Chronic prothrombotic tendency in patients with granulomatosis with polyangiitis

Abstract

Introduction: Patients with granulomatosis with polyangiitis (GPA) show increased tendency toward thromboembolic phenomena in exacerbation of their disease.

Objectives: The aim of the study was to evaluate thrombin generation potential and fibrinolytic plasma activity in patients with GPA, both in exacerbation and in remission.

Patients and methods: We included 38 patients with GPA: 18 with exacerbated GPA and 20 in remission. The control group included 39 healthy participants matched for age and sex. Plasma thrombogenic potential was assessed using calibrated automated thrombography. Plasma fibrinolytic potential was assessed using clot lysis time (CLT). We also measured levels of inflammatory markers, thrombomodulin, and fibrinolysis proteins in all participants.

Results: In the whole group of patients with GPA, endogenous thrombin potential was higher by about 25% (P <⁠0.001), while CLT was lower by about 20% (P = 0.02) when compared with controls. The endogenous thrombin potential was higher, the CLT lower, and the levels of thrombomodulin and inflammation markers (C-reactive protein, fibrinogen, factor VIII) higher both in patients with exacerbation and in remission than in the control group; no such differences were noted when comparing those with exacerbation and those in remission, however. The only parameter that differentiated patients with GPA exacerbation from those in remission was the D-dimer level (median [interquartile range], 1151 [597.2–2468.7] ng/ml vs 340.4 [255.1–500.7] ng/ml; P <⁠0.001), a marker of lysis of intravascularly formed fibrin.

Conclusions: Patients with GPA show an increased prothrombotic state, regardless of the disease phase. This is probably related to ongoing low-grade inflammation and endothelial injury. Large clinical studies are required to address the need for, and appropriate type of, antithrombotic prophylaxis during the course of GPA.

What’s new?

Granulomatosis with polyangiitis (GPA) is antineutrophil cytoplasmic antibody–associated vasculitis, primarily connected with the antineutrophil cytoplasmic antibody directed against proteinase 3. Patients in exacerbation of antineutrophil cytoplasmic antibody–associated vasculitis are at an increased risk of venous thromboembolic events. We assessed thrombin generation in patients with GPA using calibrated automated thrombography and their plasma fibrinolytic activity measured by clot lysis time both during exacerbation and remission of their disease. We have shown that the prothrombotic tendency in patients with GPA extends beyond exacerbation to remission, which might persistently increase the risk of thromboembolism.

Introduction

Granulomatosis with polyangiitis (GPA; previously known as Wegener granulomatosis) is a necrotizing granulomatosis, characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis that mainly affects small and medium vessels. GPA is an antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis (AAV), primarily linked with ANCA directed against proteinase 3 (PR3-ANCA). Symptoms of GPA include mainly granulomatous inflammation of the upper and lower respiratory tract, necrotizing vasculitis, and glomerulonephritis. It usually starts as granulomatous disease affecting the respiratory tract and then progressing to the generalized vasculitis.¹ PR3-ANCA, neutrophils, neutrophil extracellular traps (NETs), T and B cells, and vascular endothelial cells are closely involved in the GPA pathogenesis.² It has been reported that patients with exacerbated AAV are at increased risk of venous thromboembolic events: about 7 cases per 100 person-years, compared with 0.15 to 0.31 in the general population.³ Consequently, several other reports have confirmed a prothrombotic tendency, especially during the early and exacerbated GPA phase.^4-7 Moreover, an increased risk of arterial events in AAV has also been reported with an estimated prevalence between 3.1% and 18.7%.^8,9 In 2011, the EUVAS (European Vasculitis Study Group) proposed a prognostic tool to define the 5-year cardiovascular risk in patients with AVV. Using this tool, it has been shown that about 12% of patients with newly diagnosed GPA reported at least 1 cardiovascular event, defined as cardiovascular death, myocardial infarction, coronary bypass / percutaneous coronary intervention, or stroke.¹⁰ All of these observations are in line with a more general finding showing an increased tendency for venous thromboembolism in hospitalized patients with various acute systemic rheumatic diseases.¹¹ Data on the possible prothrombotic mechanisms in GPA are scarce. Endothelial cell dysfunction, a feature of AVV, can lead to an interaction between neutrophils (activated by tumor necrosis factor α and ANCA) and endothelial cells, with the subsequent massive oxidative stress finally leading to atherothrombotic complications.¹²

Neutrophils are capable of releasing extracellular nucleic acids along with histones and granular proteins which, in turn, can entrap bacterial agents. These neutrophil extracellular traps have also recently been implicated in the pathogenesis of thrombotic events which links autoimmunity with the hemostatic system.^13,14 In exacerbated AAV, neutrophils have also been shown to release tissue factor–expressing neutrophil extracellular traps.¹⁵

The association between exacerbated disease and thromboembolism suggests the involvement of mechanisms at the crossroads of thrombosis and inflammation—thromboinflammation.¹⁶ Thrombin is a key component propagating not only thrombosis but also inflammation.

