Development and Characterization of Monovalent and Bivalent RNA Aptamers Targeting the Common Pathway of Coagulation

Anticoagulant agents are commonly used drugs to reduce blood coagulation in acute and chronic clinical settings. Many of these drugs target the common pathway of coagulation because it is critical for thrombin generation and disruption of this portion of the pathway has profound effects on the hemostatic process. Currently available drugs for these indications struggle with balancing desired activity with immunogenicity and poor reversibility or irreversibility in the event of hemorrhage. While improvements are being made with the current drugs, new drugs with better therapeutic indices are needed for surgical intervention and chronic indications to prevent thrombosis from occurring.

A class of therapeutics known as aptamers may be able to meet the need for safer anticoagulant agents. Aptamer are short single-stranded RNA oligonucleotides that adopt specific secondary and tertiary structures based upon their sequence. They can be generated to both enzymes and cofactors because they derive their inhibitory activity by blocking protein-protein interactions, rather than active site inhibition. They inhibit their target proteins with a high level of specificity and bind with high affinity to their target. Additionally, they can be reversed using two different antidote approaches, specific oligonucleotide antidotes, or with cationic, “universal” antidotes. The reversal of their activity is both rapid and durable.

The ability of aptamers to be generated to cofactors has been conclusively proven by generating an aptamer targeting the common pathway coagulation cofactor, Factor V (FV). We developed two aptamers with anticoagulant ability that bind to both FV and FVa, the active cofactor. Both aptamers were truncated to smaller functional sizes and had specific point mutant aptamers developed for use as controls. The anticoagulant activity of both aptamer-mutant pairs was characterized using plasma-based clotting assays and whole blood assays. The mechanism of action resulting in anticoagulant activity was assessed for one aptamer. The aptamer was found to block FVa docking to membrane surfaces, a mechanism not previously observed in any of our other anticoagulant aptamers.

To explore development of aptamers as anticoagulant agents targeting the common pathway for surgical interventions, we fused two anticoagulant aptamers targeting Factor X and prothrombin into a single molecule. The bivalent aptamer was truncated to a minimal size while maintaining robust anticoagulant activity. Characterization of the bivalent aptamer in plasma-based clotting assays indicated we had generated a very robust anticoagulant therapeutic. Furthermore, we were able to simultaneously reverse the activity of both aptamers with a single oligonucleotide antidote. This rapid and complete reversal of anticoagulant activity is not available in the antithrombotic agents currently used in surgery.

Preservation of beam emittance in the presence of ion motion in future high-energy plasma-wakefield-based colliders.

The preservation of beam quality in a plasma wakefield accelerator driven by ultrahigh intensity and ultralow emittance beams, characteristic of future particle colliders, is a challenge. The electric field of these beams leads to plasma ions motion, resulting in a nonlinear focusing force and emittance growth of the beam. We propose to use an adiabatic matching section consisting of a short plasma section with a decreasing ion mass to allow for the beam to remain matched to the focusing force. We use analytical models and numerical simulations to show that the emittance growth can be significantly reduced.

CPAG: software for leveraging pleiotropy in GWAS to reveal similarity between human traits links plasma fatty acids and intestinal inflammation.

Meta-analyses of genome-wide association studies (GWAS) have demonstrated that the same genetic variants can be associated with multiple diseases and other complex traits. We present software called CPAG (Cross-Phenotype Analysis of GWAS) to look for similarities between 700 traits, build trees with informative clusters, and highlight underlying pathways. Clusters are consistent with pre-defined groups and literature-based validation but also reveal novel connections. We report similarity between plasma palmitoleic acid and Crohn's disease and find that specific fatty acids exacerbate enterocolitis in zebrafish. CPAG will become increasingly powerful as more genetic variants are uncovered, leading to a deeper understanding of complex traits. CPAG is freely available at www.sourceforge.net/projects/CPAG/.

Plasma miRNAs as early biomarkers for detecting hepatocellular carcinoma.

The early detection of hepatocellular carcinoma (HCC) presents a challenge because of the lack of specific biomarkers. Serum/plasma microRNAs (miRNAs) can discriminate HCC patients from controls. We aimed to identify and evaluate HCC-associated plasma miRNAs originating from the liver as early biomarkers for detecting HCC. In this multicenter three-phase study, we first performed screening using both plasma (HCC before and after liver transplantation or liver hepatectomy) and tissue samples (HCC, para-carcinoma and cirrhotic tissues). Then, we evaluated the diagnostic potential of the miRNAs in two case-control studies (training and validation sets). Finally, we used two prospective cohorts to test the potential of the identified miRNAs for the early detection of HCC. During the screening phase, we identified ten miRNAs, eight of which (miR-20a-5p, miR-25-3p, miR-30a-5p, miR-92a-3p, miR-132-3p, miR-185-5p, miR-320a and miR-324-3p) were significantly overexpressed in the HBV-positive HCC patients compared with the HBV-positive cancer-free controls in both the training and validation sets, with a sensitivity of 0.866 and specificity of 0.646. Furthermore, we assessed the potential for early HCC detection of these eight newly identified miRNAs and three previously reported miRNAs (miR-192-5p, miR-21-5p and miR-375) in two prospective cohorts. Our meta-analysis revealed that four miRNAs (miR-20a-5p, miR-320a, miR-324-3p and miR-375) could be used as preclinical biomarkers (pmeta  < 0.05) for HCC. The expression profile of the eight-miRNA panel can be used to discriminate HCC patients from cancer-free controls, and the four-miRNA panel (alone or combined with AFP) could be a blood-based early detection biomarker for HCC screening.