Anti-β₂-Glycoprotein I Autoantibody Expression as a Potential Biomarker for Strokes in Patients with Anti-Phospholipid Syndrome

Abstract

Anti-phospholipid syndrome (APS) is an autoimmune disease. Cerebral ischemia associated with APS occurs at a younger age than typical atherothrombotic cerebrovascular disease, is often recurrent, and is associated with high positive IgG anti-phospholipid (GPL) unit levels. This study sought to determine the frequency rates of anti-cardiolipin (aCL) dependent on the presence of β₂-GPI, anti-β₂-glycoprotein I (aβ₂-GPI), and anti-phosphatidyl serine (aPS) IgG autoantibodies among stroke patients, and thus demonstrate the importance of testing for aβ₂-GPI autoantibodies. For these study, stroke patients and control subjects recruited from Mosul, Erbil, and Dohuk provinces in Northeren Iraq between March 2004 and March 2005 were evaluated. All cases were under 50 years-of-age and had no recognizable risk factors. Using ELISA to evaluate the presence of IgG isotype of aCL, aβ₂-GPI, and aPS autoantibodies in their blood, the results indicated that the frequency of aβ₂-GPI was 14/50 (28%), aCL was 11/50 (22%), and aPS was 9/50 (18%) among stroke patients. In contrast, aCL was detected in 2/30 (6.7%) of control subjects; each of the other anti-phospholipid antibodies (APLA) was never observed. Of all the aβ₂-GPI⁺ cases, the incidence of stroke patients having the combined profile of aβ₂-GPI + aCL was 11/14 (78.6%) and of aβ₂-GPI + aPS was 9/14 (64.3%). Only 2/14 (14.3%) of these aβ₂-GPI⁺ patients also expressed aCL in the absence of aPS. The frequency of patients expressing all three markers was only 9/14 (64.3 %). In none of the APS/stroke patients were aCL or aPS expressed in the absence of the aβ₂-GPI. Conversely, aβ₂-GPI as a sole marker was seen in 3/14 (21.4%) of these patients (i.e., in absence of either other marker). It can be concluded from these studies that the among the three major forms of APLA examined, the presence of aβ₂-GPI IgG autoantibodies appeared to correlate best with stroke in patients who were concurrently suffering APS.

Keywords :

• Anti-β₂-glycoprotein I
• autoantibodies
• stroke

INTRODUCTION

Anti-phospholipid syndrome (APS) is an autoimmune disease. The definitive criteria for classification of patients with this syndrome were established in 1998 (Wilson et al., 1999); these included that patient must have either vascular thrombosis or fetal loss and demonstrate evidence of anti-phospholipid antibodies (APLA; either by detection of the APLA themselves) or a positive lupus anticoagulant. Furthermore, these autoantibodies must be detected on at least two occasions 6 wk apart in order to distinguish between persistent autoimmune antibody responses and transient responses caused by infection or drug exposures (Hanly, 2003).

There are a substantial number of gaps in the knowledge about the association between stroke and APS (Gatenby, 2004). Cerebral ischemia associated with APS occurs at a younger age than typical atherothrombotic cerebrovascular disease, is often recurrent, is associated with high positive IgG phospholipid (GPL) unit levels (i.e., APLA values), and is usually linked to the presence of anti-cardiolipin (aCL) antibodies (Brey et al., 1990; Nencini et al., 1992). Other data suggested that subsequent thrombo-occlusive events and death after focal cerebral ischemia are associated with aCL IgG and might occur sooner (Brey et al., 2002).

APLA have been found in 25% of patients under the age of 45 who presented with cerebrovascular accident with unclear etiology (Munts et al., 1998). Another study has reported the prevalence of APLA in 20% of stroke victims under the age of 50 years (Gromnica-Ihle and Schossler, 2000). Brey et al. (2001) sought to determine the relative risk of stroke associated with the presence of APLA and noted an increased risk of stroke associated with the presence of IgG antibodies that were dependent on the presence of β ₂-GPI.

Based on those findings, it is reasonable to conclude that in the absence of other risk factors, APS should be suspected in young patients presenting with stroke. Therefore, the aim of this study was to determine the frequency rates of anti-cardiolipin (aCL), aβ₂-GPI, and anti-phosphatidyl serine (aPS) IgG autoantibodies among select stroke patients, with an emphasis on determining the potential importance of testing for aβ₂-GPI autoantibodies in particular.

