Autoantibodies against the Fibrinolytic Receptor, Annexin A2, in Cerebral Venous Thrombosis

Abstract

Background and Purpose

Cerebral venous thrombosis (CVT) may be a manifestation of underlying autoimmune disease. Antibodies against annexin A2 (anti-A2Ab) coincide with the antiphospholipid syndrome (APS), in which antiphospholipid antibodies (aPLA) are associated with thrombosis in any vascular bed. Annexin A2, a profibrinolytic receptor and binding site for β2-glycoprotein-I (β2-GPI), the main target for aPLA, is highly expressed on cerebral endothelium. Here we evaluate the prevalence of anti-A2Ab in CVT.

Methods

Forty individuals with objectively documented CVT (33 women and 7 men) and 145 healthy controls were prospectively studied for hereditary and acquired prothrombotic risk factors, classical aPLA, and anti-A2Ab.

Results

One or more prothrombotic risk factors were found in 85% of CVT subjects, (pregnancy/puerperium in 57.5%, classical aPLA in 22.5%, and hereditary procoagulant risk factors in 17.5%). Anti-A2Ab (titer >3SD) were significantly more prevalent in patients with CVT (12.5%) than in healthy individuals (2.1%, p<0.01, OR:5.9).

Conclusions

Anti-A2Ab are significantly associated with CVT, and may define a subset of individuals with immune-mediated cerebral thrombosis.

INTRODUCTION

Cerebral venous thrombosis, a multifactorial disease that preferentially affects young adults and children, occurs without recognizable risk factors in up to 20% of subjects¹. aPLA are detected in 6%–25% of cases^{1, 2}. Though more frequent in APS than in the general population, CVT is still a rare event (0.7%/1000 patients)³. We have previously found anti-A2Ab are significantly more prevalent in patients with APS-related thrombosis (22.6%) than in healthy individuals (2.1%, P<.001), patients with non-autoimmune thrombosis (0%, P = .017), or patients with lupus but without thrombosis (6.3%, P<.001)⁴. Here we examined the prevalence of anti-A2Ab and other prothrombotic risk factors in consecutive patients with CVT.

METHODS

Subject Populations

We studied 185 adults, 40 consecutive patients with CVT and 145 with no prior history of thrombosis, who met standard criteria for blood donation. Consent was obtained per IRB approved protocols. CVT was documented by MRI (100%) and by cerebral angiography (35%). Blood samples were collected at 2–6 months following the thrombotic event. aPLA were considered positive if aCL (>5SD), anti-β2GPI (>3SD), or a positive LA were present.

Coagulation and Antibody Assays

Sera were evaluated for anti-A2Ab (IgG and IgM) by ELISA as previously described⁴. Lupus anticoagulant (LA) was determined using dRVVT (American Diagnostica kit.), and ACL and β2GPI by ELISA⁴. Functional protein C, S, and AT (Stago kits) and the PCR/Mnl-1 restriction enzyme assay for factor V Leiden mutation were determined as described⁵.

Statistical Analyses

Descriptive statistics were used to define the subject’s characteristics. Categorical variables were compared using chi-square or Fisher’s exact test. P value was set at <0.05, two-tailed. Analysis was conducted using SPSS version 17 for Windows.

RESULTS

Among patients studied, 57.5% recovered fully, while 30%, 7.5%, and 5% had mild, moderate, and severe sequelae, respectively, at discharge.

Prothrombotic risk factors are shown in Table 1. Nine patients with CVT (22.5%) had at least one positive aPLA titer, and one fulfilled diagnostic criteria for systemic lupus erythematosus. Among patients with CVT, 12.5% (IgG:7.5%;IgM:5%) were positive for anti-A2Ab (>3SD) compared to 2.1% (IgG:1.4%; IgM:0.7%; p<0.01) of healthy controls; OR 5.9 (with wide 95% CI:1.3–25.8), Table 2. Concomitant risk factors for individuals with anti-A2Ab are depicted in Table 3.

