Reduced plasma fibrin clot permeability is associated with recurrent thromboembolic events in patients with antiphospholipid syndrome

Reduced plasma fibrin clot permeability is associated with recurrent thromboembolic events in... Abstract Objectives APS is associated with arterial and venous thrombosis. The unfavourable fibrin clot phenotype, including formation of dense and poorly lysable clots, has been reported in thrombotic APS. We investigated whether abnormal plasma clot properties are predictive of recurrent thromboembolism in APS. Methods We followed 126 consecutive patients with thrombotic APS and 105 control subjects, without APS, matched for thrombotic events. Plasma fibrin clot permeability (Ks), turbidity measurements and clot lysis time were evaluated ⩾5 months after a thrombotic event. The primary composite end point was symptomatic recurrent venous thromboembolism, ischaemic stroke and/or myocardial infarction. Results During follow-up (median, 62 months; range 46–74 months; 1183.2 patient-years), the primary outcome was observed in 33 (26.2%) APS patients and 16 (15.2%) controls, including 25 (19.8%) and 14 (13.3%) subjects with recurrent venous thromboembolism, respectively. Reduced Ks and prolonged clot lysis time predicted recurrent thromboembolic events in APS patients [per 1 × 10−9 cm2: hazard ratio (HR) = 0.37; 95% CI: 0.24, 0.56; and per 10 min: HR = 1.20; 95% CI: 1.01, 1.40, respectively] and in controls (per 1×10−9 cm2: HR = 0.23; 95% CI: 0.11, 0.42; and per 10 min: HR = 1.51; 95% CI: 1.08, 2.16, respectively). A multivariate analysis showed that positive IgG and IgM anti-β2 glycoprotein I antibodies, withdrawal of anticoagulation, lower platelet count and reduced Ks predicted thromboembolic events in APS patients. Conclusion Formation of denser fibrin networks could be a novel risk factor for recurrent thromboembolism in APS, which highlights the importance of fibrin phenotype in thrombotic disorders. antiphospholipid syndrome, clot, fibrin, recurrence, thrombosis Rheumatology key messages APS is associated with increased risk of recurrent thromboembolism. Plasma clot properties were tested as potential predictors of recurrent thrombotic events in APS. Low clot permeability and lysability predicted recurrent thromboembolism in APS during 5 years of follow-up. Introduction APS is an autoimmune disease characterized by a hypercoagulable state in the presence of aPL, arterial thrombosis (AT) and/or venous thromboembolism (VTE) or obstetrical complications. APS coexists with other autoimmune diseases, mainly SLE, in 70% of patients, but it may also appear as a primary disorder [1–3]. Venous thrombosis manifests mainly as deep vein thrombosis (DVT), and ∼14% of APS patients would suffer from pulmonary embolism (PE) [4]. AT, predominantly ischaemic cerebrovascular events, occurs in ∼20% of APS patients [5–7]. The group of aPL comprises numerous types of antibodies, including LA, aCL and anti-β2 glycoprotein I (aβ2GPI) antibodies. The positive LA and high titres of aCL and IgG aβ2GPI or high positive titres of these antibodies (so-called triple positive APS) are associated with increased risk of thrombosis [3]. The prothrombotic mechanisms of aPL involve increased thrombin generation [8], endothelial cell activation [9], decreased fibrinolysis, dysregulation of anticoagulant function of β2GPI [10] and increased release of endothelium- and platelet-derived microparticles expressing tissue factor [11]. Some patients who meet the criteria for APS [12] may experience recurrent thrombotic events even when they are treated with vitamin K antagonists (VKAs) to a target international normalized ratio (INR) 2.0–3.0. The risk of recurrent VTE is lower when the INR is >3.0 [13]. The type, level and persistence of circulating aPL are the most important thrombotic risk factors, where LA and combined aPL positivity are associated with the highest risk of thrombosis. Patients with APS have additional cardiovascular risk factors, such as arterial hypertension, cigarette smoking, hypercholesterolaemia, obesity or oestrogen use [14]. In 160 APS patients with triple antibody positivity, the recurrent VTE and AT events were observed in 59 and 68%, respectively, during a mean follow-up of 6 years [14]. In a cohort of 1,000 APS patients followed for 5 years, the overall rate of recurrent thromboembolic events was estimated at 17% in patients with no treatment, 29% in patients on aspirin, 41% in individuals on warfarin with target INR 2.0–3.0 and 13% in those with INR >3.0 [15]. An additional 115 thrombotic events (14.4%) occurred during the subsequent 5 years of follow-up [16]. The Piedmont cohort study [17] showed that discontinuation of anticoagulation, diabetes and inherited thrombophilia associated with a high-risk aPL profile were the risk factors for thrombosis recurrences in APS. Looking for new prothrombotic mechanisms underlying thrombotic APS, we demonstrated the so-called prothrombotic clot phenotype, involving reduced plasma clot permeability and lysability [18]. Similar observations were made by Vikerfors et al. [19]. Reduced clot permeability and susceptibility to lysis were also found in patients with primary APS and increased intima–media thickness in the carotid arteries [20]. Recently, it has been shown that β2GPI has a strong affinity for fibrinogen and induces formation of thinner and shorter fibrin fibres [21]. β2GPI and its antibody complex also affected the viscoelastic properties of a platelet-rich clot [22]. The prothrombotic plasma clot phenotype may predispose to recurrent DVT and PE during long-term follow-up after anticoagulation withdrawal [23, 24]. It is unknown whether certain plasma fibrin clot characteristics can identify APS patients at the highest risk of thrombotic events in the future. Based on several reports on the links of venous and arterial thromboembolic events with unfavourable fibrin properties [25–28], we hypothesized that reduced clot permeability and susceptibility to lysis provide prognostic information in thrombotic APS. Methods Patients We followed 126 APS patients and 105 controls for ⩾46 months from the initial evaluation in a prospective cohort study. The inclusion and exclusion criteria were presented previously [18]. Briefly, the diagnosis of APS was established based on the modified classification criteria [12]. The exclusion criteria were as follows: a thromboembolic event within the previous 3 months, exacerbation of SLE (diagnosed based on ACR classification criteria [29]), acute infection, pregnancy, diabetes mellitus, known cancer and serum creatinine ⩾120 μM. Patients (n = 105) matched by frequency for age, sex, current smoking and the prior thromboembolic events, in whom APS was excluded, served as controls. All subjects and controls were recruited at the same time in two outpatient clinics for patients with coagulation disorders in Krakow, Poland. The study protocol was approved by the Ethics Committee of the Jagiellonian University, and informed consent was obtained from all the participants in accordance with the Declaration of Helsinki. Follow-up Follow-up started at the time of blood collection for fibrin analysis and was carried out on a 6-month basis (a visit at the centres or telephone contact). All subjects were asked about the continuation or interruption of anticoagulant treatment. The duration of anticoagulation and the type of the anticoagulant used, categorized as VKA or non-VKA oral anticoagulant, were recorded. During follow-up, the aPL and LA were assessed at least once in APS patients and yielded positive results, although some decreased substantially. The primary composite end point was the occurrence of any of the following conditions: symptomatic, documented, recurrent DVT; PE; stroke; or myocardial infarction (MI). The secondary end points were recurrent VTE, including DVT and/or PE, and AT defined as ischaemic stroke or MI. The diagnosis of VTE was established based on a positive finding in colour duplex sonography. PE was diagnosed based on clinical symptoms and positive results of high-resolution spiral CT. An unprovoked VTE episode was defined as having no history of cancer, surgery requiring general anaesthesia, major trauma, plaster cast or hospitalization in the last month, pregnancy or delivery in the last 3 months. Stroke was diagnosed based on a new and abrupt neurological