MATERIALS AND METHODS

Study Population

Individuals in this study were a subset of those who participated in a study examining the potential role of APLA in patients with cardio-cerebrovascular diseases in the region (Ali, 2006). This original population of 150 cases contained 50 patients who had presented with deep venous thrombosis of the lower extremities (32% of these patients had one or more of the three APLA) and 50 patients who had presented with premature ischemic heart diseases in the form of angina and/or myocardial infarction (30% of these patients bore one or more of the three APLA). The study reported here was carried out on 50 stroke patients from this original population and 30 healthy individuals. As with the other patients noted above, these 80 individuals were recruited from the Mosul, Erbil, and Dohuk areas of Northern Iraq during the period from March 2004 to March 2005. Within this stroke case study group, 37/50 (74%) were males and 13/50 (26%) were females. Moreover, 38/50 (76%) presented with a history of a single stroke attack and 12/50 (24%) had a history of recurrent attacks.

Fifty selected patients suffering from strokes and 30 healthy individuals were tested for APLAs in the Postgraduate Laboratory of Medical College, University of Dohuk, Kurdistan Region, Iraq in April 2005. All patients were not being previously diagnosed as SLE cases, were under 50 years of age, and had no recognizable risk factors including hypertension (systolic blood pressure > 140 mm Hg and/or diastolic of > 90 mm Hg), diabetes mellitus (fasting blood glucose of ≥ 126 mg/dl and/or 2-hr post-prandial glucose of ≥ 140 mg/dl), dyslipidemia (total cholesterol level of > 240 mg/dl and HDL of < 35 mg/dl), smoking (current or previous) and, lastly, not overweight (body mass index of < 25). The purpose of the study was discussed in details with the participants of both groups and obligatory ethical agreement was obtained from cases accepted for inclusion in this study.

A venous blood sample of ≈ 10 ml was obtained from each participant and tested for the presence of aCL, aβ₂-GPI, and aPS autoantibodies. A second blood sample was taken 6 wk later for confirmative purposes (i.e., to validate status of originally-designated APLA-positive [APLA⁺] subjects) and to fulfill the criteria (i.e., the Sapporo criteria) for positive diagnosis of APS. In each case, serum was isolated using standard procedures and frozen at -80°C until used for analyses.

Determination of APLA

Enzyme-immunoassay kits (ELISA; Orgentic Diagnostika, Mainz, Germany) that employ indirect solid phase enzyme immunoassaytechniques were used to measure levels (IgG isotypes) of aCL, aβ₂-GPI, and aPS autoantibodies in the blood samples. For IgG aCL measurements, kits were used that estimate antibodies to cardiolipin that are dependent on the presence of β₂-GPI (i.e., highly purified cardiolipin is bound to microwells saturated with β₂-GPI). For a β₂-GPI measurements, the kits used contained highly purified β₂-glycoprotein bound to the well walls. For aPS measurements, the kits used contained highly purified phosphatidyl serine bound to the well walls. All results were assessed in terms of IgG phospholipid (GPL) units, with one unit equal to 1 μ g IgG/ml. According to the manufacturer's recommendations, a sample was considered significantly positive if the titer was > 10 GPL units for aCL, > 8 GPL for aβ₂-GPI, and > 10 GPL for aPS autoantibodies.

Statistics

All data were organized into contingency tables according to the presence or absence of APLA. The relative risk factor was determined by estimation of the odd's ratio (OR) and the corresponding 95% confidence intervals (CI) wherever indicated using the statistical program, accordingly. A statistical and significant relative risk factor was considered when an OR > 1.0 was recorded. The 95% CI of proportions was calculated according to the binomial distribution. In all analyses, the level for assigning statistical significance was set at p < 0.05.

RESULTS

The age range of the studied cases (i.e., individuals with stroke events) was 25–48 yr (mean ± SD; 36.2 ± 6.7). All subjects in this group were defined by the clinical criteria of focal neurological deficits of sudden onset that lasted more than 24 hr. These cases were also clinically diagnosed by neurologists and confirmed using imaging techniques such as Computerized Tomography (CT) scanning or Magnetic Resonance Imaging (MRI). The age range of subjects in the control group was also 26–48 yr (36 ± 6.5); each control employed in the study had no history of any thromboembolic manifestations and presented with no recognizable risk factors such as smoking, hypertension, diabetes mellitus, hyperlipidemia, or obesity.

