A kallikrein-targeting RNA aptamer inhibits the intrinsic pathway of coagulation and reduces bradykinin release

SummaryBackground Plasma kallikrein is a serine protease that plays an integral role in many biological processes including coagulation, inflammation, and fibrinolysis. The main function of kallikrein in coagulation is the amplification of activated factor XIIa (FXIIa) generation which ultimately leads to thrombin generation and fibrin clot formation. Kallikrein is generated by FXIIa-mediated cleavage of the zymogen, prekallikrein which is usually complexed with the nonenzymatic cofactor, high-molecular-weight kininogen (HK). HK also serves as a substrate for kallikrein to generate the pro-inflammatory peptide, bradykinin (BK). Interestingly, prekallikrein-deficient mice are protected from thrombotic events while retaining normal hemostatic capacity. Therefore, therapeutically targeting kallikrein may provide a safer alternative to traditional anticoagulants with anti-inflammatory benefits. Objectives We sought to isolate and characterize an RNA aptamer that binds and inhibits plasma kallikrein and to elucidate its mechanism of action. Methods and Results Using convergent Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we isolated an RNA aptamer that targets kallikrein. This aptamer, Kall1-T4, specifically binds to both prekallikrein and kallikrein with similar subnanomolar binding affinities and dose-dependently prolongs fibrin clot formation in an aPTT coagulation assay. In a purified in vitro system, Kall1-T4 inhibits the reciprocal activation of prekallikrein and FXII primarily by reducing the rate of FXIIa-mediated prekallikrein activation. Additionally, Kall1-T4 significantly reduces kallikrein-mediated HK cleavage and subsequent BK release. Conclusions We have isolated a specific and potent inhibitor of prekallikrein/kallikrein activity that serves as a powerful tool for further elucidating the role of kallikrein in thrombosis and inflammation. This article is protected by copyright. All rights reserved.