deficit lasting ⩾24 h and positive CT or MRI. MI was diagnosed based on a typical chest pain and ECG changes in at least two contiguous leads, and elevated cardiac troponin levels. At the time of a recurrent event, INR was assessed in subjects receiving VKA. The end of follow-up was defined as the date of a recurrent thromboembolic event. Recurrence status was checked on December 15th, 2017. In the absence of any recurrence or death, this date was registered as the end of follow-up. Laboratory investigations At the time of blood sampling, patients previously taking VKAs for ⩾5 months were switched to enoxaparin for 14 days, and blood was collected ⩾12 h after the last injection. Lipid profiles, complete blood count, creatinine, glucose, CRP, INR, fibrinogen, D-dimer, tissue plasminogen activator antigen (tPA: Ag), plasminogen activator inhibitor-1 antigen (PAI-1: Ag) and factor (F) VIII were determined as previously described [18]. Levels of aCL and aβ2GPI antibodies were measured by INOVA kits (San Diego, CA, USA) as described previously [18]. LA was determined as recommended in 2009 [30]. Blood samples for fibrin analyses were mixed with 3.2% citrate (9:1), centrifuged for 20 min and stored at −80°C to allow batch analyses. All measurements performed at the start of the study were analysed without the knowledge of whether a recurrent event had occurred. Plasma fibrin clot parameters were measured as described previously [18]. Briefly, plasma clot permeability was assessed using a pressure-driven system and expressed as a permeation coefficient (Ks), which indicates the pore size in fibrin networks. The lag phase of the turbidity curve and the maximal absorbance at the plateau phase at 450 nm (ΔAbs) were recorded. Clot lysis time (CLT), induced by recombinant tPA added to plasma with tissue factor and phospholipids, was determined. Moreover, D-dimer levels formed during tPA-induced lysis were measured in the effluent up to the collapse of the plasma gel, and maximal rate of increase in D-dimer (D-Drate) with maximal D-dimer concentrations (D-Dmax) were estimated. For details, see the supplementary data methods section, available as at Rheumatology online. Statistical analysis Categorical variables are presented as numbers and percentages. Continuous variables are expressed as the mean (s.d.) or median and interquartile range (IQR). Normality was assessed by the Shapiro–Wilk test. Equality of variances was assessed using Levene’s test. Differences between groups were compared using Student’s or Welch’s t-test depending on the equality of variances for normally distributed variables. The Mann–Whitney U-test was used for non-normally distributed continuous variables. Categorical variables were compared by Pearson’s χ2 test or Fisher’s exact test if 20% of cells had expected count <5. Univariate and multivariate Cox proportional hazard analysis was performed to identify independent predictors of the primary outcome. The multivariate model was fitted using backward stepwise regression. Variables that were associated with the occurrence of VTE, stroke or MI with a significance level of P < 0.2 in the bivariate models were selected for possible inclusion in the multivariate logistic regression model to predict the occurrence of the primary outcome. Receiver operating characteristic curves were analysed to determine the optimal cut-off values for parameters. To analyse survival in selected risk groups, Kaplan–Meier curves were drawn. The log-rank statistic was used to test the differences in outcome between the groups. All statistical analyses were performed with JMP, version 13.1.0 (SAS Institute Inc., Cary, NC, USA). Results Patient characteristics Baseline characteristics of 124 APS patients and 105 control subjects after ⩾3.8 years of follow-up are summarized in Table 1. Of the 124 APS patients, 100 (80.6%) had primary and 24 (19.4%) secondary APS associated with SLE. In all 24 patients SLE was in remission, and in 20 subjects CS treatment was continued and the daily doses ranged between 2.5 and 5 mg of prednisone or its equivalent. Sixty-three (50.8%) APS patients had positive LA, and 42 (33.9%) had triple positive APS. Most of APS patients (n = 107, 86.3%) had a history of VTE. Table 1 Baseline characteristics of the APS patients and controls Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Values are the median (interquartile range) unless otherwise stated. aβ2GPI: anti-β2-glycoprotein antibodies; ACE-I: angiotensin-converting enzyme inhibitor; ΔAbs: maximal absorbance of fibrin gel at 405 nm; CLT: clot lysis time; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; DVT: deep vein thrombosis; FVIII: factor VIII; GPL: IgG phospholipid unit; HDL-C: high-density lipoprotein cholesterol; hsCRP: high-sensitivity CRP; Ks: permeability coefficient; LDL-C: low-density lipoprotein cholesterol; MI: myocardial infarction; MPL: IgM phospholipid unit; PAI-1: Ag: plasminogen activator inhibitor-1 antigen; PE: pulmonary embolism; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; TC: total cholesterol; TG: triglycerides; tHcy: total homocysteine; tPA: Ag: tissue-type plasminogen activator antigen; VTE: venous thromboembolism. Table 1 Baseline characteristics of the APS patients and controls Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Values are the median (interquartile range) unless otherwise stated. aβ2GPI: anti-β2-glycoprotein antibodies; ACE-I: angiotensin-converting enzyme inhibitor; ΔAbs: maximal absorbance of fibrin gel at 405 nm; CLT: clot lysis time; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; DVT: deep vein thrombosis; FVIII: factor VIII; GPL: IgG phospholipid unit; HDL-C: high-density lipoprotein cholesterol; hsCRP: high-sensitivity CRP; Ks: permeability coefficient; LDL-C: low-density lipoprotein cholesterol; MI: myocardial infarction; MPL: IgM phospholipid unit; PAI-1: Ag: plasminogen activator inhibitor-1 antigen; PE: pulmonary embolism; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; TC: total cholesterol; TG: triglycerides; tHcy: total homocysteine; tPA: Ag: tissue-type plasminogen activator antigen; VTE: venous thromboembolism. APS patients compared with controls had 12.3% lower Ks, 5% shorter lag phase, similar ΔAbs, 9.8% longer CLT, 4.5% higher D-Dmax and similar D-Drate (Table 1). Plasma fibrin clots from patients with positive LA (n = 63), compared with those from LA-negative APS patients, had 7.25% lower Ks (P = 0.005), 10.4% longer CLT (P = 0.003) and 7.14% slower rate of D-dimer release (P = 0.004). Triple antibody positivity was associated with 5.9% lower Ks (P = 0.006) and 6.7% slower D-Drate (P = 0.008), and other fibrin variables were similar to those measured in the remaining patients. Thromboembolic events during follow-up The median follow-up was 62 months (range 46–74 months), resulting in 1183.2 patient-years. One patient with APS was lost to follow-up and one died of a brain tumour, whereas no control subjects were lost. Twenty-four APS patients (19.4%) and 40 controls (38.1%) stopped anticoagulation (P = 0.0016). Twenty-eight (22.6%) APS patients and seven controls (6.7%) took aspirin, including three subjects with APS (2.4%) on aspirin in combination with VKA (Table 2). Table 2 Follow-up characteristics of APS patients vs controls Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Values are given as n (%) unless stated otherwise. DVT: deep vein thrombosis; NOAC: non-vitamin K oral anticoagulants; MI: myocardial infarction; PE: pulmonary embolism; VKA: vitamin K antagonists; VTE: venous thromboembolism. Table 2 Follow-up characteristics of APS patients vs controls Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Values are given as n (%) unless stated otherwise. DVT: deep vein thrombosis; NOAC: non-vitamin K oral anticoagulants; MI: myocardial infarction; PE: pulmonary embolism; VKA: vitamin K antagonists; VTE: venous thromboembolism. The primary composite end points, that is, VTE or AT episodes, were observed in 33 APS patients and in 16 controls [hazard ratio (HR) = 2.02; 95% CI: 1.04, 3.92; P = 0.039). Recurrent VTE was recorded in 25 APS patients (20.2%; 3.87/100 patient-years; HR = 1.64; 95% CI: 0.80, 3.35; P = 0.17) including 22 DVT (17.7%; 4.40/100 patient-years), 6 PE (4.8%; 0.93/100 patient-years) and 3 DVT combined with PE episodes (2.4%; 0.46/100 patient-years). In control subjects, we observed 