The frequency of detected APLA in the stroke patients and control subjects are shown in Table 1. All data in this table are based upon the analyses of both sets of samples (blood from first-draw and from 6 wk later). The data show that aβ₂-GPI IgG was present in 14/50 (28%) of stroke patients and completely absent in the blood of controls (OR = 11.7 [95% CI = 8.4–78.3]; p < 0.01). A presence of IgG aCL was noted in 11/50 (22%) of the stroke victims, but only in 2/30 (6.7%) of controls (OR = 4 [95% CI = 1–16]; p < 0.05). Last, while IgG aPS was detected in the samples of 9/50 (18%) stroke patients (Table 2), and completely absent in the control subjects (OR = 6.5 [95% CI = 1–40]; p < 0.05). Of all the aβ₂-GPI⁺ cases, the incidence of stroke patients having the combined profile of aβ₂-GPI + aCL was 11/14 (78.6%) and of aβ₂-GPI + aPS was 9/14 (64.3%). Only 2/14 (14.3%) of these aβ₂-GPI⁺ patients also expressed aCL in the absence of aPS. The frequency of patients expressing all three markers was only 9/14 (64.3 %). In none of the APLA⁺ stroke patients were aCL or aPS expressed in the absence of the aβ₂-GPI. Conversely, IgG aβ ₂-GPI as a sole marker was seen in 3/14 (21.4%) of these patients (i.e. in absence of either other marker [viz. aCL⁻ and aPS⁻]) These results make it clear that expression of aPS always seems to occur in tandem with aβ₂-GPI (as well as with aCL), but the reverse case(s) need not be true, in stroke victims that concurrently suffer with APS. Compared with aPS, the linkage between expression of aCL and aβ₂-GPI in these patients appears even stronger, although again, and it is likely that a presence of aβ₂-GPI need not necessarily be predictive of that for aCL.

TABLE 1 The frequencies of the different anti-phospholipid autoantibodies in stroke patients and controls.

Tested APLAs (GPL units)	Patients No. (%)	Controls No. (%)	OR	95% CI	P value
aCL
> 10	11/50 (22)	2/30 (6.7)	4	1–16	P < 0.05
≤ 10	39/50 (78)	28/30 (93.3)
aβ 2−GPI
> 8	14/50 (28)	0/30 (0.0)	11.7	8.4–78.3	P < 0.01
≤ 8	36/50 (72)	30/30 (100)
aPS
> 10	9/50 (18)	0/30 (0.0)	6.5	1–40	P < 0.05
≤ 10	41/50 (82)	30/30 (100)

*According to manufacturer's recommendations, a sample was considered significantly positive if fiter of IgG isotype was > 10 units for aCL, > 8 for aβ₂-GPL, and > 10 for aPS autoantibodies. OR-Odds ratio, CI-Confidence interval.

TABLE 2 Summary of all APLA results in aβ₂-GPI⁺ patients.

Case No.	aβ2-GPI^+	aCL	aPS
1	+	+	+
2	+	+	+
3	+	−	−
4	+	+	+
5	+	+	+
6	+	+	−
7	+	−	−
8	+	+	+
9	+	+	−
10	+	+	+
11	+	+	+
12	+	−	−
13	+	+	+
14	+	+	+

The neuroimaging findings of the brain (using CT or MRI; images not shown) among the 14 aβ₂-GPI⁺ stroke cases revealed a localized ischemic infarcted area and multiple infarcted areas in 6/14 (42.8%) and 4/14 (28.6%) subjects, respectively. In contrast, these neuroimaging techniques were unsuccessful in localizing any lesion in 4/14 (28.6%) of the patients. All cases with positive neuroimaging findings of a localized infracted area had a history of single stroke attack, while those with multiple affected areas had a history of recurrent episodes of stroke.

