Factor IXa

Factor IX (FIX) is a serine protease that plays an important role in generating the critical quantity of thrombin necessary in coagulation. The TF/FVIIa complex cleaves FIX to generate FIXa. FIXa combines with FVIIIa on the platelet surface and activates FX to FXa, which finally converts prothrombin into thrombin (Schmidt and Bajaj, 2003). An RNA selection to FIXa was conducted by Rusconi and co-workers, and after eight rounds of selection, an aptamer was identified that bound to FIXa with a Kd of 0.65 + 0.2nmol/L and exhibited greater than 5000-fold specificity for FIXa over structurally similar coagulation factors VIIa, Xa, XIa, and activated protein C (Rusconi et al., 2002). A truncated version of this FIXa aptamer (9.3t) retained high affinity for FIXa (Kd = 0.58 + 0.1nmol/L) and completely blocked FX cleavage by the enzyme complex.

The anticoagulant activity of 9.3t was tested in an activated partial thromboplastin time (aPTT) test that is sensitive to FIXa inhibition. The aptamer increased the clot time in a dose-dependent manner and was able to prolong clotting times to that of plasma that was FIX deficient (

Hemorrhage is a major cause of morbidity associated with anticoagulation therapy (Ginsberg et al., 2001; Moll and Roberts, 2002). Effective control of anticoagulation is paramount in establishing truly safe therapies for all patient populations. To achieve this, Rusconi and co-workers developed an RNA antidote that reversed 9.3t activity and ultimately produced the first rationally designed drug-antidote pair for anticoagulant therapy. By exploiting the sequence-specific structure of an aptamer, they constructed a second RNA oligonucleotide that is complimentary to a region of the 9.3t aptamer (Fig. 6.4). This antidote oligonucleotide reversed the anticoagulant activity of the FIXa aptamer within 10 min in a sustainable fashion for over 5 h (Rusconi et al., 2002). Almost 5% of the 12 million people who receive heparin annually develop heparin-induced thrombocytopenia (HIT) (Blakeman, 1999) and are therefore unable to receive the drug (Eikelboom and Hankey, 2002). Patients who require repeated anticoagulation, such has those requiring renal dialysis, require viable alternatives.

In order to evaluate the activity of the factor IXa aptamer in vivo, Rusconi and colleagues developed a cholesterol-modified aptamer (Ch-9.3t) which retained binding affinity (Kd = 5.29 + 1.1 nmol/L) and potent anticoagulation activity (Rusconi et al., 2004). The aptamer was then tested in porcine and murine plasma and found to cross-react in both species with similar efficiency to that seen in human plasma. They showed using a porcine systemic anticoagulation model that the ap-tamer increased ACT and aPTT compared with negative controls without affecting PT. The difference in duration of effect between the two compounds was striking, with a 9.3t blood half-life of ~5-10min and —60-90 min for Ch-9.3t. Antidote 5-2C reversed > 95% of aptamer function within 10 min in these animals (Rusconi et al., 2004).

In order to evaluate the antithrombotic effect of the aptamer, the drug was tested in a murine arterial injury model whereby the carotid artery was injured with ferric chloride in animals that received Ch- 9.3t or a functionally inactive form of the aptamer. All the mice in the negative control group developed an occlusive thrombi in 8.1 + 0.1 min in contrast to the aptamer-treated group where > 80% remained patent after 30 min after injury (time to occlusive thrombus > 24.4 min, P < 0.0001) (Rusconi et al., 2004). To measure the effect of antidote 5-2C to ameliorate bleeding due to anticoagulation with Ch-9.3t, a murine tail-transection bleeding model was employed where the aptamer or negative control was administered and after 1 h the tail was clipped and blood loss was measured over 15 min. Animals treated with Ch-9.3t exhibited significantly more blood loss (176 + 23.7 mL) than controls (48 + 17.8 mL, P = 0.007). Administration of 5 -2C immediately after tail transaction however, prevented the hemorrhage seen in aptamer-treated animals (blood loss 54.5 + 13.6 mL, P =0.0001) (Rusconi et al., 2004).

Regado Biosciences has developed an optimized version of this anti-FIXa aptamer-antidote pair, termed REG1, and is progressing rapidly towards phase I clinical testing.

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