14 VTE episodes [13.3%; 2.62/100 patient-years; 12 DVT (11.4%; 2.24/100 patient-years), 5 PE (4.8%; 0.94/100 patient-years) and 3 DVT combined with PE (2.9%; 0.56/100 patient-years); Table 3]. Table 3 Characteristics of APS patients and controls with and without recurrent VTE and AT episodes Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 a Median (IQR). b Mean (s.d.). aβ2GPI: anti-β2-glycoprotein antibodies; AT: arterial thrombosis; CLT: clot lysis time; ΔAbs: maximal absorbance of fibrin gel at 405 nm; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; GPL: IgG phospholipid unit; IQR: interquartile range; Ks: permeability coefficient; MPL: IgM phospholipid unit; n: number; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; VTE: venous thromboembolism. Table 3 Characteristics of APS patients and controls with and without recurrent VTE and AT episodes Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 a Median (IQR). b Mean (s.d.). aβ2GPI: anti-β2-glycoprotein antibodies; AT: arterial thrombosis; CLT: clot lysis time; ΔAbs: maximal absorbance of fibrin gel at 405 nm; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; GPL: IgG phospholipid unit; IQR: interquartile range; Ks: permeability coefficient; MPL: IgM phospholipid unit; n: number; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; VTE: venous thromboembolism. Among APS patients, 16 VTE recurrences were unprovoked (64%), and in the control group eight events were unprovoked (57%, P = 0.67). Seven APS patients (5.6%; 1.08/100 patient-years) and two controls (1.9%; 0.37/100 patient-years) experienced stroke during follow-up (P = 0.18). One stroke in an APS patient and one in a control subject was a recurrent event. One subject from the APS group (0.8%; 0.15/100 patient-years) and two of the controls (1.9%; 0.37/100 patient-years) experienced MI during follow-up (Table 3). Stroke combined with recurrent VTE was recorded in two control subjects (1.9%; 0.37/100 patient-years) and in two APS patients (1.6%; 0.31/100 patient-years). As expected, the rate of the primary end point in the APS group was higher in subjects who stopped anticoagulation (HR = 3.39; 95% CI: 1.61, 6.80; P = 0.0019). No such observation was made in the control group (HR = 1.32; 95% CI: 0.45, 3.88; P = 0.61). The controls with thromboembolic events during follow-up who discontinued anticoagulation were younger than those continuing treatment [37 (28–46) vs 45 (32–52) years; P = 0.017], and the time of anticoagulation was shorter in the former group [17 (12–20) vs 24 (19.5–28.5) months; P = 0.031]. The cessation of anticoagulant treatment increased the combined risk of VTE, stroke or MI by 47.1% at 30 months and by 58.8% at 40 months in the APS patients (P < 0.0001). In 20 (60.6%) APS patients who experienced thromboembolism during follow-up, the thromboembolic event occurred during VKA treatment, including 13 subjects in whom adequate anticoagulation was recorded at the time of this outcome. Fibrin clot properties and thromboembolic events during follow-up Baseline Ks was 11.6% lower in APS patients and 18.9% lower in controls who experienced the primary end point during follow-up (Fig. 1A), along with 13 and 11% prolonged CLT (Fig. 1B), respectively. APS patients with thrombotic events during follow-up had 4.9% higher ΔAbs than APS patients without such events. Ks and CLT predicted venous or arterial events in the APS group also after adjustment for fibrinogen (per 1 × 10−9cm2: HR = 0.37, 95% CI: 0.24, 0.56; P < 0.0001; and per 10 min: HR = 1.20, 95% CI: 1.012, 1.40; P = 0.037, respectively]. Other fibrin variables were similar in APS patients with thrombotic events during follow-up and those free of such events. Fig. 1 View largeDownload slide Fibrin clot permeability (A) and clot lysis time (B) in patients and controls during follow-up Differences were considered statistically significant for P < 0.05, according to the Mann–Whitney U-test. Fig. 1 View largeDownload slide Fibrin clot permeability (A) and clot lysis time (B) in patients and controls during follow-up Differences were considered statistically significant for P < 0.05, according to the Mann–Whitney U-test. Receiver operator characteristic analysis showed high accuracy for Ks, CLT, lag phase, ΔAbs and D-Dmax to predict VTE, stroke or MI during follow-up in APS patients (supplementary Table S1, available at Rheumatology online). Kaplan–Meier analysis showed that four of the six plasma clot variables, that is, reduced Ks, prolonged CLT, higher D-Dmax and higher ΔAbs were predictive of recurrent VTE and AT in APS patients, whereas in controls only lower Ks, prolonged CLT and higher ΔAbs had such predictive value (Fig. 2). Fig. 2 View largeDownload slide Event rates in APS patients with recurrent thromboembolic events with regard to fibrin clot properties Fibrin clot permeability (Ks; log-rank P < 0.0001; A), clot lysis time (CLT; log-rank P = 0.0002; B), maximal absorbance of fibrin gel at 405 nm (ΔAbs; log-rank P = 0.0005; C) and maximal D-dimer levels in the lysis assay (D-Dmax; log-rank P = 0.031; D). Fig. 2 View largeDownload slide Event rates in APS patients with recurrent thromboembolic events with regard to fibrin clot properties Fibrin clot permeability (Ks; log-rank P < 0.0001; A), clot lysis time (CLT; log-rank P = 0.0002; B), maximal absorbance of fibrin gel at 405 nm (ΔAbs; log-rank P = 0.0005; C) and maximal D-dimer levels in the lysis assay (D-Dmax; log-rank P = 0.031; D). Univariate analysis showed that Ks ⩽6.3 × 10−9cm2, ΔAbs >0.85, CLT >116 and D-Dmax >3.96 mg/l min were associated with thromboembolic events in APS during follow-up, also after adjustment for fibrinogen (supplementary Table S2, available at Rheumatology online). Decreased high-density lipoprotein cholesterol (HDL-C) and lower platelet count were associated with increased risk of thromboembolic events in APS patients (Table 4). A multivariate analysis showed that positive IgM and IgG aβ2GPI, anticoagulation withdrawal, lower platelet count and reduced Ks independently predicted VTE, stroke or MI in APS during follow-up (Table 4). Table 4 Cox regression models for risk factors of thromboembolic events (VTE, stroke and MI) recurrence in APS Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 a Multivariate model was fitted using backward stepwise regression, with fibrinogen locked in the model. aβ2GPI: anti-β2-glycoprotein antibodies; HR: hazard ratio; HDL-C: high-density lipoprotein cholesterol; Ks: permeability coefficient; MI: myocardial infarction; NOAC: non-vitamin K oral anticoagulants; VKA: vitamin K antagonists; VTE: venous thromboembolism. Table 4 Cox regression models for risk factors of thromboembolic events (VTE, stroke and MI) recurrence in APS Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 a Multivariate model was fitted using backward stepwise regression, with fibrinogen locked in the model. aβ2GPI: anti-β2-glycoprotein antibodies; HR: hazard ratio; HDL-C: high-density lipoprotein cholesterol; Ks: permeability coefficient; MI: myocardial infarction; NOAC: non-vitamin K oral anticoagulants; VKA: vitamin K antagonists; VTE: venous thromboembolism. In the control group, recurrent venous or arterial thromboembolic events were independently predicted by platelet count (per 10 × 103/µl: HR = 1.11; 95% CI: 1.004, 1.22; P = 0.041), Ks (per 1 × 10−9cm2: HR = 0.23, 95% CI: 0.11, 0.42; P < 0.0001) and CLT (per 10 min: HR = 1.51, 95% CI: 1.08, 2.16; P = 0.015). Discussion To our knowledge, this study is the first to demonstrate that a key feature of the prothrombotic plasma fibrin clot phenotype could predict the recurrence of thromboembolic venous and arterial events in APS patients during 5 years of follow-up, regardless of the anticoagulation status. It suggests that unfavourably altered plasma clot properties observed in APS could be new laboratory predictive risk factors of recurrent thrombosis in APS apart from the high-risk aPL profile, discontinuation of anticoagulation and cardiovascular risk factors [14–17]. Given that the architecture of the fibrin fibres reflects the impact of several genetic and environmental factors [31–34], Ks might be a good marker of a prothrombotic state, in some clinical settings better than, for example, D-dimer. The present study provides new insights into the clinical relevance of alterations to fibrin clot characteristics in APS. Fibrin