DISCUSSION

APS is an autoimmune disease characterized by the presence in a patient of one or more APLA and at least one clinical manifestation, the most common being venous/arterial thrombosis or recurrent fetal loss. Since the discovery in 1990 that β₂-GPI is involved in APS as a co-factor, interest in this protein has increases tremendously; however, crucial information is still lacking. This protein has low affinity for anionic phospholipids compared to other plasma proteins such as clotting factors; meanwhile, this affinity increases up to 100 times after interaction with aβ₂-GPI (Willems et al., 1996; Lutters et al., 2001). As a result of this increase in affinity, these proteins significantly interfere with the interaction of other plasma proteins with anionic phos-pholipid surfaces and can inhibit their coagulation function (de Laat et al., 2004).

In this study, although the number of the studied cases was relatively small, one or more of the APLA were detected in a minimum of 18% (i.e., the frequency of aPS⁺ cases) and a maximum of 28% (the frequency of aβ₂-GPI⁺ cases) of the stroke patients examined, a fact that is notable and worthwhile in this region. Moreover, the detected frequencies of these APLA in the stroke patients (as compared to controls) were significantly different enough as to suggest that their presence carries a significant risk factor for development of stroke events related to APS. Specifically, aβ₂-GPI IgG was present with the greatest frequency among all APLA⁺ cases, and it was present in all in those cases positive for APLA. Unfortunately, while strong, the co-expression linkage between aβ₂-GPI and aPS is not as strong as that between aβ₂-GPI and aCL. Therefore, the presence of aβ ₂-GPI autoantibody seems to be a useful biomarker (more so than either of the other APLA markers) of stroke in patients concurrently suffering with APS.

The term “anti-phospholipid co-factor syndrome” was used to describe cases in which clinical manifestations are related to APS in the presence of aβ₂-GPI and absence of other APLAs markers (Cabral et al., 1996). This highlights the importance of testing for aβ₂-GPI antibodies in diagnosing clinical events related to APS to avoid underestimation of cases. This category of syndrome was also reported by Picillo et al. (1998) in cases with recurrent arterial and/or venous thrombosis without SLE. Moreover, Katsarau et al. (2003) reported this phenomenon among a case with ischemic stroke.

The mechanisms by which aβ₂-GPI initiates prothrombotic states are multiple and many receptors on different cell types have been proposed to bind the β₂-GPI-antibody complex and affecting the coagulation cascade. The induction of tissue factor, intracellular adhesion molecules (ICAM) and vascular adhesion molecule (VCAM) are examples of this pathogenic activity of aβ₂-GPI on endothelial cells (Gharavi et al., 1999; Salemink et al., 2000).

Bevers et al. (1991) and Arnout et al. (1999) reported that both Lupus anti-coagulant (LA) and conventional aCL to be non-specific tests, since LA can detect antibodies to both human prothrombin and human β ₂-GPI or other antibodies. Moreover, the conventional aCL assay can detect a range of antibodies, including anti-human β₂-GPI, antibodies directly bind cardiolipin, and antibodies directed to other cardiolipin-binding plasma proteins (Roudey, 1996; Rampazzo et al., 2001). Although, in 1999, the determination of aβ₂-GPI was not included in the Sapporo laboratory criteria for diagnosis of APS (Hughes, 1998), it was suggested as an important testing marker in 2002 (Pengo, 2002). The main problem in different previous studies was the lack of standardization of aβ₂-GPI antibody ELISA kits. New ELISA assay techniques with improved sensitivity and specificity for testing aβ₂-GPI dependent methods are in use. These findings necessitate testing for this marker in stroke patients and ascertain the evidence accumulating which would assign a central role for aβ₂-GPI in stroke cases during APS.

In conclusion, the results of the current study demonstrate an increased risk, positive association, and good correlation between the presence of aβ₂-GPI and stroke cases related to APS. Moreover, the detected rate of APLA among stroke cases in this region is notable and worthwhile. On the other hand, our results revealed that testing for this autoantibody, along with other APLA, might help to identify more cases related to APS. This, therefore, justifies the use of assessment of this marker (apart from the other APLA) in serologic tests performed in potential/suspected stroke cases in patients with APS. In addition, because the neuroradiological imaging procedures performed here revealed positive findings in 71.4% (10/14) of the APLA⁺ cases, we recommend that imaging be routinely applied as a part of health examinations of APS patients who are APLA⁺ in general, and aβ₂-GPI⁺ in particular, in order to enable the earliest possible detection of any lesions/cerebrovascular hotspots in these individuals who are highly susceptible to, but have not as yet suffered, a stroke.