogen in patients with urticaria/angioedema may suggest an activation of the co- agulation pathway and fibrinolysis. Activation of the coagulation pathway leads to thrombus formation, followed by activation of anticoagulation and fibrinolysis. The interaction between inflam- mation and coagulation creates a loop that maintains and en- hances both systems (41). Generally, in our study, D-dimers were significantly increased only in patients with angioedema with wheals, with levels higher than those observed in HCs. The dif- ference was not significant for patients with isolated angioedema. It is also worth noting that D-dimer values are elevated in several patients without thrombosis (this assay has a negative predictive value to exclude thrombosis) and that an increase in D-dimer is observed in patients with extravascular fibrin deposition. In pa- tients with angioedema, the increase in D-dimer may be related, at least in part, to the increased vascular permeability and plasma extravasation into the surrounding tissues (7). In addition to autoimmune/immune-mediated cutaneous ill- nesses, several inflammatory conditions also exhibit high levels of coagulation and fibrinolysis activation indicators. The activa- tion of coagulation may have two major effects: a) it may play a local pathogenic role in causing skin lesions, and b) it may play a systemic role in raising the risk of thrombosis. 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In an uncontrolled study, four out of five chronic urticaria patients were able to achieve remission with warfarin at a therapeutic dose (53). According to Asero et al., patients with severe chronic urti- caria that are resistant to antihistamines and have high levels of D-dimer may benefit from a combination of low-molecular-weight heparin and tranexamic acid therapy, which is successful in treat- ing patients with idiopathic angioedema or angioedema brought on by a lack of C1-INH (49). All of these data, when considered collectively, point to the relevance of coagulation cascade activa- tion in chronic angioedema and chronic urticaria and support the use of anticoagulant and antifibrinolytic medications for both diseases, particularly in the presence of additional cardiovascular risk factors. Although it is novel, a limitation of this research is the small number of participants, a restricted number of examined coagu- lation parameters, and age differences among the three groups. Thus, the healthy subjects were significantly younger than the pa- tients with isolated angioedema and the patients with angioede- ma with wheals, which may affect the results obtained. Therefore, further research, encompassing a larger number of parameters examined, is needed. Conclusions According to our research, significantly higher D-dimer and fi- brinogen levels were recorded in patients with chronic angioede- ma (including those with isolated angioedema and those with angioedema with wheals) than in the HCs, with higher levels in those with angioedema with wheals. In addition, elevated co- agulation factors were recorded more often in both angioedema groups than in HCs. This indicates that one of the characteristics of angioedema is an elevated blood coagulation potential, which may help produce fibrin and may be important in controlling an- gioedema attacks. 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