architecture is a crucial factor that determines clot mechanical stability and its resistance to fibrinolysis [31–34]. We showed that CLT is a predictor of VTE, stroke or MI during follow-up of APS patients. It has been reported that prolonged CLT increased the risk of a first VTE [35]. The present study indicates that CLT can also predict recurrent thrombotic episodes in APS. Reduced Ks has been reported to be associated with the recurrence of cerebral venous sinus thrombosis [36], DVT [23] and PE [24]. It might be postulated that APS is another disease in which the plasma fibrin clot phenotype could provide prognostic information. A pilot international study on the standardization of Ks measurement showed potential benefits from a standardized protocol for this method [37], which suggests that measurement of Ks could be useful in clinical practice, especially after implementation of automated analysis [38]. Large multicentre studies are needed to validate this concept. In our earlier study, we showed that in APS clot permeability and lysis time were independently predicted by positive LA and triple antibody positivity [18]. In the present follow-up study, the only antibodies that independently increased the risk of recurrent thromboembolic events in APS were positive aβ2GPI, which is in line with the study by Neville et al. [39], who showed in a prospective cohort of 415 persons at a high risk of aPL positivity that positive IgM and IgG aβ2GPI predicted new vascular events over a median time of 7.4 years. The recent in vitro findings support our observation [21]. Lower HDL-C was also an independent risk factor for recurrent thrombosis in our APS patients. Eichinger et al. [40] showed that patients after a first VTE with high levels of apolipoprotein AI and HDL-C had a decreased risk of recurrent thrombosis. It has been speculated that IgG antibodies against HDL in APS patients might limit the antioxidant effect of HDL, favouring low-density lipoprotein oxidation [41]. We have also shown that a lower platelet count independently predicted recurrent thromboembolic events in APS patients. Thrombocytopenia might be a risk factor for developing thrombosis in aPL-positive patients without previous thrombotic manifestations [42]. We observed that thrombocytopenia also has a predictive value among APS patients with previous thromboembolism. However, Ks helps to identify APS patients at risk of recurrent thromboembolism, independently of platelet count, which remains a new observation. Withdrawal of anticoagulant treatment was associated with a 3-fold higher risk of thrombosis recurrence in APS patients. Of note, a significant proportion of the APS patients stopped the anticoagulant treatment despite our recommendations to maintain anticoagulation, which highlights everyday problems with long-term therapy associated with several limitations and an urgent need for safer anticoagulant agents. Interestingly, our controls who stopped the treatment after a median of 17 months of anticoagulation were younger, and they did not have the higher rate of VTE recurrences. It should be noted that thromboembolism recurs especially within the first 6–12 months after the initial event, and an increase in age by a decade was reported to be an independent predictor for the first overall VTE recurrence [43]. Our study confirms that APS is a potent risk factor for recurrent thrombosis, and a lifetime anticoagulation, preferentially with VKA, is recommended [44]. Given that we observed thromboembolic events during follow-up while on anticoagulation with INR values in the range of 2–3, a higher target INR of ∼3–3.5 or the combination of VKA and aspirin might be considered to some APS patients at an extremely high thromboembolic risk, in particular with AT, although such regimens are associated with increased bleeding risk [45]. Efforts to maintain long-term anticoagulation in APS after thromboembolic events and to improve patient compliance are of paramount importance [46]. This study has several limitations. First, the number of patients was limited; however, the groups were well matched, the diagnosis of APS was confirmed by the repeated assessment of aPL during the follow-up, and only one patient was lost to follow-up. The study was also adequately powered, although the subgroup analysis should be interpreted with caution. Second, our analysis was based on a determination of fibrin clot variables at a single time point, so we cannot exclude some changes in clot properties over time. We did not evaluate thrombin generation, which is a well-established marker of a hypercoagulable state and can predict VTE recurrence [47]; however, data on thrombin generation in APS are conflicting [48]. We did not measure anti-factor Xa activity to confirm that the patients switched to low-molecular weight heparin had no residual anticoagulant effects; however, such effects are rather unlikely >12 h after the last injection [49]. We realize that CS treatment might have an additional prothrombotic effect [50]; however, we did not observe any associations between the events and such therapy. The issue of the impact of CSs on thromboembolism in the context of the plasma fibrin clot phenotype deserves further studies. The present study should be perceived as hypothesis generating, which requires further investigation on a larger cohort of APS patients to be validated. To conclude, we demonstrated that impaired fibrin clot phenotype, especially lower plasma clot permeability, is associated with increased risk of recurrent thromboembolic events in APS. It might be speculated that screening for clot permeability as a key measure of the prothrombotic fibrin clot phenotype in APS might identify subjects at increased thrombotic risk and help to establish an individual therapeutic plan. 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Reduced plasma fibrin clot permeability is associated with recurrent thromboembolic events in patients with antiphospholipid syndrome

Rheumatology , Volume Advance Article (8) – Apr 17, 2018

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Abstract

Abstract Objectives APS is associated with arterial and venous thrombosis. The unfavourable fibrin clot phenotype, including formation of dense and poorly lysable clots, has been reported in thrombotic APS. We investigated whether abnormal plasma clot properties are predictive of recurrent thromboembolism in APS. Methods We followed 126 consecutive patients with thrombotic APS and 105 control subjects, without APS, matched for thrombotic events. Plasma fibrin clot permeability (Ks), turbidity measurements and clot lysis time were evaluated ⩾5 months after a thrombotic event. The primary composite end point was symptomatic recurrent venous thromboembolism, ischaemic stroke and/or myocardial infarction. Results During follow-up (median, 62 months; range 46–74 months; 1183.2 patient-years), the primary outcome was observed in 33 (26.2%) APS patients and 16 (15.2%) controls, including 25 (19.8%) and 14 (13.3%) subjects with recurrent venous thromboembolism, respectively. Reduced Ks and prolonged clot lysis time predicted recurrent thromboembolic events in APS patients [per 1 × 10−9 cm2: hazard ratio (HR) = 0.37; 95% CI: 0.24, 0.56; and per 10 min: HR = 1.20; 95% CI: 1.01, 1.40, respectively] and in controls (per 1×10−9 cm2: HR = 0.23; 95% CI: 0.11, 0.42; and per 10 min: HR = 1.51; 95% CI: 1.08, 2.16, respectively). A multivariate analysis showed that positive IgG and IgM anti-β2 glycoprotein I antibodies, withdrawal of anticoagulation, lower platelet count and reduced Ks predicted thromboembolic events in APS patients. Conclusion Formation of denser fibrin networks could be a novel risk factor for recurrent thromboembolism in APS, which highlights the importance of fibrin phenotype in thrombotic disorders. antiphospholipid syndrome, clot, fibrin, recurrence, thrombosis Rheumatology key messages APS is associated with increased risk of recurrent thromboembolism. Plasma clot properties were tested as potential predictors of recurrent thrombotic events in APS. Low clot permeability and lysability predicted recurrent thromboembolism in APS during 5 years of follow-up. Introduction APS is an autoimmune disease characterized by a hypercoagulable state in the presence of aPL, arterial thrombosis (AT) and/or venous thromboembolism (VTE) or obstetrical