GENETIC ANALYSIS OF THE ANNEXIN A5 M2 HAPLOTYPE AND ASSOCIATION WITH PREGNANCY COMPLICATIONS IN 500,000 INDIVIDUALS

Abstract Abstract 631 Background: The antiphospholipid (aPL) syndrome (APS) is a diagnostic category that is defined by non-mechanistic empirically-derived assays - the lupus anticoagulant (LA) assay and aPL-antibody immunoassays for anticardiolipin and anti-ß2 glycoprotein-I antibodies. APS patients have heterogeneous antibodies and it is likely that several mechanisms are involved in the disease process. Annexin A5 (AnxA5) is a potent anticoagulant protein that forms 2-dimensional crystals over membranes that express phosphatidyl serine and shields the phospholipid from availability for phospholipid-depended coagulation reactions. We previously demonstrated that aPL antibodies can disrupt this crystallization and accelerate coagulation reactions. We therefore investigated whether disruption of AnxA5 anticoagulant activity might mark patients with histories for aPL antibody associated thrombosis. Design: We investigated samples from 966 adult patients from a large urban academic medical center with the following categories of histories: thrombosis, including venous thromboembolism (VTE) and stroke (n=433); pregnancy complications attributable to placental insufficiency (n=218); autoimmune disease (n=279); cancer (n=63); renal disease (n=101); liver disease (n=51); HIV (n=23); and free of known disease (n=140) (total >966 because of concurrent diagnosis). An assay that detects annexin A5 resistance (A5R) was performed on all samples. Results were expressed as “% AnxA5 anticoagulant activity”. A quintile analysis was performed to obtain cutoffs for A5R values. Values among groups were compared using student t-test, one way ANOVA and odds ratio analysis. Results: aPL-antibody positive patients with thrombosis (VTE and/or stroke) have significantly lower mean A5R values (194±57, n=76) than aPL-antibody negative patients with a thrombotic event (238±41, n=175; p<0.001) and from patients who are free of known disease (262±41, n=140; p<0.001, Figure 1). 54% (41/76) of patients in the aPL-antibody positive and thrombosis group had A5R values within the lowest quintile of results (<199%), in contrast to 15% (26/175) of the aPL-antibody negative and thrombosis patients and 8% (11/140) of the disease-free control. An AnxA5 anticoagulant activity of <199% was significantly associated with aPL-antibody positivity and thrombosis (OR: 13.7, p<0.001). aPL-antibody positive patients with pregnancy complications (PCs) have significantly lower A5R values (218±45, n=111) than aPL-negative patients with pregnancy complications (248±43, n=107; p<0.001) and from patients who are free of known disease (262±41, n=140; p<0.001, Figure 1). 28% (31/111) of patients in the aPL-antibody positive and pregnancy complication group had A5R values within the lowest quintile of results (<199%), in contrast to 8% (9/107) of the aPL-antibody negative and thrombosis patients and 8% (11/140) of the disease-free control. An AnxA5 anticoagulant activity of <199% was significantly associated with aPL-antibody positivity and pregnancy complications (OR: 4.6, p<0.001). There were no significant differences in AnxA5 anticoagulant activity in patients with cancer, HIV, and renal disease, however there were significantly lower A5R values in patients with autoimmune disease and liver disease. The latter appeared to correlate with severity of liver disease. Patients with triple aPL-antibody positivity or greater had significantly lower mean A5R values (155±48%, n=23, p<0.001), than patients with double positivity (193±55%, n=44, p<0.01), or single positivity (214±47, n=111, p<0.05). All aPL-positive groups had significantly lower A5R values than patients who were negative for aPL antibodies (240±43%, p<0.001). Conclusion: A subset of ∼50% of aPL-antibody positive patients with thrombosis and ∼30% with pregnancy complications showed evidence for this mechanism - resistance to annexin A5 anticoagulant activity. The identification of specific mechanisms for aPL-antibody associated thrombosis and pregnancy complications offers the possibilities of improving diagnosis and developing targeted treatments for this enigmatic disorder. Disclosures: No relevant conflicts of interest to declare.

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Annexin A5 Resistance Is Associated with Thrombotic Risk in Patients with the Lupus Anticoagulant: The Vienna Lupus Anticoagulant and Thrombosis Study (LATS)

Blood ◽

10.1182/blood.v126.23.3555.3555 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3555-3555

Author(s):

Florian Posch ◽

Johanna Gebhart ◽

Wu Xiao-Xuan ◽

Silvia Koder ◽

Cihan Ay ◽

...

