Annexin A2 As a Potential Biomarker for Venous Thromboembolism in Sickle Cell Anemia

Introduction

Sickle cell disease (SCD) is an inherited blood disorder that affects about 100,000 individuals in the United States (U.S.). It is referred to as a hypercoagulable state and is characterized by the occurrence of multiple complications including venous thromboembolism (VTE), stroke, avascular necrosis (AVN), pulmonary hypertension, and priapism. Despite being recognized as the first molecular disease, treatment options are limited. Furthermore, a severe gap remains in the identification of prognostic biomarkers for complications of SCD.

VTE, including both deep venous thrombosis and pulmonary embolism, has a high incidence in adults with SCD, occurring in up to 12% of patients by 40-years of age. Annexin A2 (ANXA2), a calcium-regulated, phospholipid-binding protein that is expressed within and on the surface of endothelial cells, has been identified as a key component of the fibrinolytic system. Reduced expression of human ANXA2 has been implicated as a possible risk factor for VTE in a cohort of non-SCD patients. Single nucleotide polymorphisms (SNPs) in human A2 gene have been associated with more severe SCD phenotypes, such as AVN, stroke and priapism. However, there is a paucity of data on the protein expression and function in SCD patients with these genotypic variants.

This study was a pilot study conducted to evaluate the relationship between annexin A2 expression level and VTE in patients with SCD.

Methods

The study protocol was approved by the Institutional Review Board (IRB) at the University of Tennessee Health Sciences Center (UTHSC). Subjects with SCD, 18 years and above, were enrolled at the outpatient sickle cell clinic following written informed consent. Whole blood was collected in EDTA-coated vacutainer tubes and processed for isolation of PBMCs (peripheral blood mononuclear cells). ANXA2 mRNA (messenger ribonucleic acid) levels were assessed using quantitative real time polymerase chain reaction (qPCR), from RNA isolated from PBMCs. To compare ANXA2 mRNA levels in this cohort, a t-test was employed. The sample size for the study was determined based on a non-SCD patient cohort, as the expression of ANXA2 protein had not been previously evaluated in SCD patients. The sample size calculation aimed for a total of 32 subjects to achieve 80% power at a 95% confidence interval.

Results

A total of 32 subjects with homozygous SCD (HbSS) were enrolled, 16 with VTE history and 16 without VTE. There were 12 male and 20 female subjects, age range 18-74 years. In this cohort, 12 subjects had a history of AVN and 20 subjects did not.

Using qPCR, ANXA2 mRNA levels were lower in subjects with a history of VTE compared to subjects without VTE (0.8855 +/- 0.079 vs 1.058 +/- 0.087 units, SEM, p = 0.0839), although this difference was not statistically significant. In addition, ANXA2 mRNA levels were evaluated in subjects with and without AVN and were found to be lower in subjects with AVN (mean 0.8798 +/- 0.089 vs 1.034 +/- 0.079, p = 0.0831).

Conclusion

This pilot study shows decreased annexin A2 mRNA levels in SCD subjects with VTE and AVN. Although the difference between study groups did not achieve statistical significance, our study may be limited by a small sample size. Western blot studies to evaluate ANXA2 protein expression in this study cohort are underway.

To our knowledge, this is the first study evaluating ANXA2 mRNA levels/protein expression in subjects with SCD. Based on its known function, ANXA2 could serve as a useful prognostic biomarker for SCD disease severity and further studies are warranted.

Ogu:Pfizer: Speakers Bureau; Vertex Pharmaceuticals: Consultancy; Novo-Nordisk: Consultancy; Emmaus: Speakers Bureau. Ataga:GSK: Consultancy, Honoraria; Hillhurst Biopharmaceuticals: Consultancy, Honoraria; Novartis: Honoraria; Novo Nordisk: Research Funding; Sanofi: Consultancy; Vertex: Membership on an entity's Board of Directors or advisory committees; Vitalant: Membership on an entity's Board of Directors or advisory committees; Forma Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Agios Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees.