J Thromb Thrombolysis DOI 10.1007/s11239-015-1176-8

ABO blood group in primary antiphospholipid syndrome: influence in the site of thrombosis? Nata´lia Mastantuono Nascimento • Sergio Paulo Bydlowski • Rosangela Paula Silva Soares • Danieli Castro Oliveira de Andrade • Eloı´sa Bonfa´ • Luciana Parente Costa Seguro • Eduardo Ferreira Borba

Ó Springer Science+Business Media New York 2015

Abstract Antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or obstetric complications associated with presence of antiphospholipid antibodies (aPL) but additional factors would also induce thrombosis. ABO (H) blood groups are known to be closely related to thrombosis, especially non-O blood type with venous events. The aim of this study was to investigate possible role of ABO (H) blood types in the thrombotic events in primary APS (PAPS). Seventy PAPS patients were selected for the study and were divided according to ABO blood group in: O PAPS (n = 26) and non-O PAPS (n = 44). ABO blood group phenotyping was performed by indirect technique. aPL anticardiolipin (aCL) and anti-beta2 glycoprotein-1 (ab2GPI) and the concentrations and activities of von Willebrand factor (VWF) were measured with ELISA. Lupus anticoagulant (LA) was detected by coagulation assays. A significant higher frequency of venous events was observed in non-O PAPS group (72.7 vs. 46.2 %, p = 0.040). In contrast, the frequency of arterial events was significantly higher in the O PAPS compared to the non-O

N. M. Nascimento  S. P. Bydlowski (&) Laboratory of Genetics and Molecular Hematology (LIM 31), School of Medicine, University of Sa˜o Paulo (USP), Av. Dr. Ene´as de Carvalho Aguiar, 155, 1° andar, sala 43, Sa˜o Paulo, SP 05403-000, Brazil e-mail: [email protected] R. P. S. Soares Fundac¸a˜o Pro´-Sangue Hemocentro de Sa˜o Paulo, Av. Dr. Ene´as de Carvalho Aguiar, 155, 1° andar, Sa˜o Paulo, SP 05403-000, Brazil D. C. O. de Andrade  E. Bonfa´  L. P. C. Seguro  E. F. Borba Rheumatology Division, School of Medicine, University of Sa˜o Paulo (USP), Av. Dr. Arnaldo 455, 3° andar, sala 3190, Sa˜o Paulo, SP 01246-903, Brazil

PAPS group (69.2 vs. 36.4 %, respectively; p = 0.013). Frequencies of aCL, LA, ab2GPI and triple aPL positivity were similar in both groups (p [ 0.05). VWF antigen (75.54 ± 8.68 vs. 79.51 ± 7.07 IU/dl, p = 0.041) and activity (70.23 ± 11.96 vs. 77.92 ± 13.67 %, p = 0.020) were decreased in O PAPS compared to non-O blood group. VWF:CB/VWF:Ag ratio was similar among groups (p [ 0.05). This is the first report that confirms the role of ABO blood system in thrombosis of PAPS and suggests that non-O blood group was related with venous events and O blood group with arterial thrombosis. Keywords Thrombosis  ABO blood group  Antiphospholipid antibody  Primary antiphospholipid syndrome  von Willebrand factor

Introduction Antiphospholipid syndrome (APS) is an autoimmune disease characterized by vascular thrombosis and/or obstetric complications associated with presence of antiphospholipid antibodies (aPL) [1, 2]. These antibodies target protein cofactors that bind to negatively charged phospholipids and additional pathogenic mechanisms have been reported in primary APS (PAPS) [3, 4]. In fact, the ‘‘second hit’’ hypothesis was proposed since additional factors would be necessary to induce thrombosis in PAPS [4, 5]. In this regard, ABO (H) blood group could have a role in this disease since it has been related to thrombotic events [6–8]. Interestingly, non-O blood group predisposes to venous thromboembolism compared to O blood group [8] but the exact mechanism for this vascular risk is not completely understood [9]. One possible explanation could be related to plasma von Willebrand factor [10] (VWF)

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since ABO (H) blood group plays a role in its secretion [11] and clearance [11–13]. VWF is a multimeric protein [14], which is involved in several pathophysiological process where thrombosis is a feature [15]. Moreover, higher VWF levels are demonstrated in non-O blood group subjects [16]. Therefore, the aim of the present study was to investigate possible association of ABO (H) blood group and thrombotic events in PAPS, particularly venous thrombosis.