For this reason, we decided to assess thrombin generation using calibrated automated thrombography (CAT) together with plasma fibrinolytic activity measured by plasma CLT in patients with GPA. Plasma hemostatic parameters together with markers of inflammation and endothelial injury were also measured. We attempted to evaluate whether a prothrombotic tendency in GPA extends beyond exacerbation.

Patients and methods

Patients

The study included 38 consecutive adult patients with GPA recruited from the Outpatient Clinic for Autoimmune Diseases, Department of Internal Medicine, Jagiellonian University Medical College, Kraków, Poland. Patients were enrolled in the study if they fulfilled the 2012 Chapel Hill Consensus Conference GPA nomenclature criteria.¹⁷ The activity of the disease was assessed according to the Birmingham Vasculitis Activity Score (BVAS) version 3 (v.3),¹⁸ and chronic organ damage by the Vasculitis Damage Index (VDI).¹⁹ The exclusion criteria included: cancer, severe hepatic injury (Child–Pugh class 3), heart failure (New York Heart Association class III or IV), and current anticoagulant therapy. Patients were considered to be in the remission phase if their BVAS(v.3) score was 0.

This study was approved by the local ethics committee, and informed consent was obtained from all patients according to the Declaration of Helsinki.

Laboratory investigations

Venus blood samples were drawn in 3.2% (0.109 mol/l) sodium citrate tubes (1 part sodium citrate to 9 parts venous blood), then centrifuged at 2000 × g for 10 minutes within 30 minutes after drawing, and stored in aliquots at –80 °C for further analysis.

Blood from patients with exacerbation was drawn before the start of the induction immunosuppressive therapy with cyclophosphamide and / or rituximab. They were receiving low to median doses of glucocorticosteroids, at a median daily dose of 18.2 mg (range, 0–80 mg) in exacerbation and 3.8 mg (range, 0–14 mg) in remission.

Complete blood counts, biochemical parameters (glucose, creatinine), and basic coagulation tests (prothrombin time, activated partial thromboplastin time, and fibrinogen) were determined using routine laboratory assays. C-reactive protein (CRP), C3, and C4 were measured by nephelometry (Siemens, Marburg, Germany). Antineutrophil cytoplasmic antibodies were measured by indirect immunofluorescence (Euroimmun, Lubeck, Germany). Specific anti-PR3 and anti-myeloperoxidase antibodies were identified by the immunoenzymatic assays (ELISA anti-PR3 and ELISA anti-myeloperoxidase, Euroimmun, Lubeck, Germany).

Commercially available immunoenzymatic assays were used to determine plasma tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) antigens (both Hyphen Biomed, Neuville, France), thrombin-antithrombin complexes (TAT, Enzygnost TAT micro, Siemens, Marburg, Germany), thrombomodulin (Human Thrombomodulin ELISA Kit, Biorbyt LLC, San Francisco, United States). Activity of plasma α₂-antiplasmin and plasminogen were measured by chromogenic assays (Berichrom α₂-antiplasmin and Berichrom plasminogen; Siemens, Marburg, Germany). Concentrations of D-dimer were assessed using the turbidimetric method (Innovance D-dimer; Siemens, Marburg, Germany). Factor VIII activity was measured using a coagulometric assay with factor VIII–deficient plasma (Siemens, Marburg, Germany).

The activity of antithrombin and protein C were determined with chromogenic methods (Innovance Antithrombin, Berichrom Protein C; Siemens, Marburg, Germany). Concentrations of free protein S were assessed using the turbidimetric method (Innovance Free Protein S; Siemens, Marburg, Germany).

