Soluble guanylate cyclase activators as inhibitors of platelet function

Purpose: PGI2 and NO synergize with P2Y12 receptor blockade to inhibit platelet aggregation. Recently novel allosteric activators of soluble guanylyl cyclase (sGC) have been identified that can stimulate the formation of cyclic nucleotides through both heme-dependent (e.g. riociguat and BAY 41-2272) and heme-independent (e.g. cinaciguat and BAY 60-2770) pathways. Therefore, we hypothesize that in the presence of P2Y12 receptor blockade these sGC modulators could produce enhanced platelet inhibition.

Materials and methods: Blood was taken from healthy humans, aged 18-40. To assess platelet reactivity, platelet rich plasma (PRP) was obtained by centrifugation of whole blood and 96-well plate aggregometry (1) was used to measure platelet aggregation in response to TRAP-6 amide (thrombin receptor agonist peptide) 10 and 25 µM and collagen 3 and 10 µg/ml in the presence of sGC modulators and/or the P2Y12 blocker, prasugrel active metabolite (PAM 3 µM), and/or vehicle. Further studies examined the aggregation of platelets in response to TRAP-6 in whole blood, following treatment with/without sGC modulators and/or PAM, using as an end point determinant fluorescence-activated cell sorting (FACS) (2). Finally, adhesion of platelets under shear to collagen-coated surfaces was assessed by visualizing the perfusion through IBIDI chambers of mepacrine-labelled platelets in whole blood treated with sGC modulators and/or PAM and/or vehicle.

Results: sGC activators were more potent than sGC stimulators as inhibitors of platelet function in both platelet rich plasma and whole blood. In combination with a P2Y12 blocker, a powerful synergistic effect was observed in whole blood experiments (e.g. aggregation induced by TRAP-6: PAM alone, 76 ± 4%; PAM + BAY 60-2770, 22 ± 6%; n=7). This pattern was also replicated in studies examining platelet adhesion under shear, where less platelet adherence was seen when a sGC activator was included in addition to PAM: geometric mean fluorescence index (MFI) for whole blood plus vehicle 60 ± 5; PAM alone, 48 ± 3, PAM + BAY 60-2770, 39 ± 3; n=3.

Conclusion: Both allosteric activators have the potential to be anti-platelet agents when used in combination with P2Y12 receptor blockers. We suggest that providing the sGC activators in low dose combinations with P2Y12 receptor blockers could provide a focused and powerful anti-platelet effect with relatively lesser effects at other sGC sites such as the vascular smooth muscle. Thus in combination with a P2Y12 receptor blocker, sGC activators could produce a strong anti-platelet effect with reduced incidence of headache and hypotension.

References:

(1) Chan, M. V. et al (2011). Optical multichannel (optimul) platelet aggregometry in 96-well plates as an additional method of platelet reactivity testing. Platelets 22 (7), 485-494.

(2) Armstrong P.C. et al (2015) Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction. Blood; 126(10), e11-18.