Autoantibodies to Annexin A2 and cerebral thrombosis: Insights from a mouse model

Abstract

Introduction: Antiphospholipid syndrome (APS) is an autoimmune disorder manifested by thromboembolic events, recurrent spontaneous abortions and elevated titers of circulating antiphospholipid antibodies. In addition, the presence of antiphospholipid antibodies seems to confer a fivefold higher risk for stroke or transient ischemic attack. Although the major antigen of APS is β₂ glycoprotein I, it is now well established that antiphospholipid antibodies are heterogeneous and bind to various targets. Recently, antibodies to Annexin A2 (ANXA2) have been reported in APS. This is of special interest since data indicated ANXA2 as a key player in fibrinolysis. Therefore, in the present study we assessed whether anti-ANXA2 antibodies play a pathological role in thrombosis associated disease.

Materials and methods: Mice were induced to produce anti-ANXA2 antibodies by immunization with ANXA2 (iANXA2) and control mice were immunized with adjuvant only. A middle cerebral artery occlusion stroke model was applied to the mice. The outcome of stroke severity was assessed and compared between the two groups.

Results: Our results indicate that antibodies to ANXA2 lead to a more severe stroke as demonstrated by a significant larger stroke infarct volume (iANXA2 133.9 ± 3.3 mm³ and control 113.7 ± 7.4 mm³; p = 0.017) and a more severe neurological outcome (iANXA2 2.2 ± 0.2, and control 1.5 ± 0.18; p = 0.03).

Conclusions: This study supports the hypothesis that auto-antibodies to ANXA2 are an independent risk factor for cerebral thrombosis. Consequently, we propose screening for anti-ANXA2 antibodies should be more widely used and patients that exhibit the manifestations of APS should be closely monitored by physicians.

Keywords: Annexin; MCAo; antiphospholipid syndrome; autoimmunity; stroke; thrombosis.

Figure 1.

Anti-ANXA2 antibodies levels in serum. (a) Moderate but significant increased serum levels of anti-ANXA2 antibodies in APS patients (n = 4) compared to matched controls (n = 14) (t test, p = 0.0162). (b) Pronounced and significant increased serum levels of anti-ANXA2 antibodies in mice immunized with ANXA2 injection (n = 5) compared to adjuvant immunized control mice (n = 5), (t test, p value < 0.0001). (c) Western blot analysis of anti-ANXA2 mouse serum and commercial anti-ANXA2 antibody to mouse blood clot and glioma derived tissues showing a major band corresponding to ANXA2 at the predicted MW. Values are presented as mean±SEM.

Figure 2.

Assessment of anti-ANXA2 antibodies effect on blood coagulation. (a) PT (n = 4 for iANXA2 and n = 5 control) and (b) aPTT (n = 8 for each group) analysis of plasma samples from iANXA2 and controls mice. In both tests, no difference in the averages of the groups was observed. However, the variances in each group of the aPTT measurements are significantly different (F test p value = 0.0157). Representative charts of ROTEM test in the presence of (c) control mice sera and (d) iANXA2 mice sera. ROTEM measures analysis of area under the curve (e) during all reaction (AUCtotal), (f) during the initial 15 min of clot formation (AR15) and (g) during the last phase of the reaction (AUCtotal-AR15). ROTEM kinetics measures analysis of (h) the speed at which a solid clot forms (alpha-angle) and (i) clot formation time (CFT). (j) The percentage of remaining clot stability in relation to the maximum clot firmness value, at 30 min after clot starts to form (lysis index, LI30). *p = 0.05.

Figure 3.

iANXA2 mice demonstrate a more severe neurological score after stroke. Neurological function was evaluated 24 hours following MCAo. The scores in the iANXA2 group (n = 10) were significantly higher than the control group (n = 8). Values are presented as mean ± SEM. *p = 0.05 by Mann-Whitney test.

Figure 4.

Larger infract volume was observed in iANXA2 mice. (a) Brains were cut to 1mm coronal sections and numbered as demonstrated in the picture. Black - ischemic core, dark gray - ischemic penumbra, light gray - 2mm from the ischemic core. (b) Representative TTC staining of brain slice No. 3. Scale bars: 1 mm. (c) Evaluation of infarct volume measured 24 hours following MCAo in iANXA2 (n = 10) and control (n = 8) mice (d) infarct volume as a function of the distance from the ischemic core. Values are presents as mean ± SEM. *p = 0.017, **p = 0.015.