Circulating Thrombomodulin: Release Mechanisms, Measurements, and Levels in Diseases and Medical Procedures.

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 novel coronavirus disease 2019 (COVID-19) recently. In addition, microvesicles (MVs) that contain membrane TM (MV-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 MV-TM have been reported, including enzymatic, chemical, and TM mutation mechanisms. Measurements of sTM and MV-TM have been developed and explored as biomarkers in many diseases. In this review, we summarize all these advances in three categories as follows: (1) release mechanisms of circulating TM, (2) methods for measuring circulating TM in biological samples, and (3) correlation of circulating TM with diseases. Altogether, it provides a whole picture of recent advances on circulating TM in health and disease.

Buyer beware: Inexpensive, high cadmium jewelry can pose severe health risks.

The use of cadmium to produce inexpensive jewelry has recently been documented. Governments have adopted varying standards, with US states focused on either total cadmium content or extractable cadmium from children's jewelry, while the European Union has adopted a limit of 100 mg/kg cadmium for all jewelry. This study evaluated 80 items purchased at a discount jewelry store. The objective was to determine prevalence of cadmium in this jewelry, the amount of cadmium released by simulated mouthing or ingestion, and to confirm previous reports that damage to jewelry can increase cadmium release. Finally, a modified toxicity characteristic leaching procedure (TCLP) assessed the potential for jewelry to release cadmium after disposal. Thirty-two (40%) items showed detectable cadmium by X-ray fluorescence. Nine high­cadmium pendants and rings with cadmium content ranging from 31.3 to 89.2% were subjected to extractions simulating mouthing or ingestion. Seven of nine items extracted in dilute saline to simulate mouthing released more than the US recommended maximum of 18 micrograms. Damaged jewelry released more cadmium for most items tested, with one ring yielding an average of 10,600 micrograms. Two pendants small enough to be swallowed were tested using dilute HCl to simulate ingestion. While one pendant did not release cadmium in excess of the US recommended maximum of 200 micrograms even when damaged, the other released an average of 63,100 micrograms after being damaged. Fourteen of fifteen samples of two high cadmium charms extracted using a modified TCLP extraction exceeded the 1.0 mg/L TCLP limit for cadmium, averaging 13.1 and 9.6 mg/L respectively for the two charms. These results demonstrate that high­cadmium jewelry may pose a serious hazard if mouthed or ingested, and that regulatory standards that do not take into account the potential for increased release of cadmium resulting from damage to jewelry electroplating are inadequate.

End-point modification of recombinant thrombomodulin with enhanced stability and anticoagulant activity.

Thrombomodulin (TM) is an endothelial cell membrane protein that plays essential roles in controlling vascular haemostatic balance. The 4, 5, 6 EGF-like domain of TM (TM456) has cofactor activity for thrombin binding and subsequently protein C activation. Therefore, recombinant TM456 is a promising anticoagulant candidate but has a very short half-life. Ligation of poly (ethylene glycol) to a bioactive protein (PEGylation) is a practical choice to improve stability, extend circulating life, and reduce immunogenicity of the protein. Site-specific PEGylation is preferred as it could avoid the loss of protein activity resulting from nonspecific modification. We report herein two site-specific PEGylation strategies, enzymatic ligation and copper-free click chemistry (CFCC), for rTM456 modification. Recombinant TM456 with a C-terminal LPETG tag (rTM456-LPETG) was expressed in Escherichia coli for its end-point modification with NH2-diglycine-PEG5000-OMe via Sortase A-mediated ligation (SML). Similarly, an azide functionality was easily introduced at the C-terminus of rTM456-LPETG via SML with NH2-diglycine-PEG3-azide, which facilitates a site-specific PEGylation of rTM456via CFCC. Both PEGylated rTM456 conjugates retained protein C activation activity as that of rTM456. Also, they were more stable than rTM456 in Trypsin digestion assay. Further, both PEGylated rTM456 conjugates showed a concentration-dependent prolongation of thrombin clotting time (TCT) compared to non-modified protein, which confirms the effectiveness of these two site-specific PEGylation schemes.

Chemical Reactive Anchoring Lipids with Different Performance for Cell Surface Re-engineering Application.

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.