Phosphatidylserine IgG and beta-2-glycoprotein I IgA antibodies may be a risk factor for ischaemic stroke

T. Kahles1, M. Humpich1,2, H. Steinmetz1, M. Sitzer1 and E. Lindhoff-Last2

1 Department of Neurology and 2 Department of Internal Medicine, Division of Angiology, University Hospital, JW Goethe University Frankfurt, Germany.

Correspondence to: T. Kahles, Department of Neurology, University Hospital, JW Goethe University Frankfurt, Schleusenweg 2–16, ZNN, D-60528 Frankfurt/Main, Germany. E-mail: t.kahles{at}em.uni-frankfurt.de


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Objective. Antiphospholipid antibodies (APLA) are established risk factors for venous thrombosis but their role in the pathogenesis of cerebral ischaemia is unclear. The purpose of the present study was to evaluate the relevance of various APLA in patients with cryptogenic stroke (group A, n = 21) and determined causes of stroke (group B, n = 104) according to the TOAST classification in comparison with healthy volunteers without any thrombotic or ischaemic event in their history (group C, n = 84).

Methods. Median ages were 52 yr (A), 60 yr (B) and 51 yr (C). Blood samples were tested for lupus anticoagulant (LA) using phospholipid-dependent coagulation tests (activated partial thromboplastin time, diluted Russell viper venom time). Confirmatory tests were performed if necessary. Furthermore, we assessed the presence of specific APLA and their antibody subclasses against cardiolipin (AclA), phosphatidylserine (ApsA), phosphatidylinositol (ApiA) and beta-2-glycoprotein I (Aß2A) using enzyme-linked immunosorbent assay.

Results. For ApsA IgG we found a significantly higher prevalence in stroke patients (57.7%) compared with normal subjects (4.8%; P<0.001). Similarly, Aß2A IgA was significantly more prevalent in stroke patients (20.8%) in comparison with normals (3.6%; P<0.001). For all other APLAs tested, no significant differences emerged after adjustment for multiple comparisons. We did not find significant differences between stroke subtypes for any APLA.

Conclusion. The results of this study suggest a relevant role for antiphosphatidylserine IgG and anti-ß2-glycoprotein I IgA in stroke aetiology.

KEY WORDS: Ischaemic stroke, Phospholipid antibodies, Lupus anticoagulant, Anti-ß2-GP I, Antiphosphatidylserine


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Antiphospholipid antibodies (APLA) are immunoglobulins, belonging to a group of heterogeneous autoantibodies directed against phospholipids and phospholipid protein complexes. The presence of these immunoglobulins has been associated with thromboembolic events such as deep vein thrombosis, pulmonary embolism, myocardial infarction and acute cerebral ischaemia [1–3]. However, the importance of these various autoantibodies, whether they are a cause or a consequence or even only a coincidence is still a matter of debate. Several studies in the past examining the association between APLA and myocardial as well as cerebral infarction have shown controversial results [1, 2, 4–12]. These results are, at least in part, due to different patient/control selection criteria or due to the use of varying test methods. Furthermore, a substantial number of studies focused only on anticardiolipin antibodies, neglecting the possible relevance of the clinically probably more important lupus anticoagulant (reviewed by [13]).

Nevertheless, the first step towards evaluating a causal role for APLA in the pathogenesis of cerebral ischaemia is to show a higher prevalence of APLA in stroke patients in comparison with various well-defined control groups. Thus, the objective of the present study was to determine the prevalence of a variety of APLA in patients with cryptogenic vs a determined cause of stroke vs normal subjects. Positive findings may serve as the basis for prospective follow-up studies.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study population
All cryptogenic stroke (i.e. stroke of undetermined cause) patients (n = 21, group A) admitted to our stroke unit over a 2-yr period were included in the present study. Controls were 104 unselected stroke patients with a definitely determined cause of stroke according to the TOAST classification [14] (group B) and 84 healthy blood donors (group C) without any history of thrombotic or ischaemic events. Patients with non-ischaemic diseases (e.g. intraparenchymal or sub-arachnoidal haemorrhage, trauma or tumours) were not included in the present study.

All patients gave their informed consent for the analysis of the tested haematological parameters obtained from routine blood sampling. Ethical approval was not obtained.

Medical and neurological history, a neurological examination, cerebral imaging (magnetic resonance imaging or computed tomography), ECG, Doppler/duplex sonography of the vessels supplying the brain, echocardiography, routine laboratory and specialized coagulation tests were performed in all patients.

