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. 2018 Oct;22(10):5076-5082.
doi: 10.1111/jcmm.13791. Epub 2018 Jul 16.

Platelet Inhibitory Effects of the Phase 3 Anticancer and Normal Tissue Cytoprotective Agent, RRx-001

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Free PMC article

Platelet Inhibitory Effects of the Phase 3 Anticancer and Normal Tissue Cytoprotective Agent, RRx-001

Bryan Oronsky et al. J Cell Mol Med. .
Free PMC article

Abstract

The platelet inhibitory effects of the Phase 3 anticancer agent and nitric oxide (NO) donor, RRx-001, (1-bromoacetyl-3,3-dinitroazetidine) were examined ex vivo and compared with the diazeniumdiolate NO donor, diethylenetriamine NONOate (DETA-NONOate), which spontaneously releases nitric oxide in aqueous solution. In the absence of red blood cells and in a dose-dependent manner, DETA-NONOate strongly inhibited platelet aggregation induced by several stimuli (ADP, epinephrine and collagen) whereas RRx-001 only slightly inhibited platelet aggregation under the same conditions in a dose-dependent manner; these antiaggregant effects were blocked when both DETA-NONOate and RRx-001 were co-incubated with carboxy-PTIO (CPTIO 0.01-100 micromol), a widely accepted NO scavenger. However, in the presence of red blood cells from healthy human donors, RRx-001, which binds covalently to haemoglobin (Hb) and catalyses the production of NO from endogenous nitrite, more strongly inhibited the aggregation of platelets than DETA-NONOate in a dose-dependent manner likely because haemoglobin avidly scavenges nitric oxide and reduces its half-life; the RRx-001-mediated platelet inhibitory effect was increased in the presence of nitrite. The results of this study suggest that RRx-001-bound Hb (within RBCs) plays an important role in the bioconversion of NO2- to NO. , which makes RRx-001 a more physiologically relevant inhibitor of platelet aggregation than other nitric oxide donors, whose effects are attenuated in the presence of red blood cells. Therefore, RRx-001-mediated platelet inhibition is a potentially useful therapeutic property, especially in hypercoagulable cancer patients that are at an increased risk of thrombotic complications.

Keywords: RRx-001; cancer; haemoglobin; nitric oxide; red blood cells.

Figures

Figure 1
Figure 1
A Diagram, which Depicts Four Methods of NO Release, Leading to Increased Blood Flow: (a) nitric oxide is postulated to be released from RRx‐001 (formula shown below) directly via a Nef‐like reaction; (b) RRx‐001 binds to the betaCys93 residue on haemoglobin, which accelerates the formation of nitric oxide from deoxyhemoglobin in RBCs; (c) spontaneous release of nitric oxide from a NONOate in aqueous media; (d) production of NO via eNOS in the endothelium
Figure 2
Figure 2
A, Blood Smear for Aggregation Studies Obtained from Heparinized Human Blood Untreated or Treated with Vehicle, RRx‐001 (10 mg/kg) or Dextran 500 kDa (10 mg/kg in Sterile Saline, Positive Control). From left to right vehicle‐treated blood, RRx‐001‐treated blood and Dextran‐treated blood. Clumping and rouleaux formation is only seen with Dextran, the positive control; B, RBC aggregation index of human blood untreated, or treated with Vehicle, RRx‐001 (10 mg/kg) or Dextran 500 kDa (10 mg/kg in sterile saline, positive control). RBC aggregation index was unchanged between untreated blood, vehicle‐treated blood and RRx‐001 treated blood. Only Dextran, the positive control, showed a significant (P < 0.05) increase in RBC clumping and aggregation (far right panel)
Figure 3
Figure 3
Basic Coagulation and Platelet Aggregation Parameters with Untreated, Vehicle‐treated and RRx‐001‐treated Blood. RRx‐001 does not Affect APTT, PTT, Fibrinogen or Platelet Numbers Compared to Untreated Blood or Control. However, RRx‐001 appeared to attenuate platelet aggregation in response to ADP, collagen and epinephrine. ADP, adenosine diphosphate; APTT, activated partial thromboplastin time; PTT, partial thromboplastin time
Figure 4
Figure 4
Platelet Aggregation of Platelet‐rich Plasma Measured using % of Light Transition. Effects of RRx‐001 and DETANONO in the absence (left panel) and in the presence (right panel) of the nitric oxide scavenger, CPTIO. Each point represents the mean ± SEM of all experiments. The platelets were incubated with test drugs for before stimulation with ADP, and aggregation was monitored thereafter. Results are expressed as percent inhibition of aggregation relative to the untreated platelets CPTIO: 5,2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazoline 1‐oxyl 3‐oxide
Figure 5
Figure 5
Inhibition of Platelet Aggregation by DETANONO Decreased in the Presence of Increasing Haematocrit whereas Inhibition of Platelet Aggregation by RRx‐001 Increased in the Presence of Increasing Haematocrit. Platelet aggregation was induced by 8 μmol/L ADP. CPTIO completely reversed the inhibition. All experiments were performed at 37°C
Figure 6
Figure 6
Nitrite+ red blood cells (RBCs) (20% Haematocrit) Inhibited ADP‐induced Platelet Aggregation. Nitrite was incubated in PRP + RBCs in the presence or absence of CPTIO before induction of aggregation by ADP

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