Table 1.

Prothrombotic risk factors in healthy controls and CVT patients

Patient Characteristics	Healthy controls	CVT
Number	145	40
Median Age Range	36.7 (22–3)	28.2 (14–61)
Female/Male	145 (100%)/0	33 (82.5%)
Concomitant non-cerebral thrombosis	-	7 (17.5%)
No identifiable risk factors	-	5 (12.5%)
Aquired Risk Factors for CVT	-	6 (15%)
Puerperium	-	19 (47.5%)
Pregnancy	-	4 (10.0%)
Oral Contraceptives	-	1 (2.5%)
Anabolics	-	1 (2.5%)
aPLA	-	9 (22.5%)
-Systemic lupus	-	1/9
Hereditary Risk Factors	3 (2.1%)	7 (17.5%)
Factor V Leiden (heterozygous)	3 (2.1%)	1 (2.5%)
Protein C	0	3 (7.5%)
Protein S	0	1 (2.5%)
Antithrombin	0	2 (5.0%)

Table 2.

Prevalence of anti-annexin A2 antibodies in healthy controls and CVT patients

	Healthy Controls	CVT
	# 145	# 40
IgG	2 (1.38%)	3 (7.5%)
IgM	1 (0.69%)	2 (5.0%)
IgG and IgM	0	0
IgG or IgM	3 (2.07%)	5 (12.5%)*

^*(p<0.01).

Table 3.

Characteristics of CVT patients with anti-A2 antibodies.

1	Female	Idiopathic CVT
2	Female	aPLA +, recurent arterial VTE
3	Male	aPLA+
4	Female	Protein S deficiency, family history of VTE
5	Female	Post-partum

DISCUSSION

As opposed to US and European series where CVT is rare, it comprises 8% of individuals (166 of 2045) with Mexican Mestizo ancestry at the National Neurology and Neuropsychiatry Institute’s Stroke Registry. Nutritional deficiency may account for this high incidence².

Although the prevalence of known prothrombotic risk factors was similar to other series, the factor V Leiden mutation was not associated with CVT. Interestingly, anti-A2Ab was strongly associated with CVT, independently of classical aPLA’s, A limitation to our cross-sectional design is that the stability of anti-A2Ab titers over time is unknown.

Annexin A2 localizes fibrinolytic activity to the cell surface and is also the high affinity receptor for β2GPI, the main target antigen for pathogenic aPLAs⁶. Upon binding to endothelial cells (ECs), aPLAs induce nuclear factor kappa B (NF-κB) translocation, possibly by signaling through toll-like receptors in complex with A2⁷. Cultured human cerebral ECs express higher amounts of A2 and generate more plasmin (P<0.0001) when compared to those from skin, lung, iliac artery or vein, aorta, and coronary artery. Blockade of A2 inhibits tPA-induced cerebral EC plasmin generation, suggesting a key role for A2 in maintaining cerebral vascular patency⁸. Of related interest, A2 polymorphisms are a risk factor for stroke in sickle cell disease⁹

Recent studies^{10, 11} confirm our previous finding that anti-A2Ab are significantly associated with APS-related thrombosis and that patient-derived anti-A2Ab promote thrombosis by blocking EC surface fibrinolysis, and by inducing tissue factor expression⁴ . A2 may offer a novel therapeutic target, as current efforts at preventing cellular activation by aPLAs are being sought through inhibition of binding of aPLA to β2GPI, inhibition of antibody/β2GPI complex binding to cell surfaces, downregulation of intracellular signaling and proteasome inhibition to suppress NF-κB activation¹².

In conclusion, anti-A2Ab are significantly associated with CVT in our patient population, a finding which requires confirmation in an independent sample. Anti-A2Ab may play a role in the pathogenesis of CVT, offer novel therapeutic strategies, and define a new subset of individuals with immune-mediated thrombosis.