Cerebrovascular risk factors were defined as follows: blood pressure greater than 140/90 mmHg at repeated measurements (arterial hypertension), fasting serum glucose level above 120 mg/dl on more than one occasion or positive oral glucose tolerance test (diabetes), serum total cholesterol or fasting serum triglyceride level of 200 mg/dl or above (hyperlipidaemia). Patients consuming 10 or more cigarettes daily were defined as smokers. A diagnosis of systemic lupus erythematosus (SLE) was based on the criteria of the American College of Rheumatology [15, 16].

Table 1 summarizes the demographic characteristics and the cardiovascular risk factors of the study population. The median (mean) age was 52 (51) yr in group A, 60 (59) yr in B and 51 (49) yr in group C. The three groups did not differ to a relevant degree in current smoking habits. Hyperlipidaemia was found significantly more frequently in patients with cerebral ischaemia compared with blood donors (P ≤ 0.001) and also among ischaemic stroke patients with a determined cause compared with those without (P = 0.017). Arterial hypertension showed a similar distribution (Table 1). Blood donors were free of diabetes when selected.


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TABLE 1. Characteristics of stroke patients and controls

 
All blood samples were obtained within a median time of 4 days after admission, almost 90% of them within 10 days. After centrifugation and processing, plasma samples were aliquoted, frozen and stored at –70°C until the APLA immunoassays were performed.

Phospholipid-dependent coagulation tests
For detection of lupus anticoagulant (LA), a lupus-sensitive activated partial thromboplastin time (aPTT SP, IL, Kirchheim, Germany) and LAC-Screen (IL, Kirchheim, Germany) based on the principle of diluted Russell viper venom time (dRVVT) were performed. Mixing studies (patient plasma:normal plasma = 1:1) were performed in order to rule out lack of coagulation factors if the results of the above described screening tests were abnormal. If the mixing evaluations were still outside our laboratory reference range, we tested for confirmation of LA: LAC Confirm (IL, Kirchheim, Germany) and MixCon-LA (IL, Kirchheim, Germany). Patients and controls were considered LA-positive if at least one confirmatory test showed abnormal values. Patients positive for LA in the acute phase of stroke were retested for LA ≥ 2 months later.

Assays for the detection of antibodies to phospholipids
Enzyme-linked immunosorbent assays (ELISA) were performed according to the manufacturer's operating instructions using commercial kits for the following APLA: anticardiolipin (AclA) IgG, IgM, IgA and anti-beta-2-glycoprotein I (Aß2A) IgG, IgM, IgA from Pharmacia (P) (Freiburg, Germany). In addition anticardiolipin (AclA) IgG and IgM, antiphosphatidylserine (ApsA) IgG and IgM, and antiphosphatidylinositol (ApiA) IgG and IgM from Orgentec (O) (Mainz, Germany) were analysed. Briefly, for Pharmacia (P) kits, plasma samples were diluted 1:101 with sample buffer containing phosphate-buffered saline (PBS) and bovine serum albumin provided by the manufacturer. One hundred µl of the diluted sample was added to each well of the microtitre plates, incubated for 30 min and washed with wash buffer containing PBS. These plates were coated with bovine cardiolipin and bovine beta-2-glycoprotein I (ß2-GP I) as its cofactor in the case of AclA (P) and with human ß2-GP I in the case of Aß2A (P). Then, a total of 100 µl horseradish peroxidase-conjugated anti-human IgG, IgM and IgA was dispensed, incubated for 30 min and washed again three times with wash buffer. One hundred ml of enzyme substrate solution (TMB) was added to the wells and incubated for 10 min in the dark at room temperature. Finally, 50 µl of stop solution (0.5 M sulphuric acid) was added and the absorbance (i.e. optical density) was read within 10–30 min of adding the stop solution at 450 nm and 620 nm as reference.

Briefly for Orgentec (O) kits, plasma samples were diluted 1:100 with sample buffer provided by the manufacturer. One hundred µl of the diluted sample was added to each well of the microtitre plates and incubated for 30 min at room temperature and then washed three times with wash buffer. These plates were coated with bovine cardiolipin and human ß2-GP I as its cofactor, phosphatidylserine or phosphatidylinositol. A total of 100 µl peroxidase-conjugated anti-human IgG and IgM was added and incubated for 15 min at room temperature and washed again with wash buffer three times. One hundred ml of 3,3',5,5'-tetramethylbenzidine (TMB) solution was dispensed to each well and incubated for 15 min at 20–28°C. Finally 100 ml of the stopping solution (HCl) was added and the absorbance of the plates at 450 nm was read. In two patients with a determined cause of stroke, plasma was not sufficient for the additional determination of all ELISAs from Orgentec (see Tables 2 and 3).