Lupus anticoagulant was determined in a 3-step procedure according to the guidelines of the International Society on Thrombosis and Haemostasis.²⁰ Anticardiolipin and anti-β2-glycoprotein I antibodies of immunoglobulins G and M isotype were assessed using enzyme-linked immunosorbent assays QUANTA Lite (Inova Diagnostics, Saint Louis, Missouri, United States) according to the International Society on Thrombosis and Haemostasis guidelines.²¹

Genotyping for factor V Leiden R506Q (mutation rs6025) and prothrombin G20210A (mutation rs1799963) was performed as previously described^22,23 or by using TaqMan assays (Applied Biosystems). All the tests were performed in both study patients and controls.

Plasma thrombogenic potential

Plasma thrombogenic potential was assessed according to the manufacturer’s instructions using CAT with a computational model of thrombin dynamics (Thrombinoscope BV, Maastricht, the Netherlands). Duplicate plasma samples were analyzed in the 96-well plate fluorimeter (Ascent Reader, Thermo Lab Systems OY, Helsinki, Finland) equipped with the 390/460 filter at 37 °C. Briefly, 80 µl platelet-poor plasma was diluted with 20 µl of a tissue factor–based activator (Diagnostica Stago, Asnieres, France) and 20 µl of FluCa solution (Diagnostica Stago, Asnieres, France). We assessed peak thrombin generation (maximum concentration of thrombin formed during the recording time), time to peak, lag time, and endogenous thrombin potential (ETP; area under curve).^24,25

Clot lysis time

Clot lysis time was measured as previously described.²⁶ Briefly, citrated plasma was mixed with 15 nM of calcium chloride, human thrombin (Merck, Kenilworth, New Jersey, United States) at a final concentration of 0.5 U/ml, 10 µM of phospholipid vesicles, and 18 ng/ml of recombinant t-PA (Boehringer Ingelheim, Ingelheim, Germany). The mixture was transferred to a microtitre plate and its turbidity was measured at 405 nm at 37 ºC. CLT was defined as time from the midpoint of the clear-to-maximum-turbid transition, which represents clot formation, to the midpoint of the maximum–turbid-to-clear transition. Intra-assay and interassay coefficients of variation were 6% to 8%. The assay was performed in triplicate.

Statistical analysis

The results were obtained using the STATISTICA Tibco 13.3 software. Data distribution was evaluated by the Shapiro–Wilk test. All continuous variables were non-normally distributed and were presented as medians with interquartile ranges (IQRs) and compared by the Mann–Whitney test, the Kruskal–Wallis test, and the multiple comparison test with the Bonferroni correction, as appropriate. Categorical variables were compared by the χ² test. Spearman rank correlation analysis was used to evaluate the relationship between continuous variables. A P value of less than 0.05 was considered statistically significant.

Results

We included 38 patients with GPA: 18 in the exacerbation phase and 20 in remission (BVAS, 0). Demographic and laboratory characteristics of the study patients are presented in Table 1. The median (IQR) disease duration was 1.5 years (1 month to 8 years) measured since the diagnosis. The control group included 39 healthy volunteers matched for age and sex who had no history of any thromboembolic complications and were not taking any antithrombotic drugs. They were also matched for body mass index and smoking status.