complications. APS coexists with other autoimmune diseases, mainly SLE, in 70% of patients, but it may also appear as a primary disorder [1–3]. Venous thrombosis manifests mainly as deep vein thrombosis (DVT), and ∼14% of APS patients would suffer from pulmonary embolism (PE) [4]. AT, predominantly ischaemic cerebrovascular events, occurs in ∼20% of APS patients [5–7]. The group of aPL comprises numerous types of antibodies, including LA, aCL and anti-β2 glycoprotein I (aβ2GPI) antibodies. The positive LA and high titres of aCL and IgG aβ2GPI or high positive titres of these antibodies (so-called triple positive APS) are associated with increased risk of thrombosis [3]. The prothrombotic mechanisms of aPL involve increased thrombin generation [8], endothelial cell activation [9], decreased fibrinolysis, dysregulation of anticoagulant function of β2GPI [10] and increased release of endothelium- and platelet-derived microparticles expressing tissue factor [11]. Some patients who meet the criteria for APS [12] may experience recurrent thrombotic events even when they are treated with vitamin K antagonists (VKAs) to a target international normalized ratio (INR) 2.0–3.0. The risk of recurrent VTE is lower when the INR is >3.0 [13]. The type, level and persistence of circulating aPL are the most important thrombotic risk factors, where LA and combined aPL positivity are associated with the highest risk of thrombosis. Patients with APS have additional cardiovascular risk factors, such as arterial hypertension, cigarette smoking, hypercholesterolaemia, obesity or oestrogen use [14]. In 160 APS patients with triple antibody positivity, the recurrent VTE and AT events were observed in 59 and 68%, respectively, during a mean follow-up of 6 years [14]. In a cohort of 1,000 APS patients followed for 5 years, the overall rate of recurrent thromboembolic events was estimated at 17% in patients with no treatment, 29% in patients on aspirin, 41% in individuals on warfarin with target INR 2.0–3.0 and 13% in those with INR >3.0 [15]. An additional 115 thrombotic events (14.4%) occurred during the subsequent 5 years of follow-up [16]. The Piedmont cohort study [17] showed that discontinuation of anticoagulation, diabetes and inherited thrombophilia associated with a high-risk aPL profile were the risk factors for thrombosis recurrences in APS. Looking for new prothrombotic mechanisms underlying thrombotic APS, we demonstrated the so-called prothrombotic clot phenotype, involving reduced plasma clot permeability and lysability [18]. Similar observations were made by Vikerfors et al. [19]. Reduced clot permeability and susceptibility to lysis were also found in patients with primary APS and increased intima–media thickness in the carotid arteries [20]. Recently, it has been shown that β2GPI has a strong affinity for fibrinogen and induces formation of thinner and shorter fibrin fibres [21]. β2GPI and its antibody complex also affected the viscoelastic properties of a platelet-rich clot [22]. The prothrombotic plasma clot phenotype may predispose to recurrent DVT and PE during long-term follow-up after anticoagulation withdrawal [23, 24]. It is unknown whether certain plasma fibrin clot characteristics can identify APS patients at the highest risk of thrombotic events in the future. Based on several reports on the links of venous and arterial thromboembolic events with unfavourable fibrin properties [25–28], we hypothesized that reduced clot permeability and susceptibility to lysis provide prognostic information in thrombotic APS. Methods Patients We followed 126 APS patients and 105 controls for ⩾46 months from the initial evaluation in a prospective cohort study. The inclusion and exclusion criteria were presented previously [18]. Briefly, the diagnosis of APS was established based on the modified classification criteria [12]. The exclusion criteria were as follows: a thromboembolic event within the previous 3 months, exacerbation of SLE (diagnosed based on ACR classification criteria [29]), acute infection, pregnancy, diabetes mellitus, known cancer and serum creatinine ⩾120 μM. Patients (n = 105) matched by frequency for age, sex, current smoking and the prior thromboembolic events, in whom APS was excluded, served as controls. All subjects and controls were recruited at the same time in two outpatient clinics for patients with coagulation disorders in Krakow, Poland. The study protocol was approved by the Ethics Committee of the Jagiellonian University, and informed consent was obtained from all the participants in accordance with the Declaration of Helsinki. Follow-up Follow-up started at the time of blood collection for fibrin analysis and was carried out on a 6-month basis (a visit at the centres or telephone contact). All subjects were asked about the continuation or interruption of anticoagulant treatment. The duration of anticoagulation and the type of the anticoagulant used, categorized as VKA or non-VKA oral anticoagulant, were recorded. During follow-up, the aPL and LA were assessed at least once in APS patients and yielded positive results, although some decreased substantially. The primary composite end point was the occurrence of any of the following conditions: symptomatic, documented, recurrent DVT; PE; stroke; or myocardial infarction (MI). The secondary end points were recurrent VTE, including DVT and/or PE, and AT defined as ischaemic stroke or MI. The diagnosis of VTE was established based on a positive finding in colour duplex sonography. PE was diagnosed based on clinical symptoms and positive results of high-resolution spiral CT. An unprovoked VTE episode was defined as having no history of cancer, surgery requiring general anaesthesia, major trauma, plaster cast or hospitalization in the last month, pregnancy or delivery in the last 3 months. Stroke was diagnosed based on a new and abrupt neurological deficit lasting ⩾24 h and positive CT or MRI. MI was diagnosed based on a typical chest pain and ECG changes in at least two contiguous leads, and elevated cardiac troponin levels. At the time of a recurrent event, INR was assessed in subjects receiving VKA. The end of follow-up was defined as the date of a recurrent thromboembolic event. Recurrence status was checked on December 15th, 2017. In the absence of any recurrence or death, this date was registered as the end of follow-up. Laboratory investigations At the time of blood sampling, patients previously taking VKAs for ⩾5 months were switched to enoxaparin for 14 days, and blood was collected ⩾12 h after the last injection. Lipid profiles, complete blood count, creatinine, glucose, CRP, INR, fibrinogen, D-dimer, tissue plasminogen activator antigen (tPA: Ag), plasminogen activator inhibitor-1 antigen (PAI-1: Ag) and factor (F) VIII were determined as previously described [18]. Levels of aCL and aβ2GPI antibodies were measured by INOVA kits (San Diego, CA, USA) as described previously [18]. LA was determined as recommended in 2009 [30]. Blood samples for fibrin analyses were mixed with 3.2% citrate (9:1), centrifuged for 20 min and stored at −80°C to allow batch analyses. All measurements performed at the start of the study were analysed without the knowledge of whether a recurrent event had occurred. Plasma fibrin clot parameters were measured as described previously [18]. Briefly, plasma clot permeability was assessed using a pressure-driven system and expressed as a permeation coefficient (Ks), which indicates the pore size in fibrin networks. The lag phase of the turbidity curve and the maximal absorbance at the plateau phase at 450 nm (ΔAbs) were recorded. Clot lysis time (CLT), induced by recombinant tPA added to plasma with tissue factor and phospholipids, was determined. Moreover, D-dimer levels formed during tPA-induced lysis were measured in the effluent up to the collapse of the plasma gel, and maximal rate of increase in D-dimer (D-Drate) with maximal D-dimer concentrations (D-Dmax) were estimated. For details, see the supplementary data methods section, available as at Rheumatology online. Statistical analysis Categorical variables are presented as numbers and percentages. Continuous variables are expressed as the mean (s.d.) or median and interquartile range (IQR). Normality was assessed by the Shapiro–Wilk test. Equality of variances was assessed using Levene’s test. Differences between groups