Inactivation of Plasminogen Activator Inhibitor-1 by RNA Aptamer Molecules

The serine protease inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1), binds and inhibits the following plasminogen activators: tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). This decreases plasmin production and triggers the dissolution of fibrin clots. Elevated levels of PAI-1 have been correlated with an increased risk for cardiovascular disease, as well as obesity and metabolic syndrome. Consequently, pharmacologically suppressing PAI-1 might prevent, or successfully treat vascular disease. Several PAI-1 small molecule inhibitors have recently been studied (PAI-039 is the best characterized). Since PAI-1 is a multifunctional protein, completely inhibiting PAI-1 may hinder its other functions. Therefore, it is important to independently develop inhibitors to the various regions of PAI-1. This can be accomplished by using small RNA molecules (aptamers) that bind with high affinity and specificity to individual protein domains. We recently published a paper showing how PAI-1 specific RNA aptamers bind to the heparin/vitronectin binding site of PAI-1 (Blake et al., 2009). We demonstrated that PAI-1 specific aptamers prevent cancer cells from detaching from vitronectin (in the presence of PAI-1), resulting in increased cell adhesion. These aptamers had no effect on PAI-1's other functions, particularly its antiproteolytic activity.

This study's goal was to develop RNA aptamers to the active site of PAI-1; thereby, preventing the ability of PAI-1 to interact with plasminogen activators (tPA and uPA).

The aptamers were generated by the systematic evolution of ligands by exponential enrichment (SELEX). Adopting the SELEX in vitro selection technique ensures the creation of nuclease-resistant RNA molecules that will bind to target proteins. We used in vitroassays to determine the effect of the aptamers on the interaction of PAI-1 with both tPA and uPA.

We isolated a family of aptamers that bind to wild-type PAI-1 with affinities in the nanomolar range. From this family, two aptamer clones (10–2 and 10–4) exhibited reduced binding to elastase cleaved PAI-1 and the PAI-1/tPA complex. This suggests that they bind to, or in the vicinity of, the active site. Using a chromogenic assay, we showed that the aptamer clone 10–4, and (to a lesser extent) the aptamer clone 10–2, inhibited PAI-1's antiproteolytic activity against tPA, further suggesting that these clones bind to PAI-1 within its active site region. Interestingly, neither clone was able to prevent PAI-1 from inhibiting uPA activity. Both aptamer clones disrupted PAI-1's ability to form a stable covalent complex with tPA. Increasing aptamer concentrations positively correlated with an increase in cleaved PAI-1, suggesting that these aptamer clones convert PAI-1 from an inhibitor to a substrate. Furthermore, we showed that both aptamer clones are able to inhibit PAI-1's activity in the presence of vitronectin.

We have shown that we are able to inhibit one of PAI-1's functions without hindering its other functions. To our knowledge, this is the first report of an RNA molecule that is able to inhibit the antiproteolytic activity of PAI-1. We have generated two specific RNA aptamer molecules that hinder the ability of PAI-1 to interact with tPA, which has the potential to be used as an antithrombotic agent.

No relevant conflicts of interest to declare.