Functional Modulation of Activated Protein C using DNA-Aptamers

Abstract

Aptamers are single stranded DNA or RNA oligonucleotides which are able to interact with their designated target molecules with high affinity and specificity. The in-vitro procedure used for aptamer selection from a randomly designed oligonucleotide library is named Systematic Evolution of Ligands by EXponential enrichment (SELEX). Since aptamers, in comparison to antibodies, bind to larger surface structures, these molecules have the potential to better discriminate between the enzymatically inactive zymogen and active enzymes. Indeed, it has been previously shown that a DNA-aptamer selected against activated protein C (APC) possesses a high selectivity over zymogenic protein C.<br /> APC is a serine protease which is generated from zymogenic protein C by thrombin-mediated proteolytic activation on the surface of endothelial cells. APC performs its anticoagulant activity by proteolytic inactivation of activated factors V (FVa) and VIII (FVIIIa) which act as procoagulant cofactors within the blood coagulation cascade. Besides its anticoagulant functions, APC shows anti-inflammatory and anti-apoptotic activities which lead to endothelial barrier stabilization. Mild and moderate forms of inherited PC-deficiency predispose patients to an increased risk of venous thromboembolism while severe forms are associated with the development of purpura fulminans, a severe and potentially life-threatening thromboinflammatory disease comparable to severe sepsis. In severe sepsis acquired PC-deficiency plays a central role in the development of microvascular thrombosis leading to multiorgan failure. Substitution of septic patients with plasma-purified PC or a recombinant version of APC has been shown to improve outcome in terms of mortality rates. An increased incidence of bleedings, however, restricted the substitution of APC in patients with septicemia. Recombinant APC variants with impaired anticoagulant activity but intact cytoprotective properties are potentially safer drugs for the treatment of severe septicemia. Alternatively, specific ligands that selectively inhibit the anticoagulant activity of APC might offer several advantages over genetically engineered APC-variants.<br /> To increase the probability to select distinct APC binding sequences with divergent functional activities we used a capillary electrophoresis (CE)-based SELEX strategy. In addition different randomized ssDNA-libraries were applied, including a G-rich library to increase the likelihood of selecting G-quadruplex containing aptamers. The SELEX technology was further improved by developing a novel method for single-stranded DNA (ssDNA) production, allowing the convenient and rapid purification of ssDNA.<br /> A previously identified consensus motif dominated the selected aptamer pools despite using two differently structured randomized DNA-libraries during CE-SELEX. However, a G-quadruplex forming sequence raised up when using a G-rich DNA-library. Evaluation of the impact of identified aptamers on the amidolytic activity of APC combined with competition experiments using heparin as competitor revealed the so-called basic exosite of APC, which mediates its anticoagulant functions, as exclusive binding site. Further functional analysis clarified that, despite sharing the same binding site, different aptamers alter the functions of APC in different ways. Most interestingly, the G-quadruplex-based aptamer protected APC from inactivation by plasma protein C inhibitor while the other APC-specific aptamers rather accelerated this process.<br /> To conclude, the described aptamers may be useful for fast and efficient inhibition of APC under APC-mediated bleeding situations. Since the anti-apoptotic and anti-inflammatory functions of APC are most likely not influenced by aptamer-binding, such aptamers may be used as an adjuvant therapy in hemophilia in which APC inhibition might at least partially compensate the absence or reduced activity of FVIII or FIX. Furthermore, the availability of specific ligands with high discriminatory power between the zymogenic and active form of APC might be useful for the quantification of the active enzyme in biological fluids.