**Table 1**. Characteristics of patients with granulomatosis with polyangiitis and the control group
Variable		GPA			Controls (n = 39)
Variable		All patients (n = 38)	Exacerbation (n = 18)	Remission (n = 20)	Controls (n = 39)
Sex	Female	21	10	11	19
Sex	Male	17	8	9	20
Age, y		55 (39.5–61.7)	53 (39–64.7)	56 (40.5–61.2)	44 (40–58.5)
BVAS (v. 3), points		–	18 (16–25)	–	NA
VDI, points		–	–	5 (4–6)	NA
Basic laboratory tests
RBC, × 10⁶/μl		4.3 (3.8–4.7)^a	4.1 (3.6–4.5)^b	4.4 (4.2–5.1)	4.7 (4.5–5)
Hemoglobin, g/dl		12.5 (10.9–13.9)^a	11.5 (10.2–13.5)^b	13.1 (12.1–14.6)^c,d	14.3 (13.8–14.7)
Hematocrit, %		37.5 (33.6–42.3)^a	35.3 (31.6–40.5)^b	39.8 (35.6–43.5)^c,d	43.1 (41.6–43.8)
WBC, × 10³/μl		8.4 (6.1–10.9)^a	10.0 (6.5–11.7)^b	7.1 (5.8–9.6)^c	5.8 (4.6–7)
Platelets, × 10³/μl		260 (226–334)^a	278.5 (253–445)^b	232 (206–295)	224 (208–247)
Glucose, mmol/l		5.3 (5–6.4)^a	5.7 (5.1–6.4)^b	5.3 (4.9–6.4)^c	4.6 (4.4–5.4)
Urea, mmol/l		6.8 (4.9–12.5)^a	8.4 (4.6–16.3)^b	5.4 (5–10.2)	5.1 (4.3–5.4)
Creatinine, μmol/l		79.7 (70.4–205.3)^a	104.3 (70.4–285.6)^b	77.7 (70.3–163.6)	71.1 (67.2–82.3)
Aspartate transaminase, U/l		16 (14–21)	15 (12–21)	17.5 (14.5–21)	16.4 (13.7–18.8)
Alanine transaminase, U/l		19 (13–25)	14.5 (12–29)	21.5 (16–24)	14.7 (12.6–22.3)
PT, s		11.4 (10.9–12.2)	12.1 (11.4–12.9)^a	11.3 (10.7–11.7)^d	11.1 (10.7–12)
aPTT, s		26.1 (23.1–28)	27.1 (23.5–30)	25.6 (22.9–27.6)	26.6 (24.9–28.4)
Fibrinogen, g/l		4.6 (3.9–5.7)^a	5 (3.8–5.8)^b	4.3 (3.9–5.2)^c	2.4 (2.2–3.3)
Factor VIII, %		202.4 (140.2–291)^a	257.1 (205.4–310.7)^b	159.4 (131.5–218.8)^c,d	113.4 (93.9–139.1)
CRP, mg/l		6.5 (3.5–14.3)	12.1 (5.6–84.8)	4.3 (2.4–8.9)^d	ND
C3, g/l		1.21 (1.13–1.35)	1.23 (1.15–1.32)	1.21 (1.12–1.36)	ND
C4, g/l		0.26 (0.23–0.34)	0.24 (0.21–0.3)	0.27 (0.24–0.34)	ND
Thrombomodulin, pg/ml		7321.5 (5 618.1–11 252.7)^a	8480.5 (5 349.6–11 682)^b	6528.9 (5 618.1–11 078.7)^c	5204.5 (4752–5987.3)
Data are presented as median (interquartile range). a Significant differences between the whole GPA group vs controls b Significant differences between GPA exacerbation vs controls c Significant differences between GPA remission vs controls d Significant differences between GPA exacerbation vs GPA remission Abbreviations: aPTT, activated partial thromboplastin time; BVAS, Birmingham Vasculitis Activity Score; CRP, C-reactive protein; GPA, granulomatosis with polyangiitis, NA, not applicable; ND, not determined; PT, prothrombin time; RBC, red blood cells; WBC, white blood cells; VDI, Vasculitis Damage Index

All patients in remission had completed the induction immunosuppressive therapy at least 6 months before the blood drawing and were receiving low doses of glucocorticosteroids; 3 patients were on maintaining therapy with methotrexate, and 3 patients received azathioprine. Six patients (15.8%) received low-dose aspirin (3 with exacerbation and 3 in remission). None of them had any thromboembolic complications in the past nor was treated with anticoagulant drugs.

Out of 18 exacerbated patients, 11 (61.1%) presented with a generalized form of GPA and 7 (38.9%) with its limited form. The most frequently affected organs were the ear, nose, and throat (n = 13; 72.2%), lungs (n = 13; 72.2%), and kidneys (n = 11; 61.1%). Out of 20 patients in remission, 7 (35%) presented with a generalized form of the disease, and 13 (65%) with its limited form. The most frequently chronically damaged organs were lungs (n = 13; 65%) and kidneys (n = 11; 55%).

Positive PR3-ANCA were detected in all patients during exacerbation (median [IQR], 40 [17–71] IU/ml) and in 13 (65%) of those in remission (median [IQR], 7.5 [1.6–52.5] IU/ml).

Patients with vasculitis, both during exacerbation and in remission, showed typically lower values of hematological parameters as compared with healthy controls. Parameters of kidney function tended to be higher in both groups of patients. As expected, all patients presented with elevated serum inflammatory markers (CRP, fibrinogen, factor VIII activity) as compared with healthy controls. As expected, all of those parameters were higher in those with exacerbation.