Keyword(s):

Pregnancy Complications ◽

Lupus Anticoagulant ◽

Pregnancy Complication ◽

Mean Difference ◽

Annexin A5 ◽

Glycoprotein I ◽

Spearman's Rho ◽

Spearman’S Rho ◽

History Of ◽

Thrombotic Complications

Abstract INTRODUCTION: Antiphospholipid (aPL) antibody mediated interference with the anticoagulant effect of annexin A5 (A5) - designated "annexin A5 resistance" (A5R) - has been implicated in adverse clinical outcomes. In this study, we ask whether A5R is associated with anamnestic thrombotic complications and adverse pregnancy outcomes in patients with the lupus anticoagulant (LA). PATIENTS & METHODS: We conducted a cross-sectional study including 143 patients (median age: 40.5 years, interquartile range (IQR): 31.8-60.0; female: n=119) who tested persistently positive for LA according to ISTH criteria (Miyakis et al. J Thromb Haemost 2006; 4: 295). Thrombotic complications were defined as a composite of arterial and/or venous thromboembolic events. Pregnancy complications were defined according to the modified Sapporo criteria. Out of the 143 patients, 94 (65.7%) had a history of a thrombembolic event (arterial: n=21, venous: n=80, both: n=7). Ninety-three women had at least one pregnancy, and 40 (43.0%) out of these women had at least one pregnancy complication as defined by the above mentioned criteria. It is important to mention that on average, these pregnancy complications had occurred more than ten years before study inclusion. A5R assays were done using phospholipid that was first exposed to patient plasmas as previously described; A5R results were expressed as the ratio of coagulation times with and without annexin A5. In a subsample (n=109), a blinded external validation using two separate measurements of the A5R was performed. RESULTS: In blinded analysis, the concordance between the two A5R measurements was very high (Liu's concordance correlation coefficient=0.83, average difference=12.2 units (95% limits of agreement from Bland-Altman analysis: -37.0 - 61.3). The A5R was inversely correlated with levels of antibodies against cardiolipin (Spearman's rho for IgG and IgM: -0.65 and -0.24, respectively), and against beta2-glycoprotein I (Spearman's rho for IgG and IgM: -0.57 and -0.28). The median A5R level was 205 units (IQR: 205-250, range: 130-336). Patients with a prior history of a thrombotic complications had significantly lower median levels of A5R (193.1 vs. 238.7 units, mean difference: 26.7 units (95% CI: 11.0-42.4), p=0.001). This difference was independent of both IgG and IgM isotype antibody levels against cardiolipin and beta2-glycoprotein I (adjusted mean difference: 20.7 units (95% CI: 6.4-35.0), p=0.005), and of whether patients were on anticoagulation or not (adjusted mean difference: 24.8 units (95% CI: 4.3 - 45.2), p=0.018). In the 95 women with at least one pregnancy, average A5R were lower in women with (n=40) compared to those without (n=53) at least one pregnancy complication (median: 200.9 vs. 231.9 units), however, this difference was not statistically significant (t-test p=0.255). CONCLUSION: A5R is associated with a history of thromboembolic complications in LA positive patients, suggesting that the ex vivo measurement of the A5R may reflect a prothrombotic phenotype in this patient population. Interestingly, we found no clear association between the A5R and anamnestic pregnancy complications was observed, however, the generalizability of this finding is hampered by the long time interval between anamnestic pregnancy complications and inclusion in this study. Disclosures No relevant conflicts of interest to declare.

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Absence of Distinct Immunohistochemical Distribution of Annexin A5, C3b, C4d, and C5b-9 in Placentas From Patients With Antiphospholipid Antibodies, Preeclampsia, and Systemic Lupus Erythematosus

Pediatric and Developmental Pathology ◽

10.1177/1093526619836025 ◽

2019 ◽

Vol 22 (5) ◽

pp. 431-439 ◽

Cited By ~ 1

Author(s):

Cathleen E Matrai ◽

Jacob H Rand ◽

Rebecca N Baergen

Keyword(s):

Systemic Lupus Erythematosus ◽

Lupus Erythematosus ◽

Pregnancy Complications ◽

Tissue Injury ◽

Obstetric Outcomes ◽

Annexin A5 ◽

Antiphospholipid Antibody ◽

Antiphospholipid Antibody Syndrome ◽

Intrinsic Variability ◽

Systemic Lupus

Introduction In pregnancy, the presence of preeclampsia (PEC), systemic lupus erythematosus (SLE), and/or antiphospholipid antibody syndrome (APLS) is characterized by poor obstetric outcomes, with potential adverse effects for both mother and fetus. Although the histopathologic changes observed in these entities have been well established, the pathogenic mediators associated with tissue injury are poorly understood. Methods Forty placentas were evaluated, including 10 patients with preeclampsia, 9 with SLE, 11 with APLS, and 10 disease-free controls. Each case was subjected to a panel of immunohistochemical markers including C3b, C4d, Annexin A5, and C5b-9. Staining was graded on intensity and distribution. Results C4d staining was distinctly different among disease groups and controls. Moreover, 6/10 PEC cases, 3/9 SLE cases, and 4/11 APLS cases showed at least focal staining for C4d. All controls were negative. Annexin A5 (AnxA5) staining showed intrinsic variability in all disease groups, while 10/10 controls showed diffuse, strong staining (2+ or 3+). C3b staining was heterogeneous among groups. Discussion Previously, antiphospholipid antibody (aPLA)-associated pregnancy complications have been thought to be a consequence of a unique aPLA-mediated pathogenic mechanism. However, the immunohistochemical similarity (increased complement and decreased AnxA5 staining) observed in placentas from patients with APLS, PEC, and SLE suggests that aPLA-associated pregnancy complications may reflect a more general autoimmune mechanism.

