Introduction of selectively chemical reactive groups at the cell surface enables site-specific cell surface labeling and modification opportunity, thus facilitating the capability to study the cell surface molecular structure and function and the molecular mechanism it underlies. Further, it offers the opportunity to change or improve a cell’s functionality for interest of choice. In this study, two chemical reactive anchor lipids, phosphatidylethanolamine–poly(ethylene glycol)–dibenzocyclooctyne (DSPE–PEG2000–DBCO) and cholesterol–PEG–dibenzocyclooctyne (CHOL–PEG2000–DBCO) were synthesized and their potential application for cell surface re-engineering via lipid fusion were assessed with RAW 264.7 cells as a model cell. Briefly, RAW 264.7 cells were incubated with anchor lipids under various concentrations and at different incubation times. The successful incorporation of the chemical reactive anchor lipids was confirmed by biotinylation via copper-free click chemistry, followed by streptavidin-fluorescein isothiocyanate binding. In comparison, the cholesterol-based anchor lipid afforded a higher cell membrane incorporation efficiency with less internalization than the phospholipid-based anchor lipid. Low cytotoxicity of both anchor lipids upon incorporation into the RAW 264.7 cells was observed. Further, the cell membrane residence time of the cholesterol-based anchor lipid was evaluated with confocal microscopy. This study suggests the potential cell surface re-engineering applications of the chemical reactive anchor lipids.

Thrombomodulin (TM) is a type I transmembrane protein that is mainly expressed on endothelial cells and plays important roles in many biological processes. Circulating TM of different forms are also present in biofluids, such as blood and urine. Soluble TM (sTM), comprised of several domains of TM, is the major circulating TM, which is generated by either enzymatic or chemical cleavage of the intact protein under different conditions. Under normal conditions, sTM is present in low concentrations (<10 ng/mL) in the blood, but is elevated in several pathological conditions associated with endothelial dysfunction such as cardiovascular, inflammatory, infection, and metabolic diseases. Therefore, sTM level has been examined for monitoring disease development, such as disseminated intravascular coagulation (DIC), sepsis and multiple organ dysfunction syndrome in patients with coronavirus disease-2019 (COVID-19) recently. In addition, microvesicles that contain membrane TM (microvesicle-TM) have been found to be released from activated cells, which also contribute to levels of circulating TM in certain diseases. Several release mechanisms of sTM and microvesicle-TM have been reported, including enzymatic, chemical and TM mutation mechanisms. Measurements of sTM and microvesicle-TM have been developed and explored as biomarkers in many diseases. In this review, we summarize all these advances in three categories: (i) release mechanisms of circulating TM, (ii) methods for measuring circulating TM in biological samples, and (iii) correlation of circulating TM with diseases. Altogether, it provides a whole picture of recent advances on circulating TM in health and disease.

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