were compared using Student’s or Welch’s t-test depending on the equality of variances for normally distributed variables. The Mann–Whitney U-test was used for non-normally distributed continuous variables. Categorical variables were compared by Pearson’s χ2 test or Fisher’s exact test if 20% of cells had expected count <5. Univariate and multivariate Cox proportional hazard analysis was performed to identify independent predictors of the primary outcome. The multivariate model was fitted using backward stepwise regression. Variables that were associated with the occurrence of VTE, stroke or MI with a significance level of P < 0.2 in the bivariate models were selected for possible inclusion in the multivariate logistic regression model to predict the occurrence of the primary outcome. Receiver operating characteristic curves were analysed to determine the optimal cut-off values for parameters. To analyse survival in selected risk groups, Kaplan–Meier curves were drawn. The log-rank statistic was used to test the differences in outcome between the groups. All statistical analyses were performed with JMP, version 13.1.0 (SAS Institute Inc., Cary, NC, USA). Results Patient characteristics Baseline characteristics of 124 APS patients and 105 control subjects after ⩾3.8 years of follow-up are summarized in Table 1. Of the 124 APS patients, 100 (80.6%) had primary and 24 (19.4%) secondary APS associated with SLE. In all 24 patients SLE was in remission, and in 20 subjects CS treatment was continued and the daily doses ranged between 2.5 and 5 mg of prednisone or its equivalent. Sixty-three (50.8%) APS patients had positive LA, and 42 (33.9%) had triple positive APS. Most of APS patients (n = 107, 86.3%) had a history of VTE. Table 1 Baseline characteristics of the APS patients and controls Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Values are the median (interquartile range) unless otherwise stated. aβ2GPI: anti-β2-glycoprotein antibodies; ACE-I: angiotensin-converting enzyme inhibitor; ΔAbs: maximal absorbance of fibrin gel at 405 nm; CLT: clot lysis time; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; DVT: deep vein thrombosis; FVIII: factor VIII; GPL: IgG phospholipid unit; HDL-C: high-density lipoprotein cholesterol; hsCRP: high-sensitivity CRP; Ks: permeability coefficient; LDL-C: low-density lipoprotein cholesterol; MI: myocardial infarction; MPL: IgM phospholipid unit; PAI-1: Ag: plasminogen activator inhibitor-1 antigen; PE: pulmonary embolism; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; TC: total cholesterol; TG: triglycerides; tHcy: total homocysteine; tPA: Ag: tissue-type plasminogen activator antigen; VTE: venous thromboembolism. Table 1 Baseline characteristics of the APS patients and controls Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Variable APS group (n = 124) Controls (n = 105) P-value Male, n (%) 26 (21.0) 21 (20.0) 0.9 Age, years 40.0 (28.25, 52.75) 41.0 (29.0, 51.0) 0.6 BMI, kg/m2 25.38 (23.51, 28.03) 24.6 (22.7, 26.25) 0.02 Current smokers, n (%) 25 (20.2) 21 (20.0) 1.0 Aspirin, n (%) 37 (29.8) 14 (13.33) 0.004 Statins, n (%) 14 (11.3) 12 (11.4) 1.0 ACE-I, n (%) 29 (23.4) 11 (10.5) 0.008 Stroke, n (%) 28 (22.6) 15 (14.3) 0.1 MI, n (%) 8 (6.5) 7 (6.7) 1.0 DVT, n (%) 91 (73.4) 79 (75.2) 0.8 PE, n (%) 38 (30.7) 40 (38.1) 0.3 Arterial hypertension, n (%) 41 (33.1) 26 (24.8) 0.2 Laboratory parameters     TC, mmol/l 4.96 (4.33, 5.60) 5.05 (4.22, 5.59) 0.7     LDL-C, mmol/l 2.82 (0.93) 2.96 (0.76) 0.2     HDL-C, mmol/l 1.42 (1.1, 1.66) 1.35 (1.1, 1.56) 0.4     TG, mmol/l 1.29 (0.89, 1.88) 1.18 (0.84, 1.68) 0.2     Glucose, mmol/l 4.75 (4.4, 5.10) 4.62 (4.35, 5.0) 0.1     Creatinine, μmol/l 62.5 (55.0, 77.75) 66.8 (61.8, 72.5) 0.09     Fibrinogen, g/l 3.09 (2.44, 3.65) 3.22 (2.67, 3.66) 0.3     hsCRP, mg/l 1.35 (0.88, 2.6) 1.55 (0.9, 2.13) 0.7     D-Dimer, ng/ml 244 (208.5, 315.25) 231 (196, 305) 0.08     tPA: Ag, ng/ml 10.8 (9.2, 12.6) 12.6 (10.4, 13.85) 0.0004     PAI-I: Ag, ng/ml 26.17 (8.76) 27.46 (5.12) 0.1     tHcy, μmol/l 10.05 (8.27, 12.1) 9.2 (7.55, 11.25) 0.02     FVIII, % 125.1 (108.1, 147.05) 119 (102.6, 137.3) 0.09 APS parameters     LA positive, n (%) 63 (50.8) 0 <0.0001     aCL IgG, GPL 34.36 (10.4, 78.6) 5.2 (4.0, 7.45) <0.0001     aCL IgM, MPL 17.56 (11.22, 42.8) 9.4 (6.85, 11.0) <0.0001     aβ2GPI, IgG, SGU 24.1 (3.8, 88.2) 3.0 (2.4, 5.1) <0.0001     aβ2GPI, IgM, SMU 11.2 (4.2, 29.9) 3.4 (2.3, 5.25) <0.0001 Clot characteristics     Ks, 10−9 cm2 6.5 (6.0, 7.0) 7.3 (6.5, 7.75) <0.0001     Lag phase, s 40.0 (38.0, 42.0) 42 (40.0, 45.0) <0.0001     ΔAbs (405 nm) 0.83 (0.77, 0.88) 0.81 (0.78, 0.85) 0.09     CLT, mean (s.d.), min 102.12 (19.34) 92.1 (15.33) <0.0001     D-Dmax, mg/l 4.21 (3.88, 4.52) 4.02 (3.82, 4.3) 0.02     D-Drate, mg/l/min 0.069 (0.065, 0.075) 0.070 (0.068, 0.075) 0.05 Values are the median (interquartile range) unless otherwise stated. aβ2GPI: anti-β2-glycoprotein antibodies; ACE-I: angiotensin-converting enzyme inhibitor; ΔAbs: maximal absorbance of fibrin gel at 405 nm; CLT: clot lysis time; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; DVT: deep vein thrombosis; FVIII: factor VIII; GPL: IgG phospholipid unit; HDL-C: high-density lipoprotein cholesterol; hsCRP: high-sensitivity CRP; Ks: permeability coefficient; LDL-C: low-density lipoprotein cholesterol; MI: myocardial infarction; MPL: IgM phospholipid unit; PAI-1: Ag: plasminogen activator inhibitor-1 antigen; PE: pulmonary embolism; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; TC: total cholesterol; TG: triglycerides; tHcy: total homocysteine; tPA: Ag: tissue-type plasminogen activator antigen; VTE: venous thromboembolism. APS patients compared with controls had 12.3% lower Ks, 5% shorter lag phase, similar ΔAbs, 9.8% longer CLT, 4.5% higher D-Dmax and similar D-Drate (Table 1). Plasma fibrin clots from patients with positive LA (n = 63), compared with those from LA-negative APS patients, had 7.25% lower Ks (P = 0.005), 10.4% longer CLT (P = 0.003) and 7.14% slower rate of D-dimer release (P = 0.004). Triple antibody positivity was associated with 5.9% lower Ks (P = 0.006) and 6.7% slower D-Drate (P = 0.008), and other fibrin variables were similar to those measured in the remaining patients. Thromboembolic events during follow-up The median follow-up was 62 months (range 46–74 months), resulting in 1183.2 patient-years. One patient with APS was lost to follow-up and one died of a brain tumour, whereas no control subjects were lost. Twenty-four APS patients (19.4%) and 40 controls (38.1%) stopped anticoagulation (P = 0.0016). Twenty-eight (22.6%) APS patients and seven controls (6.7%) took aspirin, including three subjects with APS (2.4%) on aspirin in combination with VKA (Table 2). Table 2 Follow-up characteristics of APS patients vs controls Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Values are given as n (%) unless stated otherwise. DVT: deep vein thrombosis; NOAC: non-vitamin K oral anticoagulants; MI: myocardial infarction; PE: pulmonary embolism; VKA: vitamin K antagonists; VTE: venous thromboembolism. Table 2 Follow-up characteristics of APS patients vs controls Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Variable Patients (n = 124) Controls (n = 105) P-value Follow-up time, mean (s.d.), months 62.6 (4.1) 61.1 (5.0) 0.013 VTE, stroke or MI 33 (26.6) 16 (15.2) 0.04 VTE 25 (20.2) 14 (13.3) 0.17 DVT alone 22 (17.7) 12 (11.4) 0.18 PE alone 6 (4.8) 5 (4.8) 0.99 PE + DVT 3 (2.4) 3 (2.9) 0.99 Unprovoked VTE 16 (15.2) 8 (7.6) 0.19 Ischaemic stroke 7 (5.6) 2 (1.9) 0.18 MI 1 (0.8) 2 (1.9) 0.59 VKA 65 (52.4) 20 (19.0) <0.0001 NOAC 30 (24.2) 44 (41.9) 0.0043 Aspirin 28 (22.6) 7 (6.7) 0.0009 Values are given as n (%) unless stated otherwise. DVT: deep vein thrombosis; NOAC: non-vitamin K oral anticoagulants; MI: myocardial infarction; PE: pulmonary embolism; VKA: vitamin K antagonists; VTE: venous thromboembolism. The primary composite end points, that is, VTE or AT episodes, were observed in 33 APS patients and in 16 controls [hazard ratio (HR) = 2.02; 95% CI: 1.04, 3.92; P = 0.039). Recurrent VTE was recorded in 25 APS patients (20.2%; 3.87/100 patient-years; HR = 1.64; 95% CI: 0.80, 3.35; P = 0.17) including 22 DVT (17.7%; 4.40/100 patient-years), 6 PE (4.8%; 0.93/100 patient-years) and 3 DVT combined with PE episodes (2.4%; 0.46/100 patient-years). In control subjects, we observed 14 VTE episodes [13.3%; 