Thrombin generation assay and fibrinolytic activity

Patients with GPA had a significantly altered thrombin generation profile compared with the control group (Table 2; Supplementary material, Figure S1). Higher thrombin generation parameters clearly differentiated patients with GPA from controls (P <⁠0.001 for all parameters) but did not significantly differentiate between exacerbation and remission phases of the disease. Nevertheless, a certain uniform trend towards higher values of ETP, peak thrombin, and TAT during exacerbation could be seen, as compared with patients in remission. Parameters of CAT in individual patients correlated with TAT, a plasma thrombin generation marker. In the entire GPA group, there was a negative association between TAT and time to peak as well as lag time (ρ = –0.55, P <⁠0.001 and ρ = –0.47, P = 0.003, respectively). In the exacerbation group, negative association was found between TAT and ETP, and time to peak (ρ = –0.56, P = 0.02 and ρ = –0.50, P =0.04). In the remission group, a significant positive correlation was observed between TAT and peak thrombin generation (ρ = 0.47, P = 0.04). In addition, ETP was positively correlated with fibrinogen levels (ρ = 0.50, P <⁠0.001). Otherwise, we did not find any significant associations between parameters describing thrombin generation and the remaining hemostatic parameters, as well as disease activity (BVAS) or measures of organ damage (VDI)—a result which is most probably related to the small number of subjects in the study groups.

**Table 2**. Comparison of thrombin generation and fibrinolytic parameters between patients with granulomatosis with polyangiitis and the control group
Variable	GPA			Controls (n = 39)
Variable	All patients (n = 38)	Exacerbation (n = 18)	Remission (n= 20)	Controls (n = 39)
ETP, nM × min	1994.7 (1700.2–2261.4)^a	2027.9 (1792–2338.9)^b	1878 (1676.8–2156.3)^c	1600.7 (1397.1–1714)
Lag time, min	3.5 (3.1–4.1)^a	3.4 (3.1–3.7)^b	3.7 (3.1–3.7)^c	3 (2.7–3.6)
Peak TG, nM	382 (338.6–466.5)^a	423.6 (367.3–494.5)^b	369 (331.4–432.8)^c	303.7 (260.5–339.9)
Time to peak, min	5.7 (5.3–6.7)	5.7 (5.3–6.1)	6.2 (5.4–7)^c	5.3 (5.1–6.7)
TAT, μg/l	5.1 (3.4–9.7)^a	7.8 (3.7–12.6)^b	4.7 (3.4–8)	4.2 (3.1–5.2)
CLT, min	119 (89–173)^a	119 (92–173)^b	115.9 (87.9–195.2)^c	95.8 (85.9–109.9)
D-dimer, ng/ml	550.6 (309–1260.4)	1151 (597.2–2468.7)^b	340.4 (255.1–500.7)^c,d	231 (178.2–362)
α₂-antiplasmin, %	106.5 (102.6–115.2)	105.4 (101.8–115.2)	107.7 (103.8–113.9)	109.7 (102.1–115.1)
Plasminogen, %	109.9 (98.3–125.5)^a	109.1 (102.1–125.4)^b	111.6 (93.9–124.5)^c	98.9 (88.2–105.6)
t-PA antigen, ng/ml	9.7 (6.3–11.9)^a	8.4 (6.7–11.7)	10.3 (5.9–11.9)^c	7 (4–9.6)
PAI-1 antigen, ng/ml	18.3 (11.2–61.8)	14.6 (11.3–74.6)	19.1 (13.3–32.8)	15.4 (8.9–23.6)
Data are presented as median (interquartile range). a Significant differences between the whole GPA group vs controls b Significant differences between GPA exacerbation vs controls c Significant differences between GPA remission vs controls d Significant differences between GPA exacerbation vs GPA remission Abbreviations: CLT, clot lysis time; ETP, endogenous thrombin potential, PAI-1, plasminogen activator inhibitor-1; TG, thrombin generation; TAT, thrombin-antithrombin complex; t-PA, tissue-type plasminogen activator; others, see Table 1

