A fully automated immunoassay for quantifying tissue-nonspecific alkaline phosphatase activity on calcified extracellular vesicles for cardiovascular risk assessment.

Vascular calcification in atherosclerosis is a key process underlying cardiovascular disease (CVD) and chronic kidney disease (CKD), strongly predicting acute cardiovascular events. Macrocalcification stabilizes plaques, whereas microcalcification destabilizes them, making the assessment of active microcalcifications important for predicting plaque rupture. Although imaging methods such as 18F-NaF positron emission tomography can detect active microcalcifications, their clinical utility is limited by cost and accessibility. Therefore, there is a potential need for simple, minimally invasive blood biomarkers to assess microcalcification activity. Recent studies have reported that calcified extracellular vesicles (EVs) are crucial mediators of microcalcification and we hypothesized that asymptomatic plaque rupture may lead to the release of calcified EVs. In this study, we developed a fully automated immunoassay to quantify tissue-nonspecific alkaline phosphatase (TNAP) activity on calcified EVs in serum as a new biomarker of vascular microcalcification. The assay demonstrated high reproducibility and rapid turnaround (17 min), requiring only 25 μL of serum. It successfully detected calcified EVs in the culture media of primary human aortic smooth muscle cells that differentiated into osteoblast-like cells under high-phosphorus conditions, as well as in suspensions of their ultracentrifugation-derived precipitates. Feasibility testing revealed increased TNAP activity on calcified EVs in the serum of patients with a history of coronary artery disease and CKD compared with that in non-disease controls, supporting its potential as a biomarker. This study suggested that our assay could serve as a standardized tool for evaluating active microcalcification and asymptomatic plaque rupture, potentially enabling early detection of high-risk patients and informing therapeutic development.