2.62/100 patient-years; 12 DVT (11.4%; 2.24/100 patient-years), 5 PE (4.8%; 0.94/100 patient-years) and 3 DVT combined with PE (2.9%; 0.56/100 patient-years); Table 3]. Table 3 Characteristics of APS patients and controls with and without recurrent VTE and AT episodes Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 a Median (IQR). b Mean (s.d.). aβ2GPI: anti-β2-glycoprotein antibodies; AT: arterial thrombosis; CLT: clot lysis time; ΔAbs: maximal absorbance of fibrin gel at 405 nm; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; GPL: IgG phospholipid unit; IQR: interquartile range; Ks: permeability coefficient; MPL: IgM phospholipid unit; n: number; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; VTE: venous thromboembolism. Table 3 Characteristics of APS patients and controls with and without recurrent VTE and AT episodes Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 Variable APS patients with VTE or AT (n = 33) APS patients without VTE or AT (n = 91) P-value Controls with VTE or AT (n = 16) Controls without VTE or AT (n = 89) P-value Male, n (%) 6 (18.2) 20 (22.0) 0.80 6 (37.5) 15 (16.9) 0.09 Age, median (IQR), years 36 (26–46.5) 42 (29–53) 0.14 45.5 (30.5–53.75) 41 (29.5–51) 0.61 BMI, kg/m2 25.3 (22.6–27.0)a 25.50 (23.8–28.5)a 0.61 25.0 (2.9)b 24.5 (2.5)b 0.41 Current smokers, n (%) 6 (18.2) 20 (20.9) 0.81 6 (37.5) 15 (16.9) 0.09 Interruption of treatment, median (IQR), months 5 (1.25–34.5) 30 (19.5–49.25) 0.08 10 (8–12) 18 (13.5–23.5) 0.02 Platelets, 103/µl 163 (108.5–190.5)a 216 (171–260)a 0.0001 252.9 (44.2)b 224.5 (51.4)b 0.04 APS parameters LA positive, n (%) 18 (54.6) 45 (49.5) 0.69 0 (0) 0 (0) – aCL IgG, GPL, median (IQR) 44.8 (9.3–90.3) 33.1 (10.5–75.2) 0.69 5.1 (2.4–7.0) 5.2 (4.0–7.6) 0.29 aCL IgM, MPL 22.4 (10.8–72.7)a 15.2 (11.2–40.2)a 0.09 8.7 (2.7)b 9.1 (2.8)b 0.65 aβ2GPI, IgG, SGU, median (IQR) 44.7 (4.0–102.1) 19.55 (3.8–84.5) 0.28 3.1 (2.0–5.1) 3.0 (2.4–5.1) 0.97 aβ2GPI, IgM, SMU, median (IQR) 27.6 (4.7–50.5) 8.9 (3.9–21.0) 0.011 3.9 (2.1–4.9) 3.4 (2.3–5.4) 0.73 Clot characteristics Ks, 10−9 cm2 5.96 (0.75)b 6.74 (0.75)b <0.001 6.00 (5.65–6.68)a 7.40 (6.90–7.90)a <0.001 Lag phase, median (IQR), s 39.0 (37.5–41.0) 40.0 (38.0–41.0) 0.12 41.0 (39.3–45.0) 42.0 (40.0–45.0) 0.49 ΔAbs, 405 nm, mean (s.d.) 0.86 (0.07) 0.82 (0.06) 0.0035 0.83 (0.06) 0.81 (0.05) 0.10 CLT, min 112 (89.5–124)a 99 (86–110)a 0.016 101 (14)b 91 (15)b 0.015 D-Dmax, mg/l 4.17 (0.58)b 4.25 (0.45)b 0.42 4.00 (3.80–4.27)a 4.02 (3.84–4.31)a 0.95 D-Drate, mg/l/min 0.069 (0.064–0.075)a 0.069 (0.065–0.075)a 0.45 0.072 (0.005)b 0.071 (0.005)b 0.63 a Median (IQR). b Mean (s.d.). aβ2GPI: anti-β2-glycoprotein antibodies; AT: arterial thrombosis; CLT: clot lysis time; ΔAbs: maximal absorbance of fibrin gel at 405 nm; D-Dmax: maximal D-dimer levels in the lysis assay; D-Drate: maximal rate of increase in D-dimer levels in the lysis assay; GPL: IgG phospholipid unit; IQR: interquartile range; Ks: permeability coefficient; MPL: IgM phospholipid unit; n: number; SGU: standard IgG β-2 glycoprotein unit; SMU: standard IgM β-2 glycoprotein unit; VTE: venous thromboembolism. Among APS patients, 16 VTE recurrences were unprovoked (64%), and in the control group eight events were unprovoked (57%, P = 0.67). Seven APS patients (5.6%; 1.08/100 patient-years) and two controls (1.9%; 0.37/100 patient-years) experienced stroke during follow-up (P = 0.18). One stroke in an APS patient and one in a control subject was a recurrent event. One subject from the APS group (0.8%; 0.15/100 patient-years) and two of the controls (1.9%; 0.37/100 patient-years) experienced MI during follow-up (Table 3). Stroke combined with recurrent VTE was recorded in two control subjects (1.9%; 0.37/100 patient-years) and in two APS patients (1.6%; 0.31/100 patient-years). As expected, the rate of the primary end point in the APS group was higher in subjects who stopped anticoagulation (HR = 3.39; 95% CI: 1.61, 6.80; P = 0.0019). No such observation was made in the control group (HR = 1.32; 95% CI: 0.45, 3.88; P = 0.61). The controls with thromboembolic events during follow-up who discontinued anticoagulation were younger than those continuing treatment [37 (28–46) vs 45 (32–52) years; P = 0.017], and the time of anticoagulation was shorter in the former group [17 (12–20) vs 24 (19.5–28.5) months; P = 0.031]. The cessation of anticoagulant treatment increased the combined risk of VTE, stroke or MI by 47.1% at 30 months and by 58.8% at 40 months in the APS patients (P < 0.0001). In 20 (60.6%) APS patients who experienced thromboembolism during follow-up, the thromboembolic event occurred during VKA treatment, including 13 subjects in whom adequate anticoagulation was recorded at the time of this outcome. Fibrin clot properties and thromboembolic events during follow-up Baseline Ks was 11.6% lower in APS patients and 18.9% lower in controls who experienced the primary end point during follow-up (Fig. 1A), along with 13 and 11% prolonged CLT (Fig. 1B), respectively. APS patients with thrombotic events during follow-up had 4.9% higher ΔAbs than APS patients without such events. Ks and CLT predicted venous or arterial events in the APS group also after adjustment for fibrinogen (per 1 × 10−9cm2: HR = 0.37, 95% CI: 0.24, 0.56; P < 0.0001; and per 10 min: HR = 1.20, 95% CI: 1.012, 1.40; P = 0.037, respectively]. Other fibrin variables were similar in APS patients with thrombotic events during follow-up and those free of such events. Fig. 1 View largeDownload slide Fibrin clot permeability (A) and clot lysis time (B) in patients and controls during follow-up Differences were considered statistically significant for P < 0.05, according to the Mann–Whitney U-test. Fig. 1 View largeDownload slide Fibrin clot permeability (A) and clot lysis time (B) in patients and controls during follow-up Differences were considered statistically significant for P < 0.05, according to the Mann–Whitney U-test. Receiver operator characteristic analysis showed high accuracy for Ks, CLT, lag phase, ΔAbs and D-Dmax to predict VTE, stroke or MI during follow-up in APS patients (supplementary Table S1, available at Rheumatology online). Kaplan–Meier analysis showed that four of the six plasma clot variables, that is, reduced Ks, prolonged CLT, higher D-Dmax and higher ΔAbs were predictive of recurrent VTE and AT in APS patients, whereas in controls only lower Ks, prolonged CLT and higher ΔAbs had such predictive value (Fig. 2). Fig. 2 View largeDownload slide Event rates in APS patients with recurrent thromboembolic events with regard to fibrin clot properties Fibrin clot permeability (Ks; log-rank P < 0.0001; A), clot lysis time (CLT; log-rank P = 0.0002; B), maximal absorbance of fibrin gel at 405 nm (ΔAbs; log-rank P = 0.0005; C) and maximal D-dimer levels in the lysis assay (D-Dmax; log-rank P = 0.031; D). Fig. 2 View largeDownload slide Event rates in APS patients with recurrent thromboembolic events with regard to fibrin clot properties Fibrin clot permeability (Ks; log-rank P < 0.0001; A), clot lysis time (CLT; log-rank P = 0.0002; B), maximal absorbance of fibrin gel at 405 nm (ΔAbs; log-rank P = 0.0005; C) and maximal D-dimer levels in the lysis assay (D-Dmax; log-rank P = 0.031; D). Univariate analysis showed that Ks ⩽6.3 × 10−9cm2, ΔAbs >0.85, CLT >116 and D-Dmax >3.96 mg/l min were associated with thromboembolic events in APS during follow-up, also after adjustment for fibrinogen (supplementary Table S2, available at Rheumatology online). Decreased high-density lipoprotein cholesterol (HDL-C) and lower platelet count were associated with increased risk of thromboembolic events in APS patients (Table 4). A multivariate analysis showed that positive IgM and IgG aβ2GPI, anticoagulation withdrawal, lower platelet count and reduced Ks independently predicted VTE, stroke or MI in APS during follow-up (Table 4). Table 4 Cox regression models for risk factors of thromboembolic events (VTE, stroke and MI) recurrence in APS Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 a Multivariate model was fitted using backward stepwise regression, with fibrinogen locked in the model. aβ2GPI: anti-β2-glycoprotein antibodies; HR: hazard ratio; HDL-C: high-density lipoprotein cholesterol; Ks: permeability coefficient; MI: myocardial infarction; NOAC: non-vitamin K oral anticoagulants; VKA: vitamin K antagonists; VTE: venous thromboembolism. Table 