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TABLE 2. Prevalence of different APLA in stroke patients vs healthy controls. The cut-off (U/ml) was calculated as the 95th percentile of the values obtained from the blood donors

 

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TABLE 3. Prevalence of different APLA in patients with cryptogenic vs determined cause of stroke. The cut-off (U/ml) was calculated as the 95th percentile of the values obtained from the blood donors

 
Cut-off points were calculated as the 95th percentile of our healthy controls (group C) considering values above as positive and equal or below as negative.

Statistical methods
The SPSS statistical software package was used for data analysis. We performed {chi}2 analysis and Fisher's exact test for comparison of categorical variables and the Mann–Whitney U-test for continuous variables. Age- and sex-adjusted odds ratio (OR) and 95% confidence intervals (CI) were carried out to determine any association between APLA and stroke or stroke subtype, respectively. Additionally, adjustment for multiple tests was performed by modified Bonferroni correction. Moreover, we performed correlation analysis (Spearman rho) between the prevalence of ApsA IgG and Aß2A IgA and the severity of the neurological deficit (modified Rankin scale, mRS) on the one hand, and the time of blood sampling on the other hand.


    Results
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
APLAs in stroke patients vs normal subjects
For ApsA IgG (O), we found a significantly higher prevalence in stroke patients (57.7%) in comparison with normal subjects (4.8%; P<0.001; see Table 2). Similarly, Aß2A IgA (P) was also significantly more prevalent in stroke patients (20.8%) in comparison with normals (3.6%; P<0.001; see Table 2). Furthermore, we found a trend towards a potential association of lupus anticoagulant in the acute phase after ischaemic stroke, which did not reach the level of significance and completely disappeared at the time of retesting. There was also a non-significant trend for ApsA IgM (O). For all other APLAs tested, no significant differences emerged after adjustment for multiple comparisons (see Table 2).

There was no significant correlation between the severity of the neurological deficit and presence of ApsA IgG or Aß2A IgA, respectively (Spearman-rho correlation coefficient: 0.051, P = 0.575 and 0.094, P = 0.297, respectively). Furthermore no significant correlation emerged between these antibodies and the time interval between hospital admission and blood sampling (correlation coefficient: 0.019, P = 0.837 and 0.097, P = 0.283, respectively).

APLAs in stroke patients with cryptogenic vs determined cause of stroke
Overall, we did not find any significant differences between patients with a determined vs undetermined cause of stroke (see Table 3). Only persisting LA was more frequent in cryptogenic stroke patients, but the two patients harbouring LA fulfilled ARA criteria for systemic lupus erythematosus. Furthermore, we found ApiA IgM (O) to be more frequent in cryptogenic stroke patients, but none of these findings reached the level of significance after the adjustment for multiple comparisons (Table 3).


    Discussion
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Our results indicate a potential role for ApsA IgG and for Aß2A IgA in stroke aetiology with an age- and sex-adjusted odds ratio of 26.4 (95% CI, 8.8–78.7; P<0.001), and 6.5 (95% CI, 1.9–22.8; P = 0.003), respectively. Additional adjustment for stroke risk factors like arterial hypertension, diabetes, hyperlipidaemia and smoking did not substantially change these odds ratios. With respect to cryptogenic stroke, there was a trend towards a positive but not significant association for lupus anticoagulant and ApiA IgM. In long-term follow-up, LA persisted only in patients with systemic lupus erythematosus.

