Inhibiting the intrinsic pathway of coagulation with a factor XII-targeting RNA aptamer

Abstract

Background: Exposure of the plasma protein factor XII (FXII) to an anionic surface generates activated FXII that not only triggers the intrinsic pathway of blood coagulation through the activation of FXI but also mediates various vascular responses through activation of the plasma contact system. While deficiencies of FXII are not associated with excessive bleeding, thrombosis models in factor-deficient animals have suggested that this protein contributes to stable thrombus formation. Therefore, FXII has emerged as an attractive therapeutic target to treat or prevent pathological thrombosis formation without increasing the risk for hemorrhage.

Objectives: Using an in vitro directed evolution and chemical biology approach, we sought to isolate a nuclease-resistant RNA aptamer that binds specifically to FXII and directly inhibits FXII coagulant function.

Methods and results: We describe the isolation and characterization of a high-affinity RNA aptamer targeting FXII/activated FXII (FXIIa) that dose dependently prolongs fibrin clot formation and thrombin generation in clinical coagulation assays. This aptamer functions as a potent anticoagulant by inhibiting the autoactivation of FXII, as well as inhibiting intrinsic pathway activation (FXI activation). However, the aptamer does not affect the FXIIa-mediated activation of the proinflammatory kallikrein-kinin system (plasma kallikrein activation).

Conclusions: We have generated a specific and potent FXII/FXIIa aptamer anticoagulant that offers targeted inhibition of discrete macromolecular interactions involved in the activation of the intrinsic pathway of blood coagulation.

Keywords: RNA aptamers; anticoagulant agents; blood coagulation; factor XII; factor XIIa.

Figure 1. Aptamer R4cXII-1 binds with high affinity to FXII and FXIIa

Nitrocellulose filter binding assays of aptamer to A) FXII (■) and FXIIa (▲) and B) indicated proteins. C) Linear sequence and dissociation constants of full length R4cXII-1 and truncated R4cXII-1t. In A, the data represent the mean ± SEM of triplicate measures and in B, the data are representative of at least two independent experiments.

Figure 2. Aptamer R4cXII-1t dose dependently anticoagulates human plasma in an aPTT, but not a PT clotting assay

Effect of R4cXII-1t (■) and scrambled control RNA (▲) in an aPTT and R4cXII-1t (▼) in a PT. The data were normalized to the baseline clot time without aptamer present and represent the mean ± SEM of duplicate measures.

Figure 3. Aptamer R4cXII-1t dose dependently impairs thrombin generation in a TGA assay initiated with ellagic acid

Thrombograms of normal pooled plasma activated with ellagic acid with various concentrations of A) R4cXII-1t and B) scrambled control RNA. C) Lag time, D) Peak thrombin generation, E) Endogenous thrombin potential, and F) Rate of thrombin generation of R4cXII-1t (■) and scrambled control RNA (▲). Data are representative of three independent experiments.

Figure 4. Aptamer R4cXII-1 does not inhibit FXIIa cleavage of a small peptide substrate

FXIIa was incubated with varying amounts of R4cXII-1 (■), scrambled control RNA (▲), and CTI (▼), and active site activity was measured with a chromogenic substrate. The data were normalized to the rate in the absence of compound and represent the mean ± SEM of duplicate measures.

Figure 5. Aptamer R4cXII-1 does not induce autoactivation of FXII but blocks the activities of several autoactivators of FXII

A) FXII was incubated with varying amounts of dextran sulfate (■), R4cXII-1 (▲), or scrambled control RNA (▼), and assayed for the amount of FXIIa formed. The data represent the mean ± SEM of triplicate measures. B) FXII was pre-incubated with R4cXII-1 (black bars) or scrambled control RNA (grey bars) before addition of the indicated autoactivator, and assayed for the amount of FXIIa formed. The data were normalized to the rate in the absence of aptamer and represent the mean ± SEM of triplicate measures.

Figure 6. Aptamer R4cXII-1 dose dependently inhibits FXIIa-catalyzed activation of FXI

A) FXI was activated by FXIIa, the reaction was quenched at various time-points and assayed for the amount of FXIa formed in the presence of R4cXII-1 (■), scrambled control RNA (▼), or buffer (▲). The data represent the mean ± SEM of triplicate measures. B) The amount of FXIa formed was determined in the presence of R4cXII-1 at concentrations of 0nM(▲), 1nM (■), 3nM (•), 6nM (◆), 10nM (▼), 20nM (□), 50nM (○). The data are representative of three independent measures. C) Fractional activity of R4cXII-1 versus the ratio of R4cXII-1 to FXII present in the reaction. The data represent the mean ± SEM of triplicate measures.

Figure 7. Aptamer R4cXII-1 does not inhibit the ability of FXII to activate prekallikrein

Prekallikrein was activated by FXIIa, the reaction was quenched at various time points and assayed for the amount of kallikrein formed in the presence of R4cXII-1 (▼), scrambled control RNA (▲), or buffer (■).The data represent the mean ± SEM of triplicate measures.