4 Cox regression models for risk factors of thromboembolic events (VTE, stroke and MI) recurrence in APS Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 Variable HR (for subgroups or per unit change) Univariate HR (95% CI) P-value Multivariate HR (95% CI)a P-value Male Yes/No 0.77 (0.29, 1.74) 0.55 0.45 (0.15, 1.13) 0.09 Positive IgM+IgG aβ2GPI Yes/No 3.34 (1.66, 6.65) 0.0010 2.32 (1.08, 4.89) 0.03 VKA or NOAC Yes/No 0.57 (0.28, 1.22) 0.14 Interruption of treatment Yes/No 3.39 (1.61, 6.80) 0.0019 3.32 (1.55, 6.85) 0.0027 Platelets 10×103/µl 0.90 (0.85, 0.95) 0.0002 0.93 (0.87, 0.99) 0.0087 HDL-C 1 mmol/l 0.33 (0.12, 0.82) 0.020 Fibrinogen 1 g/l 1.42 (1.00, 1.9) 0.051 1.06 (0.75, 1.51) 0.73 D-dimer 100 ng/ml 0.96 (0.78, 1.01) 0.24 Ks 1×10−9 cm2 0.37 (0.24, 0.56) <0.0001 0.46 (0.28, 0.75) 0.0020 a Multivariate model was fitted using backward stepwise regression, with fibrinogen locked in the model. aβ2GPI: anti-β2-glycoprotein antibodies; HR: hazard ratio; HDL-C: high-density lipoprotein cholesterol; Ks: permeability coefficient; MI: myocardial infarction; NOAC: non-vitamin K oral anticoagulants; VKA: vitamin K antagonists; VTE: venous thromboembolism. In the control group, recurrent venous or arterial thromboembolic events were independently predicted by platelet count (per 10 × 103/µl: HR = 1.11; 95% CI: 1.004, 1.22; P = 0.041), Ks (per 1 × 10−9cm2: HR = 0.23, 95% CI: 0.11, 0.42; P < 0.0001) and CLT (per 10 min: HR = 1.51, 95% CI: 1.08, 2.16; P = 0.015). Discussion To our knowledge, this study is the first to demonstrate that a key feature of the prothrombotic plasma fibrin clot phenotype could predict the recurrence of thromboembolic venous and arterial events in APS patients during 5 years of follow-up, regardless of the anticoagulation status. It suggests that unfavourably altered plasma clot properties observed in APS could be new laboratory predictive risk factors of recurrent thrombosis in APS apart from the high-risk aPL profile, discontinuation of anticoagulation and cardiovascular risk factors [14–17]. Given that the architecture of the fibrin fibres reflects the impact of several genetic and environmental factors [31–34], Ks might be a good marker of a prothrombotic state, in some clinical settings better than, for example, D-dimer. The present study provides new insights into the clinical relevance of alterations to fibrin clot characteristics in APS. Fibrin architecture is a crucial factor that determines clot mechanical stability and its resistance to fibrinolysis [31–34]. We showed that CLT is a predictor of VTE, stroke or MI during follow-up of APS patients. It has been reported that prolonged CLT increased the risk of a first VTE [35]. The present study indicates that CLT can also predict recurrent thrombotic episodes in APS. Reduced Ks has been reported to be associated with the recurrence of cerebral venous sinus thrombosis [36], DVT [23] and PE [24]. It might be postulated that APS is another disease in which the plasma fibrin clot phenotype could provide prognostic information. A pilot international study on the standardization of Ks measurement showed potential benefits from a standardized protocol for this method [37], which suggests that measurement of Ks could be useful in clinical practice, especially after implementation of automated analysis [38]. Large multicentre studies are needed to validate this concept. In our earlier study, we showed that in APS clot permeability and lysis time were independently predicted by positive LA and triple antibody positivity [18]. In the present follow-up study, the only antibodies that independently increased the risk of recurrent thromboembolic events in APS were positive aβ2GPI, which is in line with the study by Neville et al. [39], who showed in a prospective cohort of 415 persons at a high risk of aPL positivity that positive IgM and IgG aβ2GPI predicted new vascular events over a median time of 7.4 years. The recent in vitro findings support our observation [21]. Lower HDL-C was also an independent risk factor for recurrent thrombosis in our APS patients. Eichinger et al. [40] showed that patients after a first VTE with high levels of apolipoprotein AI and HDL-C had a decreased risk of recurrent thrombosis. It has been speculated that IgG antibodies against HDL in APS patients might limit the antioxidant effect of HDL, favouring low-density lipoprotein oxidation [41]. We have also shown that a lower platelet count independently predicted recurrent thromboembolic events in APS patients. Thrombocytopenia might be a risk factor for developing thrombosis in aPL-positive patients without previous thrombotic manifestations [42]. We observed that thrombocytopenia also has a predictive value among APS patients with previous thromboembolism. However, Ks helps to identify APS patients at risk of recurrent thromboembolism, independently of platelet count, which remains a new observation. Withdrawal of anticoagulant treatment was associated with a 3-fold higher risk of thrombosis recurrence in APS patients. Of note, a significant proportion of the APS patients stopped the anticoagulant treatment despite our recommendations to maintain anticoagulation, which highlights everyday problems with long-term therapy associated with several limitations and an urgent need for safer anticoagulant agents. Interestingly, our controls who stopped the treatment after a median of 17 months of anticoagulation were younger, and they did not have the higher rate of VTE recurrences. It should be noted that thromboembolism recurs especially within the first 6–12 months after the initial event, and an increase in age by a decade was reported to be an independent predictor for the first overall VTE recurrence [43]. Our study confirms that APS is a potent risk factor for recurrent thrombosis, and a lifetime anticoagulation, preferentially with VKA, is recommended [44]. Given that we observed thromboembolic events during follow-up while on anticoagulation with INR values in the range of 2–3, a higher target INR of ∼3–3.5 or the combination of VKA and aspirin might be considered to some APS patients at an extremely high thromboembolic risk, in particular with AT, although such regimens are associated with increased bleeding risk [45]. Efforts to maintain long-term anticoagulation in APS after thromboembolic events and to improve patient compliance are of paramount importance [46]. This study has several limitations. First, the number of patients was limited; however, the groups were well matched, the diagnosis of APS was confirmed by the repeated assessment of aPL during the follow-up, and only one patient was lost to follow-up. The study was also adequately powered, although the subgroup analysis should be interpreted with caution. Second, our analysis was based on a determination of fibrin clot variables at a single time point, so we cannot exclude some changes in clot properties over time. We did not evaluate thrombin generation, which is a well-established marker of a hypercoagulable state and can predict VTE recurrence [47]; however, data on thrombin generation in APS are conflicting [48]. We did not measure anti-factor Xa activity to confirm that the patients switched to low-molecular weight heparin had no residual anticoagulant effects; however, such effects are rather unlikely >12 h after the last injection [49]. We realize that CS treatment might have an additional prothrombotic effect [50]; however, we did not observe any associations between the events and such therapy. The issue of the impact of CSs on thromboembolism in the context of the plasma fibrin clot phenotype deserves further studies. The present study should be perceived as hypothesis generating, which requires further investigation on a larger cohort of APS patients to be validated. To conclude, we demonstrated that impaired fibrin clot phenotype, especially lower plasma clot permeability, is associated with increased risk of recurrent thromboembolic events in APS. It might be speculated that screening for clot permeability as a key measure of the prothrombotic fibrin clot phenotype in APS might identify subjects at increased thrombotic risk and help to establish an individual therapeutic plan. 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RheumatologyOxford University Press

Published: Apr 17, 2018

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