Among plasma components of the fibrinolytic system, only plasminogen and t-PA antigen levels were significantly higher in patients with GPA as compared with healthy controls (P = 0.001 and P = 0.008, respectively). These differences also influenced, at least in part, the results of the test which generally assessed in vitro plasma fibrinolytic potential, namely CLT. Impaired fibrinolysis in patients with GPA was reflected by a prolonged CLT both during exacerbation (P = 0.02) and remission (P = 0.04), but again, with no difference between patients with GPA during exacerbation and in remission (Table 2; Supplementary material, Figure S2). In the entire GPA group, CLT showed positive correlation with all plasma components of the fibrinolytic system measured: α₂-antiplasmin, t-PA, PAI-1, and plasminogen (ρ = 0.54, ρ = 0.45, ρ = 0.66, ρ = 0.42 respectively; P <⁠0.001 for all). These associations were even stronger in the remission group (ρ = 0.58, ρ = 0.57, ρ = 0.71, ρ = 0.57 respectively; P <⁠0.001 for all). Interestingly, in the exacerbation group, CLT was positively associated only with α₂-antiplasmin and with PAI-1 (ρ = 0.52, P = 0.003 and ρ = 0.8, P <⁠0.001), both inhibitors of fibrinolysis. These findings were accompanied by elevated levels of D-dimer, especially marked in patients in the exacerbation phase of the disease. D-dimer levels showed a positive association with one of the main inflammation markers, CRP (ρ = 0.51, P <⁠0.001).

Endothelial dysfunction

Thrombomodulin levels, an indirect marker of endothelial injury, were significantly elevated both during exacerbation (P <⁠0.001) and remission (P = 0.002) of GPA as compared with controls but with no difference between these subgroups (Table 1). Thrombomodulin levels positively correlated with the levels of D-dimer and activity of factor VIII in all patients with GPA (ρ = 0.63 and ρ = 0.53, respectively; P <⁠0.001 for both) and in exacerbation subgroup (ρ = 0.75 and ρ = 0.64, respectively; P <⁠0.001 for both). In the remission subgroup, positive correlation of thrombomodulin levels with D-dimer was maintained (ρ = 0.63; P <⁠0.001) and, in addition, a new association was shown with plasma fibrinogen levels (ρ = 0.54; P <⁠0.001). In the exacerbation phase, there was also a weak association between thrombomodulin and the BVAS score (ρ = 0.48; P = 0.04), paralleled by a similar association with the VDI score in remission (ρ = 0.52; P = 0.02).

Renal function

Among 38 GPA patients, 11 (28.9%) had estimated glomerular filtration rate (eGFR) of 30 ml/min/1.73 m² or less. There were no differences in the majority of thrombotic and fibrinolytic parameters measured between patients with eGFR of 30 ml/min/1.73 m² or less and the rest of the patients. Only levels of thrombomodulin, D-dimer, and factor VIII were significantly higher in patients with eGFR of 30 ml/min/1.73 m² or less (P <⁠0.001, P = 0.008, P = 0.01, respectively).

Thrombophilia

Testing for hereditary thrombophilia showed heterozygosity for prothrombin mutation (G20210A) in 3 patients and no factor V Leiden mutation (G1691A). Natural anticoagulants (antithrombin, protein C, free protein S) were within normal limits. Antiphospholipid antibody testing (lupus anticoagulant, anticardiolipin, and anti-β2-glycoprotein I antibodies) was negative in all patients. There were no thrombophilic defects in any of the controls.

Discussion

To the best of our knowledge, our study is the first to show that patients with GPA have elevated thrombin generation as well as impaired fibrinolysis, both in exacerbation and in remission. Those 2 phenomena may explain, at least in part, increased prothrombotic tendency and higher incidence of thromboembolic complications in this population.

In comparison with healthy controls, our patients were also characterized by increased levels of inflammation markers and indicators of endothelial dysfunction (Table 1), with an insignificant although visible trend towards higher values in exacerbated GPA. Increased thrombin generation parameters and decreased fibrinolytic activity in patients with GPA showed some significant associations with markers of inflammation and endothelial dysfunction (as discussed in detail below).

Thrombin generation potential

Plasma thrombogenic potential assessed using CAT is recommended to provide a comprehensive insight into prothrombotic plasma properties.²⁷ Hilhorst et al²⁸ using the CAT assay showed increased thrombin potential in patients with AAV in remission, which, to our knowledge, was the only study regarding this issue. The majority of patients were diagnosed with GPA. The authors also demonstrated a significant correlation of ETP with the plasma activity of factor VIII, interpreted as a marker of endothelial cell activation and dysfunction. In our study, ETP showed positive correlation with fibrinogen level—one of the acute phase reactants.

