Introduction Sickle cell disease (SCD) is an inherited blood disorder that affects millions of individuals worldwide. It is referred to as a hypercoagulable state and patients are at risk for multiple thrombotic complications including venous thromboembolism (VTE). Despite being recognized as the first molecular disease, treatment options remain 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. At the endothelial cell surface, ANXA2 and S100A10 (p11) form a heterotetramer, which accelerates tPA (tissue plasminogen activator)-dependent activation of the fibrinolytic protease, plasmin, thereby helping to maintain blood in the fluid state. Overexpression of ANXA2 has been associated with severe, sometimes life-threatening hemorrhage, as seen in acute promyelocytic leukemia. 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 the human A2 gene have been associated with more severe SCD phenotypes, such as avascular necrosis (AVN), ischemic stroke, and priapism. However, there is a paucity of data on the protein expression and function of ANXA2 in SCD patients with these genotypic variants.

This pilot study was conducted to evaluate the relationship between ANXA2 expression and a history of (H/O) 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 older, were enrolled at the Comprehensive Sickle Cell Clinic following written informed consent. Whole blood was collected in Ethylenediaminetetraacetic acid (EDTA)-coated vacutainer tubes and processed for isolation of peripheral blood mononuclear cells (PBMCs). 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. A sample size of 32 subjects would achieve 80% power at a 95% confidence interval. ANXA2 mRNA levels were assessed using quantitative real time PCR from RNA isolated from PBMCs. Total ANXA2 protein expression was determined by Western blotting using protein isolated from PBMCs. ANXA2 protein expression was normalized to β-actin for each sample. Statistical comparisons in ANXA2 mRNA and protein expression between patients with and without histories of VTE were performed using an unpaired t-test in GraphPad Instat (v3.1).

Results Thirty-two subjects with homozygous SCD (HbSS) (females = 20 [62.5%]) with a median age of 37 years [interquartile range (IQR): 30.0-47.5] were enrolled. Sixteen (50%) of the study subjects had a history of VTE. Western blot analysis showed a significantly lower ANXA2 protein expression in subjects with H/O VTE vs. those with no H/O VTE (0.48 +/- 0.01 vs. 1.02 +/- 0.13 fold actin, p<0.01). ANXA2 mRNA by qPCR was also lower in subjects with H/O VTE compared to those without H/O VTE (0.8855 +/- 0.079 vs 1.058 +/- 0.087 units, SEM, p = 0.084), although this was not statistically significant.

Conclusion This pilot study is the first to demonstrate significantly lower ANXA2 protein expression in SCD patients with VTE compared to those without. We also observed a non-significant, but lower mRNA levels in subjects with H/O VTE. The discrepancy between protein and transcript levels suggests potential post-transcriptional regulation or increased protein degradation in the VTE group. Given ANXA2's critical role in endothelial function and fibrinolysis, these findings point to its potential as a prognostic biomarker for VTE risk and disease severity in SCD. Larger studies are warranted to confirm these results and elucidate the mechanistic role of ANXA2 in VTE and thrombotic complications in SCD.

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2025
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