Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates

Abstract

Objective: During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk.

Approach and results: We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding.

Conclusions: These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

Keywords: antisense oligonucleotide; factor XI; platelets; thrombus formation; vascular graft.

Figure 1

Diagram of the baboon AV shunt thrombosis model showing interposition of a 2.0 cm-long, 4.0 mm i.d., thrombogenic vascular graft segment (A), and representative gamma camera images of an acutely developing thrombus within the thrombogenic segment, measured in a baboon with normal FXI levels (B). Circulating ¹¹¹Indium-labeled platelets accumulate at sites of thrombus formation. Platelet deposition on the collagen segment (i.e., accumulated ¹¹¹Indium-activity that is measurable above circulating background levels) appears within 10 minutes after restoring blood flow through the shunt. Images obtained at later times (30 min, 60 min) show propagation of the thrombus into the distal shunt tubing. Images were obtained in real time and acquired at 5 minute intervals. The blood flow rate was 100 mL/min (initial wall shear rate: 265/sec).

Figure 2

An intravenous injection of aXIMab (doses ranging from 40-70 ug/kg) was given to 4 baboons and blood samples were collected into citrate anticoagulant. Inhibition of FXI plasma activity by ~50% produced ~50% inhibition of platelet deposition in propagating thrombus, as assessed from gamma camera images taken at the study endpoint, 60 mins after the intiation of blood flow (A), or by dynamic imaging of platelet deposition over the entire 0-60 min study interval (B).

Figure 3

FXI protein and activity measurements. Four baboons were given FXI ASO subcutaneously, 3 times per week, at a dose of 25 mg/kg. After dosing for various lengths of time (shaded areas, days 39, 49, 60, 53), FXI plasma protein levels and activity (A) and aPTT levels (B) were measured. Inhibition of FXI plasma activity by 50% was achieved in all animals by day 25, and reached maximum inhibition (~80%) by day 35.

Figure 4

Effect of FXI ASO-mediated inhibition on thrombus formation in individual study animals. Platelet deposition was quantitated within the components of the thrombogenic device that was inserted into AV shunts in baboons, including (A) the proximal, 2 cm-long, collagen-coated graft segment (designated the “head” of thrombus in Figure 1), and (B) the 10 cm-long shunt segment containing thrombus that propagated immediately distal to the collagen segment (designated the “tail” of thrombus in Figure 1). FXI depletion with FXI ASO had little effect on thrombus formation on the collagen segment (panel A). In contrast, propagated thrombus, which is dependent on upstream thrombin generation, was reduced by ASO depletion of FXI (panel B).

Figure 5

Effect of ASO-mediated inhibition of FXI on thrombus formation in baboons. After 60 minutes of blood exposure, platelet accumulation in propagated thrombus in the four ASO-treated animals averaged 2.40 ± 0.33 × 10⁹ platelets deposited (n = 18; Figure 4B), a value that was reduced significantly (by 41%; p = 0.037) vs. the control group results (4.06 ± 0.83 × 10⁹ platelets deposited; n = 10). The control values included measurements in the four study animals that were taken before ASO treatment (data in Figure 4B), plus results in six additional untreated control animals.

Figure 6

Samples taken from FXI ASO treated animals were evaluated for their ability to generate thrombin upon stimulation with small amounts of TF. As plasma levels of FXI were reduced by ASO treatment, thrombus propagation (A), and peak thrombin generation (PTG) levels (B) were also reduced. When FXI plasma levels were inhibited by > 50%, thrombin generation was virtually abolished.

Figure 7

FXI ASO treatment did not impair hemostasis in baboons. A total of 25 bleeding time measurements were performed in 9 untreated baboons, and averaged (3.9 ± 0.3 minutes). Twenty-one measurements were taken in 4 treated baboons beginning at least two weeks after FXI ASO administration had begun and averaged 4.0 ± 0.3 min (p > 0.5 vs. controls). Average bleeding time measurements in each of the four ASO treatment animals were also similar (~4 minutes).