Polyphosphate Expression by Cancer Cell Extracellular Vesicles Mediates Binding of Factor XII and Contact Activation

Extracellular vesicles (EV) have been implicated in diverse biological processes, including intracellular communication, transport of nucleic acids, and regulation of vascular function. Levels of EV are elevated in cancer, and studies suggest that EV may stimulate thrombosis in cancer patients through expression of tissue factor. However, limited data also implicates EV in activation of the contact pathway of coagulation through activation of factor XII (FXII) to factor XIIa (FXIIa). To better define the ability of EV to initiate contact activation, we compared the ability of EV derived from different cancer cell lines to activate FXII. EV from all cell lines activated FXII, with those derived from pancreatic and lung cancer cell lines demonstrating the most potent activity. Concordant with activation of FXII, EV induced the cleavage of high molecular weight kininogen to cleaved kininogen. We also observed that EV from cancer patients stimulated FXII activation and HK cleavage. To define the mechanisms of FXII activation by EV, EV were treated with calf intestinal alkaline phosphatase or E. coli exopolyphosphatase to degrade polyphosphate; this treatment blocked binding of FXII to EV and the ability of EV to mediate FXII activation. In vivo, EV induced pulmonary thrombosis in wild-type mice, with protection conferred by deficiency of FXII, HK, or prekallikrein. Moreover, pre-treatment of EV with calf intestinal alkaline phosphatase inhibited their prothrombotic effect. These results indicate that polyphosphate mediates binding of contact factors to EV, and that EV-associated polyphosphate may contribute to the prothrombotic effects of EV in cancer.

Facile Comparison of Nonspecific Adsorptions of Proteins on Magnetic Submicron Particles with Alkaline Phosphatases as the Probes

The soluble form of Escherichia coli alkaline phosphatase (ECAP), commercialized calf intestinal alkaline phosphatase (CIAP) and biotinylated CIAP (Biotin-AP) were tested as probes for nonspecific adsorptions of proteins on micro/nano materials represented by magnetic submicron particles (MSP). The nonspecific adsorptions of three candidate probes were evaluated by directly measuring the activities of the adsorbed probes with 4-nitrophenylphosphate. The activities of those three candidate probes exhibited reasonable resistance to detergents, but the presences of > 0.1% glycerol in the adsorption systems greatly reduced their nonspecific adsorptions on MSPs. The nonspecific adsorptions of ECAP and Biotin-AP on some MSPs were significant and saturable but became negligible after such MSPs were coated with small zwitterions, bovine serum albumin and/or streptavidin. Only MSPs applicable to chemiluminescence immunoassays showed negligible nonspecific adsorptions of Biotin-AP and ECAP, but all of the tested MSPs exhibited negligible nonspecific adsorptions of CIAP. Therefore, Biotin-AP and ECAP were suitable probes for nonspecific adsorptions of proteins on micro/nano materials.

Developing a phosphodiesterase-5 inhibitor assay to detect counterfeit drugs containing phosphodiesterase-5 inhibitors using spectrophotometry

Background: Phosphodiesterase-5 inhibitors (PDE5i) are the first line of drugs used for the treatment of erectile dysfunction. PDE5i inhibits the activities of phosphodiesterase-5 (PDE5) found in corpus cavernosum reducing production of guanosine monophosphate (GMP) that results in vasodilation of blood vessels thereby prolonging penile erection. Various counterfeit drugs adulterated with unknown levels of PDE5i pose a danger with reported undesirable adverse effects on patients. Methods: We developed an assay to detect counterfeit drugs containing PDE5i using spectrophotometry based on a colour change of malachite green from blue to green when exposed to inorganic phosphate (Pi) measured at 630 nm. Initially, a standard graph of a known Pi concentration was established ranging from 0 to 20 µM followed by a dual biochemical reaction system of converting GMP from cGMP by PDE5, generating Pi from the first reaction’s product by calf intestinal alkaline phosphatase (CIAP), and then measuring Pi using an equation derived from the generated standard curve. Dilutions of CX1A, a counterfeit drug sample adulterated with the PDE5i sildenafil, were prepared via ultrasonication and filtration for assay testing and evaluation. Results: Sil was detected from the two separate samples comprised of low and high dilutions of CX1A quantified as 0.673% and 0.407%, respectively, based on a standard curve of pure sildenafil established from 0.1 nM to 300 µM. Conclusions: This PDE5i assay that we developed has the potential to become an alternative analytical method for detecting PDE5i showing comparable results when evaluated using HPLC.

Aptamers in Education: Undergraduates Make Aptamers and Acquire 21st Century Skills Along the Way

Aptamers have a well-earned place in therapeutic, diagnostic, and sensor applications, and we now show that they provide an excellent foundation for education, as well. Within the context of the Freshman Research Initiative (FRI) at The University of Texas at Austin, students have used aptamer selection and development technologies in a teaching laboratory to build technical and 21st century skills appropriate for research scientists. One of the unique aspects of this course-based undergraduate research experience is that students develop and execute their own projects, taking ownership of their experience in what would otherwise be a traditional teaching lab setting. Of the many successes, this work includes the isolation and characterization of novel calf intestinal alkaline phosphatase (anti-CIAP) RNA aptamers by an undergraduate researcher. Further, preliminary survey data suggest that students who participate in the aptamer research experience express significant gains in their self-efficacy to conduct research, and their perceived ability to communicate scientific results, as well as organize and interpret data. This work describes, for the first time, the use of aptamers in an educational setting, highlights the positive student outcomes of the aptamer research experience, and presents the research findings relative to the novel anti-CIAP aptamer.

The effect of the branched-chain amino acids on the in-vitro activity of bovine intestinal alkaline phosphatase

Branched-chain amino acids (BCAA) are used as nutritional support for patients with a range of conditions including liver cirrhosis and in-born errors of amino acid metabolism, and they are commonly used “sports” or exercise supplements. The effects of the BCAA on the in-vitro activity of calf intestinal alkaline phosphatase (EC. 3.1.3.1) were studied. All three BCAA were found to be uncompetitive inhibitors of the enzyme with L-leucine being the most potent ([Formula: see text] = 24.9 mmol/L) and L-valine, the least potent ([Formula: see text] = 37 mmol/L). Mixed BCAA are able to act in combination to inhibit the enzyme. Given the important role of intestinal alkaline phosphatase in gut homeostasis, these findings have potential implications for those taking high levels of BCAA as supplements.