Our findings confirm former studies revealing an independent association between ischaemic stroke and ApsA [6] and Aß2A [17], respectively. For ApsA IgG and IgM, Tuhrim et al. [6] found a risk factor-adjusted OR of 3.2 (95% CI, 1.8–5.5) in stroke patients of various aetiology vs normal subjects. Furthermore, in young adults with cerebrovascular disease of undetermined aetiology Toschi et al. [18] reported a high prevalence of ApsA of 18.2% [18]. Whether this finding is significant could not be answered by the authors due to a missing control group. Fiallo et al. [17] found a significant association between Aß2A (subtype not specified) for unselected stroke patients in comparison with normal controls. In contrast, Caso et al. [19] found no association for Aß2A (IgG and IgM) for unselected consecutive stroke patients but the authors did not test Aß2A IgA for which we found an association. Confirming the finding that probably only Aß2A IgA may play a role, the Honolulu Heart Program found that serum positivity for Aß2A IgG antibodies was not predictive for either stroke or myocardial infarction over a follow-up period of 20 yr [5]. Thus, in conclusion, there is at least some evidence for an independent association between the presence of ApsA IgG and Aß2A IgA and stroke in general, respectively.

For anticardiolipin antibodies, some prospective follow-up studies showed an independent predictive value with respect to future thrombo-occlusive events including stroke and transient ischaemic attacks (TIA) [5, 20, 21], but others did not [1, 11, 12, 22]. Unfortunately, we could not confirm any association between AclA (IgG or IgM) and stroke.

To answer the question whether the above mentioned APLAs may play a causal role in the pathogenesis of stroke, the presence and concentrations of APLAs should be compared between patients with an ischaemic stroke of undetermined cause (i.e. cryptogenic) and patients with a definitely determined cause. In the present examination, we did not find a relevant difference for ApsA IgG or Aß2A IgA between these groups. We found a trend towards a significant difference for persisting lupus anticoagulant and ApiA IgM, but this was closely linked to the clinical diagnosis of SLE in two patients. After the adjustment for multiple comparisons or the exclusion of the SLE patients from the cryptogenic group, there was no significant difference between undetermined and determined cause of stroke (see Table 3). To our knowledge, a direct comparison of various APLAs between groups of stroke patients using a uniform test battery has not been performed before. Thus, we could not find a specific pattern of APLAs which can reliably differentiate between a cryptogenic stroke and a stroke of determined cause.

In the situation of SLE it is probably not reasonable to classify a cerebral ischaemic event as ‘cryptogenic’, because it is well known that SLE is a disease with a significantly elevated risk of stroke due to various pathogenic mechanisms [23]. Furthermore, it has been clearly shown that the occurrence of APLA in SLE patients is associated with a higher risk of thrombo-occlusive events, including stroke and TIA [24]. Thereby, previous investigators examining the potential role of APLAs in stroke, prospectively excluded SLE patients from their analyses, e.g. Metz et al. [10].

Pathophysiologically, it is unlikely that the occurrence of APLAs in stroke patients is merely an epiphenomenon. Camerlingo and co-workers [25] found a significantly higher prevalence of anticardiolipin antibodies in stroke patients within just 6 h of symptom onset. This result supports a causative role rather than a consequential one for APLA in stroke. As APLAs are also involved in apoptosis following ischaemia, we looked at ApsA IgG and Aß2A IgA with regard to the severity of the neurological deficit (mRS) and the time point of blood sampling. Likewise, our data do not support the hypothesis that the occurrence of APLAs in serum is a consequence of cerebral ischaemia. Furthermore, several prospective follow-up studies revealed a significantly higher risk of thromboembolic events in the case of serum positivity [5, 20, 21]. On the other hand, the lack of a stronger association of APLAs in patients with cryptogenic stroke in comparison with patients with stroke of determined cause may suggest that APLAs constitute a cofactor rather than an independent cause of stroke (see Table 3). APLAs may link inflammation and thrombosis and thereby result in a destabilization of conditions leading to stroke (e.g. atherosclerosis, cardio-embolism, small vessel disease) [26].

The present study harbours some relevant limitations. The three groups of subjects compared were not completely equal with respect to age, sex distribution and the presence of stroke risk factors (see Table 1). For example, there were no diabetics in the normal control group. Repeated serum measurements were only performed for lupus anticoagulant. In particular the number of patients with cryptogenic stroke was relatively small. Despite these limitations we were able to look at a considerable number of relevant APLAs, their prevalence, their association with stroke subtypes and the odds ratios to suffer from stroke. Thus, we believe that the findings of our pilot study may serve as the basis for future prospective studies which should be focused on the specific APLA found to be associated.


    Acknowledgments
 
This work was supported by Pharmacia, Freiburg, Germany and Orgentec, Mainz, Germany.

The authors have declared no conflicts of interest.


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Submitted 29 December 2004; revised version accepted 29 April 2005.



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