Patients and methods Seventy PAPS patients according to Sydney criteria [2] were selected for this study. All patients were regularly followed at outpatient clinic of Rheumatology Division, University of Sa˜o Paulo, Brazil. Inclusion criteria were: age [18 years and previous thrombosis confirmed by imaging exams. All patients were tested for protein S, protein C and antithrombin III deficiencies, and for prothrombin and factor V Leiden gene mutations. Exclusion criteria were: systemic lupus erythematosus and other autoimmune diseases, thrombotic diseases, current thrombosis or bleeding event, acute infection, alcohol intake, hepatic disorders, pregnancy, and lactation. All PAPS patients were under warfarin therapy with stable prothrombin time (PT). PAPS patients were divided according to ABO blood group in: O PAPS (n = 26) and non-O PAPS (n = 44). Local Ethical Committee approved the study (CAPPesq# 0614/10) and all subjects signed the written informed consent.

defined by the concomitant presence of aCL (IgG and/or IgM), ab2GPI (IgG and/or IgM) and LA [18]. von Willebrand factor antigen concentration (VWF:Ag) and activity (VWF:CB) were determined by ELISA (Diagnostica Stago, France) and data were recorded in IU/dl and percentage, respectively. The reference range was 50–160 IU/dl for VWF:Ag and 55–160 % for VWF:CB. Each aliquot was tested for PT, expressed as the international normalized ratio (INR) and activated partial thromboplastin time (APTT) were measured in an automated coagulometer (ACL 3000 IL) with the use of bovine thromboplastin and silica (APTT-silica IL). C-reactive protein (CRP) was measured with commercial kit (Instrumentation Laboratory, Milan, Italy). Plasma total cholesterol (TC) and tryglicerides (TG) levels were measured enzymatically (Boehringer Mannheim, Buenos Aires, Argentina and Merck, Darmstadt, Germany, respectively) using an RA 1,000 analyzer (Technicon Instruments Corp., Tarrytown, NY, USA) [19, 20]. HDL levels were obtained after precipitating very lowdensity lipoprotein cholesterol (VLDL) and LDL with phosphotungstic acid and magnesium chloride [21]. VLDL and LDL levels were estimated for samples with TG levels that were lower than 400 mg/dl [22]. VLDL levels were calculated by multiplying the TG level by 0.45, and LDL levels were determined using the Friedewald formula [22]. Statistical analysis

Laboratorial evaluation Blood samples were collected after 12-h fasting in 3.2 % sodium citrate, in EDTA and in tubes without additives. Serum and plasma were stored at -80 °C, for specific assays.ABO blood group phenotyping was performed by indirect technique. Antiphospholipid antibodies IgG and IgM anticardiolipin (aCL) and anti-beta2 Glycoprotein-1 (ab2GPI) antibodies were measured using ELISA (QUANTA Lite ACA IgM III and IgG III, INOVA Diagnostics, USA, and QUANTA Lite b2GPI IgM and IgG, INOVA Diagnostics, USA). Assays were considered positive when results were greater than 40 GPL or MPL units for aCL and greater than 20 SGU or SMU units for ab2GPI. Lupus anticoagulant (LA) was detected by coagulation assays according to the updated guidelines of the International Society on Thrombosis and Haemostasis (Scientific Subcommittee on Lupus Anticoagulants/Phospholipid-Dependent Antibodies) [17]. Triple positivity was

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Results were expressed as mean ± standard deviation or percentage. The comparison of results between two groups was made using student t test or Mann–Whitney U test for non-parametric data. The comparison of categorical variable, clinical events and risk factors between ‘‘O PAPS’’ and ‘‘non-O PAPS’’ were examined using Fisher’s exact test. The level of statistical significance was set at p \ 0.05.

