The PI3Kδ Inhibitor Idelalisib Diminishes Platelet Function and Shows Antithrombotic Potential

Abstract

Background: Clinical management of ischemic events and prevention of vascular disease is based on antiplatelet drugs. Given the relevance of phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) as a candidate target in thrombosis, the main goal of the present study was to identify novel antiplatelet agents within the existing inhibitors blocking PI3K isoforms.

Methods: We performed a biological evaluation of the pharmacological activity of PI3K inhibitors in platelets. The effect of the inhibitors was evaluated in intracellular calcium release and platelet functional assays, the latter including aggregation, adhesion, and viability assays. The in vivo drug antithrombotic potential was assessed in mice undergoing chemically induced arterial occlusion, and the associated hemorrhagic risk evaluated by measuring the tail bleeding time.

Results: We show that PI3K Class IA inhibitors potently block calcium mobilization in human platelets. The PI3K p110δ inhibitor Idelalisib inhibits platelet aggregation mediated by ITAM receptors GPVI and CLEC-2, preferentially by the former. Moreover, Idelalisib also inhibits platelet adhesion and aggregation under shear and adhesion to collagen. Interestingly, an antithrombotic effect was observed in mice treated with Idelalisib, with mild bleeding effects at high doses of the drug.

Conclusion: Idelalisib may have antiplatelet effects with minor bleeding effects, which provides a rationale to evaluate its antithrombotic efficacy in humans.

Keywords: Idelalisib; PI3K inhibitors; platelets.

Figure 1

Effect of PI3K inhibitors on platelet activation based on intracellular calcium mobilization and aggregation assays. (A) Schematic representation of the intracellular calcium mobilization assay. For detailed information please see the Methods section. (B) TGX-221, Idelalisib, and PIK-75 block intracellular calcium release mediated by Collagen-Related Peptide (CRP)-induced GPVI-activation with micromolar potency, whereas IC-87114 and BYL-719 have a weaker effect. (C–E) Representative aggregation curves of platelets treated with TGX-221, and Idelalisib, and PIK-75 from two independent experiments. Inhibitors completely block CRP-mediated platelet aggregation with a less pronounced effect inhibiting collagen-mediated aggregation and no effect on thrombin-mediated aggregation. In the case of PIK-75, it blocks, totally or moderately, platelet aggregation induced by either CRP, collagen, or thrombin. (F) Calcein-AM-based viability assay demonstrating platelets are viable following 10 µM Idelalisib treatments.

Figure 2

Idelalisib inhibits GPVI-and CLEC-2-mediated platelet aggregation in a dose-response manner. Aggregation profiles upon platelet activation, in presence and absence of Idelalisib, with the following agonists at the indicated doses: (A) CRP (washed platelets); (B) CRP (platelet-rich-plasma (PRP)); (C) collagen (washed platelets); (D) collagen (PRP); (E) Rhodocytin (washed platelets); (F) thrombin (washed platelets); (G) thrombin receptor activating peptide (TRAP) (PRP). Effect on aggregation percentage was evaluated in N = 22 (CRP, 1 μg/mL), N = 13 (Collagen, 5 μg/mL), N = 6 (Rhodocytin, 200 nM), and N = 11 (Thrombin, 0.1 U/mL), in washed platelets; and N = 7 (CRP, 0.15 μg/mL), N = 7 (Collagen, 0.75 μg/mL), and N = 4 (TRAP, 30 μM) in PRP. H-K) Dose-response efficacy of Idelalisib at inhibiting platelet aggregation following platelet activation in washed platelets with both CRP and Rhodocytin, leading to the determination of IC₅₀ values. *** p < 0.001; **** p < 0.0001. (H,I) show representative aggregation curves, while (J,K) plots represent for each concentration used the average of 6 independent measurements from 6 different donors and the error bars are the standard error of the mean.

Figure 3

Idelalisib significantly inhibits platelet adhesion to collagen and impairs platelet adhesion and aggregation in the Impact-R test. (A) Schematic representation of the adhesion assay. For further information please see the Methods section. (B) Inhibition effect of Idelalisib on platelet adhesion to collagen. **** p < 0.0001. (C) Impact-R test: Plots show the mean values plus standard deviation of the plate surface covered by adhered platelets (%SC) and the average size of the platelet aggregates (AS μm²), obtained in blood samples under shear stress, assayed in duplicates, from five different healthy individuals, comparing vehicle samples and samples treated with 10 μM Idelalisib. (D) Representative images of the Impact-R evaluation test of untreated blood (vehicle) or blood incubated with 10 μM Idelalisib.

Figure 4

Idelalisib reduces thrombus formation with minor bleeding effects in mice. (A) Picture representing the tail bleeding assay. (B) The bleeding time was higher in the treated animals although values were always below 10 min. (C) The hemoglobin content of blood collected during bleeding was higher in the treated animals. (D) Picture representing the FeCl₃-induced arterial thrombosis model. (E) Idelalisib-treated mice display significantly longer occlusion times than control mice. * p < 0.05; ** p <0.01.