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A Functional Assay for Annexin A5 Resistance Identifies a Thrombogenic Mechanism In a Subset of Patients with Antiphospholipid Antibodies

Blood ◽

10.1182/blood.v116.21.153.153 ◽

2010 ◽

Vol 116 (21) ◽

pp. 153-153

Author(s):

Lucia Wolgast ◽

Xiao Xuan Wu ◽

Jessica Niakan ◽

Alan Arslan ◽

Jacob Rand

Keyword(s):

Venous Thromboembolism ◽

Pregnancy Complications ◽

Conflicts Of Interest ◽

Anticoagulant Activity ◽

Thrombotic Event ◽

Laboratory Data ◽

Annexin A5 ◽

Functional Assay ◽

Predictive Values

Abstract Abstract 153 Background: The antiphospholipid (aPL) syndrome (APS) is an autoimmune, thrombotic condition reported to be responsible for approximately 10% of patients with thromboembolic disease. APS is characterized by autoantibodies against phospholipids or plasma proteins bound to phospholipids that inhibit phospholipid-dependent coagulation reactions in vitro. The diagnostic criteria for APS are defined by the presence of an aPL antibody and the occurrence of one or more thrombotic events, including arterial or venous thromboses, multiple fetal losses and thrombocytopenia. The current assays for diagnosing APS are empirically-derived and include the lupus anticoagulant assay and aPL immunoassays for anticardiolipin and anti-ß2 glycoprotein-I antibodies. These assays do not test report on disease mechanisms and have poor specificities, sensitivities and predictive values. To date, several different mechanisms have been proposed for APS, including aPL antibody-mediated disruption of a potent anticoagulant protein, annexin A5. Annexin A5 forms two-dimensional crystal shields over membranes that expose anionic phospholipids; the crystallization and shielding of phospholipid from availability for coagulation reactions is disrupted by aPL antibodies. Therefore, we investigated the prevalence of evidence for this mechanism in blood samples from aPL-positive and negative patients. Methods: Clinical histories and laboratory data were obtained from adult patients with histories for venous thromboembolism (n=223), stroke (n=73) and pregnancy complications (n=178) that were evaluated for thrombophilia and from patients who were free of known disease (n=76). A novel mechanistic assay was performed for “annexin A5 resistance” (A5R), an assay that detects antibody-mediated interference with annexin A5 anticoagulant activity. Results were expressed as a percent prolongation of coagulation times by annexin A5. Differences between groups were compared using nonparametric Kruskal-Wallis test. Result: At a cut off A5R value at 210% - the lowest quartile value of A5R - 48% (72 out of 150) of patients in the aPL-positive thrombosis/stroke/pregnancy complication group had A5R values within the lowest quartile, in contrast to 15% (33 out of 221) of the aPL-negative thrombosis patients and 8% (6 out of 76) of the disease-free control. In aPL-positive patients, the A5R values (median±SD: 208±58%, n=196) were found to be significantly lower than in aPL-negative patients (247±46%, n=284, p<0.001). Furthermore, aPL-positive patients with any thrombotic event, including histories for venous thromboembolism, stroke and pregnancy complications, had significantly lower A5R values (213±57%, n=150) than aPL-negative patients with any thrombotic event (258±42%, n=221; p<0.001) and from patients who were free of known disease (275±42%, n=76; p<0.001). Specifically, aPL-positive patients with a history of either venous thromboembolism (185±59, n=40) or pregnancy complications (233±73, n=25) had significantly lower A5R values than aPL-negative patients with either venous thromboembolism (250±62, n=62; p<0.01) or pregnancy complications (245±91, n=91; p<0.001). Conclusion: This novel mechanistic assay identifies a significant subset of patients currently defined as aPL-positive as having evidence for aPL antibody-mediated resistance to annexin A5 anticoagulant activity. A5R may mark patients who have a propensity for venous thromboembolism and/or pregnancy complications. The identification of specific thrombogenic mechanisms in patients with APS through mechanistic assays opens the possibilities of improved diagnosis and targeted therapies for patients with this condition. Disclosures: No relevant conflicts of interest to declare.

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