Plasma fibrinolytic potential

To assess overall plasma fibrinolytic activity, CLT was used, showing clearly prolonged lysis time in all patients with GPA as compared with healthy controls. The only other study in the literature examining clot lysis in AAV involved patients with eosinophilic GPA and showed denser fibrin clots and prolonged lysis time in patients with vasculitis.²⁹ In GPA, such prothrombotic clot phenotype with impaired clot lysis may be, at least partially, explained by the presence of anti-plasminogen autoantibodies in patients positive for anti–PR3-ANCA.^30,31 These antibodies were shown to delay conversion of plasminogen to plasmin and increase lysis time of fibrin clots.²⁹ At the same time, however, elevated circulating levels of D-dimer indicate constant ongoing fibrin lysis in patients with GPA, significantly more pronounced in exacerbation. It may indicate increased fibrin deposition and turnover with a resulting hypercoagulable state, most pronounced in GPA exacerbation, as already suggested by Ma et al.³²

Endothelial dysfunction

Patients with AAV are at increased risk of venous thromboembolism especially when the disease is exacerbated.^3-7 Such an increased risk of arterial events in AVV has also been reported.^8,9 Our findings may explain major prothrombotic mechanisms leading to thrombosis in GPA involving both increased thrombin generation and impaired fibrinolysis. Exact mechanisms probably involve multiple pathways, activated by autoimmune inflammation and tightly related to the activation and dysfunction of endothelial cells with an initiation and propagation of thrombosis.¹⁶ In keeping with the concept of immunothrombosis, our results indirectly indicate endothelial dysfunction in patients with GPA (eg, increased factor VIII activity and thrombomodulin levels), both in the exacerbation phase and in remission of their disease. Positive associations shown between thrombomodulin levels and D-dimers, activity of factor VIII, fibrinogen, and BVAS indicate an interplay between endothelial dysfunction, disease activity, inflammation, and hypercoagulability all typical of the process of thromboinflammation.¹⁶ It also concurs with findings presented in our previous study³³ and those of others³⁴ supporting activation and disturbed endothelial function in patients with GPA.

Limitations

Our study has several limitations. First, the number of patients included in the study was low both in the exacerbation and remission subgroups. Secondly, both subgroups differed regarding their clinical characteristics. Increasing the number and clinical uniformity of study patients would make conclusions more solid. Particularly, more convincing evidence could have been offered for the correlation between hypercoagulability and activity of the disease and other possible associations between parameters of thrombin generation and fibrinolysis, and inflammation markers or other biochemical markers. It is also impossible to assess the real risk of venous thromboembolic events associated with the increase in thrombin generation and impairment of measured fibrinolysis as there was no follow-up. In addition, the possible influence of treatment on measured parameters has not been analyzed. These limitations arise mainly from the fact that GPA is a rare disease. For this reason, the Polish registry for AAV has been created³⁵ to facilitate research in this field. Regardless of all these limitations, the results of the present study clearly show prothrombotic and hypofibrinolytic changes associated with inflammation and endothelial dysfunction in patients with GPA.

In summary, our study shows that in patients with GPA, there is evidence of hypercoagulability which extends from exacerbation to its more prolonged remission phase, which might persistently increase the risk of thromboembolism. It may call for more extended antithrombotic prophylactic measures.

Supplementary material.pdf

Correspondence to

Teresa Iwaniec, PhD, Department of Hematology, Jagiellonian University Medical College, ul. Kopernika 17, 31-501 Kraków, Poland, phone: +48 12 424 76 00, email: teresa.iwaniec@uj.edu.pl

Received

May 3, 2021.

Revision accepted

June 7, 2021.

Published online

June 8, 2021.

Acknowledgments

This study was supported by the National Centre of Science in Poland (no. 2015/17/B/NZ6/03459; to JM).

Contribution statement

TI conceived the concept of the study and contributed to its design as well as to the analysis and interpretation of data: MCL, KWA, JKW, and KW contributed to clinical evaluation of the patients. LZ contributed to the interpretation of data and statistical analyses. MZ analyzed and interpreted data. JM contributed to critical writing and revision of the intellectual content; final approval of the manuscript.

Conflict of interest

None declared.

How to cite

Iwaniec T, Celińska-Löwenhoff M, Zaręba L, et al. Chronic prothrombotic tendency in patients with granulomatosis with polyangiitis. Pol Arch Intern Med. 2021; 131: 666-672. doi:10.20452/pamw.16027

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