Results PAPS patients had a predominance of female gender (75.7 %) with a mean age of 42.43 ± 12.85 years and disease duration of 7.46 ± 5.15 years. All of them had previous thrombotic events and 33.9 % of female patients also had obstetric morbidity. O blood group was identified in 26 PAPS patients (37 %) and the remaining 44 (63 %) patients were classified as non-O blood group. The main clinical and laboratorial characteristics of O PAPS and non-O PAPS groups are shown in Table 1. The

ABO blood group in PAPS

groups were similar in mean age (p = 0.194), disease duration (p = 0.054), proportion of females (p = 0.252), white race (p = 1.0), and mean body mass index (p = 0.455). A significant higher frequency of venous events was observed in non-O PAPS group (72.7 vs. 46.2 %, p = 0.040). In contrast, the frequency of arterial events was significantly higher in the O PAPS compared to the non-O PAPS group (69.2 vs. 36.4 %, respectively; p = 0.013) (Table 1). In O PAPS group, ischemic stroke was observed in 12 patients, deep venous thrombosis in 9, pulmonary embolism in 6, limb arterial ischemia in 2, central venous sinus thrombosis in 1, myocardial infarction in 1, abdominal aortic thrombosis in 1, ischemic nephropathy in 1, and skin thrombosis in 1 patient. In nonO PAPS group, deep venous thrombosis was observed in 31 patients, ischemic stroke in 11, pulmonary embolism in 8, limb arterial ischemia in 3, myocardial infarction in 2, and central venous sinus thrombosis in 1 patient. Twelve O PAPS patients (46.2 %) and 20 non-O PAPS patients (45.5 %) had more than one thrombotic event (p = 1.0). Frequencies of aCL, LA, anti-b2GPI and triple aPL positivity were similar in both groups (Table 1). O and non-O PAPS groups showed similar frequencies of current smoking (7.7 vs. 11.4 %, p = 1.0), hypertension (38.5 vs. 34.1 %, p = 0.798), and diabetes mellitus (3.8 vs. 4.5 %, p = 1.00). Moreover, no significant differences were observed in the levels of TC (186.15 ± 31.61 vs. 181.43 ± 34.09 mg/dl, p = 0.567), HDL (50.11 ± 14.37 vs. 47.57 ± 14.35 mg/dl, p = 0.476), LDL (107.00 ± 25.92 vs. 109.39 ± 22.18 mg/ dl, p = 0.684), triglycerides (135.12 ± 66.36 vs. 129.68 ± 98.82 mg/dl, p = 0.804), or blood glucose (87.73 ± 8.76 vs. 92.48 ± 25.27 mg/dl, p = 0.359).

Levels of VWF:Ag in the O PAPS group were lower than those in the non-O PAPS group (75.54 ± 8.68 vs. 79.51 ± 7.07 IU/dl, p = 0.041) (Table 2). In addition, VWF:CB was decreased in the O PAPS compared to the nonO PAPS groups (70.23 ± 11.96 vs. 77.92 ± 13.67 %, p = 0.020). In contrast, the VWF:CB to VWF:Ag ratio was similar between the groups (0.93 ± 0.16 vs. 0.99 ± 0.19, p = 0.246) (Table 2). No patient of both groups had VWF:Ag levels or VWF:CB above the normal range. CRP levels, platelet counts, and PTs (INR) were similar between O PAPS and non-O PAPS groups (Table 2). Twenty-three O PAPS patients (88.5 %) and 41 non-O PAPS patients (93.2 %) were under anticoagulation therapy (p = 0.664).

Discussion The present study describes for the first time the influences of ABO (H) blood group in the site of thrombosis of PAPS since non-O blood group was associated with venous and O blood group with arterial thrombotic events. The great advantage of our study was to include only patients with PAPS diagnosis according to Sydney criteria and previous thrombotic event. The restricted exclusion criteria was established to eliminate conditions that could enhance the risk of thrombosis. Current thrombotic or bleeding event, acute infection and pregnancy were avoided since they could interfere with proteins related with homeostasis [23, 24]. Conditions that alter hepatic function such as alcohol intake and hepatic disorders were also excluded because they decrease coagulation factors synthesis [25].

Table 1 Demographics, clinical and laboratorial characteristics of primary antiphospholipid syndrome patients of O (O PAPS) and non-O blood type (non-O PAPS) O PAPS (n = 26)

Non-O PAPS (n = 44)

p 0.194

Age, years

45.04 ± 13.06

40.89 ± 12.62

Female, n (%)

22 (84.6)

31 (70.5)

0.252

White race, n (%)

26 (100)

44 (100)

1.000

Body mass index (kg/m2)

29.18 ± 8.38

27.91 ± 5.69

0.455

Disease duration (years)

5.92 ± 3.83

8.38 ± 5.65

0.054

DVT and/or PE, n (%)

12 (46.2)

32 (72.7)

0.040*

Arterial events, n (%)

18 (69.2)

16 (36.4)

