Ticagrelor Induced Platelet Dysfunction Can be Assessed Under Shear Conditions and Correction By Platelets Is Influenced By Storage Temperature

Introduction:

The use of antiplatelet (anti-PLT) drugs for prophylaxis and treatment of cardiovascular disease continues to rise. A significant problem with these medications is increased bleeding, which can be life threatening. Strategies to manage bleeding whilst on anti-PLT medication are limited and often include PLT transfusion. Evidence of efficacy is lacking, and it is not known whether storage conditions affect outcome. We sought to study this clinical problem using an in vitro system that simulates in vivo shear forces. We assessed PLT adhesion to collagen under arterial shear conditions in whole blood (WB) treated with Ticagrelor, a P2Y12 inhibitor. We tested the hypothesis that the storage temperature of PLTs would affect their ability to correct Ticagrelor-induced platelet dysfunction.

Methods:

WB was collectedand treated with a therapeutic concentration of Ticagrelor (1.0µM). After a 15 min incubation the treated WB was dosed with apheresis platelets (AP) to simulate PLT transfusion. The AP used were collected as hyperconcentrated double units from different healthy donors (n=3), diluted with T-PAS+ (65% T-PAS+, 35% plasma), and split equally amongst two bags. One bag was stored at room temperature (22±2°C; RTP) with agitation, and the other bag was cold-stored (4±2°C; CSP) without agitation. Ticagrelor-treated WB was dosed with RTP and CSP in increments equivalent to 1, 2, and 3 times the adult therapeutic doses (ATD) of PLTs (calculated using a 70kg person with a circulating blood volume of 5000ml) stored for 0 (fresh), 3, 7 and 10 days. Random WB donors were selected who were ABO RhD compatible with the AP unit.

Bioflux 1000 (Fluxion) was used to evaluate PLT function of the treated and dosed WB. Briefly, WB (treated or dosed) was labelled with calcein and perfused through a collagen-coated chamber under arterial shear (19.83 dyn/cm²) for 10 min. PLT adhesion to collagen was recorded for 10 min, with images obtained every 30s. Fluorescence intensity and the percentage of area covered was determined using BioFlux Montage Software. Data was analysed and the t-test performed using GraphPad Prism version 7.

Prior to dosing, RTP and CSP bags were analysed at each timepoint visually for aggregates and 'swirl', PLT count (HORIBA MICROS 60), light transmission aggregometry (Chronolog 700, agonists; ADP [10µM] and AA [0.5mM]) and chemistry (iSTAT, CG4+ cartridge).

Results:

PLT counts dropped in both CSP and RTP but were not significantly different at any timepoint (Fig. 1A). PLT aggregation to ADP negligible after day 3 in RTP but was well maintained in the CSP out to 10 days (p<0.03) (Fig. 1B). pH decreased in CSP and increased in RTP but did not reach significance (p=0.07). Lactate was significantly higher in RTP compared to CSP at day 10 (p=0.01) (Fig. 1D). Increments in PLT count in treated WB were lower with doses of CSP than RTP, with the mean increases of 10x10⁶/ml and 26x10⁶/ml per ATD respectively.

For Bioflux runs, Ticagrelor treatment lowered PLT adhesion to collagen by 40-66% compared to control (WB treated with DMSO 0.1%) after 10 min of perfusion. On Day 0, PLT adhesion increased with increasing doses of fresh PLTs. At Day 3 of storage, both CSP and RTP performed equally. By day 7, CSP showed enhanced adhesion and aggregation to collagen compared to RTP, both continuing to exhibit a dose effect, with 1 dose of CSP being superior to 3 ATD RTP in all 3 donors. CSP-dosed samples effectively rescued Ticagrelor-treated WB out to 10 days storage. Addition of RTP stored beyond 7 days had a detrimental effect (i.e. lower adhesion than Ticagrelor treatment alone).

Conclusions:

This study has demonstrated that the anti-PLT effects of Ticagrelor can be observed and quantified under physiologic flow conditions using a BioFlux system and that these effects can be overcome using PLT transfusion in a dose-dependent manner. The storage temperature of AP affects their ability to rescue Ticagrelor-induced PLT dysfunction under shear conditions with cold storage providing better rescue of dysfunction than RTP even out to 10 days of storage. Further work is needed to assess the ability of PLT concentrates to rescue other causes of PLT dysfunction and to reduce bleeding.

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