0.013*

aCL, n (%)

8 (30.8)

15 (34.1)

1.000

LA, n (%)

19 (73.1)

33 (75.0)

1.000

ab2GPI, n (%)

8 (30.8)

18 (40.9)

0.451

Triple aPL positivity, n (%)

3 (11.5)

10 (22.7)

0.345

Data expressed in mean ± standard deviation or percentage DVT deep venous thrombosis, PE pulmonary embolism, aCL anticardiolipin, LA lupus anticoagulant, ab2GPI anti-b2 glycoprotein I, aPL antiphospholipid antibodies *p \ 0.05

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N. M. Nascimento et al. Table 2 Laboratorial parameters of primary antiphospholipid syndrome patients of O (O PAPS) and non-O blood type (non-O PAPS) O PAPS (n = 26)

Non-O PAPS (n = 44)

p

VWF:Ag (IU/dL)

75.54 ± 8.68

79.51 ± 7.07

0.041*

VWF:CB (%)

70.23 ± 11.96

77.92 ± 13.67

0.020*

0.93 ± 0.16

0.99 ± 0.19

0.246 0.608

VWF:CB/VWF:Ag CRP (mg/L) Platelet count (9109/l) PT (INR)

4.60 ± 3.86

5.48 ± 8.07

256.35 ± 93.40

256.02 ± 178.84

0.993

2.37 ± 0.76

2.21 ± 0.79

0.398

Data expressed in mean ± standard deviation (SD) VWF:Ag von Willebrand factor antigen concentration, VWF:CB von Willebrand factor activity, CRP C-reactive protein, PT prothrombin time, INR international normalized ratio *p \ 0.05

The reported association between non-O blood type and venous thrombosis for the general population and other conditions [8, 26, 27] was confirmed herein for PAPS. The link between vascular disorders and non-O group has been associated with higher VWF plasma levels in these patients [9]. In fact, ABO system is considered the most important genetic determinant of VWF–FVIII complex plasma levels [28]. Reinforcing these findings, higher VWF levels and an increased frequency of thrombotic events were detected in the non-O PAPS group in our study. In fact, the prevalence of non-O blood group was significantly higher in patients with venous thromboembolism compared to controls with a resulting pooled odds ratio (OR) of 2.09 (95 % CI 1.83–2.38; p \ 0.00001) in a recent systematic review of 38 studies with 10,305 cases [27]. Interestingly, the present study identified a close association of arterial thrombosis with O blood group in PAPS. Frequencies of aPL antibodies were similar among our PAPS groups and therefore the higher frequency of arterial thrombosis in O blood group cannot be explained by the previously identified aPL-mediated cell activation induced arterial events in PAPS [4, 18]. Reinforcing this lack of association, the frequency of triple positive aPL antibodies, a strong independent risk factor for arterial or venous thrombosis in APS [29], was alike in both groups. Moreover, other known risk factors for thrombosis such as hypertension, dyslipidemia and smoking do not explain our findings since they were similar in both groups. VWF would be another possible candidate since high levels have been associated to arterial thrombosis [28]. Moreover, decreased levels prevent thrombosis in von Willebrand disease [30]. Despite these observations, VWF could not explain the higher frequency of arterial thrombosis in our O PAPS patients since this group had significantly lower VWF antigenic levels and activity than non-O group. These findings support the notion that O blood type is, in fact, a relevant associated prothrombotic condition for arterial thrombosis in PAPS patients.

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In conclusion, this report was the first to demonstrate the influence of ABO blood system in primary antiphospholipid syndrome thrombosis. Non-O blood group was associated with venous events and O blood group with arterial thrombosis. Long-term prospective studies are necessary to clarify the importance of blood group status in predicting thrombosis site in PAPS patients. Acknowledgments This study was supported by the Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico, CNPq Grants 479161-2012-7 (SPB), 301411/2009-3 (EB), and 306963/2011-6 (EFB), the Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo FAPESP, Grant 2013/02785-5 (RPSS), the Federico Foundation (EB and EFB), and the Instituto Nacional de Cieˆncia e Tecnologia de Fluı´dos Complexos (SPB). Conflicts of interest of interest.

The authors declare that they have no conflict

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ABO blood group in primary antiphospholipid syndrome: influence in the site of thrombosis?

Antiphospholipid syndrome (APS) is characterized by vascular thrombosis and/or obstetric complications associated with presence of antiphospholipid an...
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