Application of Auxiliary VerifyNow Point-of-Care Assays to Assess the Pharmacodynamics of RUC-4, a Novel αIIbβ3 Receptor Antagonist

Introduction Prehospital therapy of ST-elevation myocardial infarction (STEMI) with αIIbβ3 antagonists improves clinical outcomes, but they are difficult to use in prehospital settings. RUC-4 is a novel αIIbβ3 antagonist being developed for prehospital therapy of STEMI that rapidly achieves high-grade platelet inhibition after subcutaneous administration. Standard light transmission aggregometry (LTA) is difficult to perform during STEMI, so we applied VerifyNow (VN) assays to assess the pharmacodynamics of RUC-4 relative to aspirin and ticagrelor. Methods Blood from healthy volunteers was anticoagulated with phenylalanyl-prolyl-arginyl chloromethyl ketone (PPACK) or sodium citrate, treated in vitro with RUC-4, aspirin, and/or ticagrelor, and tested with the VN ADP + PGE1, iso-TRAP, and base channel (high concentration iso-TRAP + PAR-4 agonist) assays. The results were correlated with both ADP (20 µM)-induced LTA and flow cytometry measurement of receptor occupancy and data from individuals treated in vivo with RUC-4. Results RUC-4 inhibited all three VN assays, aspirin did not affect the assays, and ticagrelor markedly inhibited the ADP + PGE1 assay, slightly inhibited the iso-TRAP assay, and did not inhibit the base channel assay. RUC-4's antiplatelet effects were potentiated in citrate compared with PPACK. Cut-off values were determined to correlate the results of the VN iso-TRAP and base channel assays with 80% inhibition of LTA. Conclusion The VN assays can differentiate the early potent anti-αIIbβ3 effects of RUC-4 from delayed effects of P2Y12 antagonists in the presence of aspirin. These pharmacodynamic assays can help guide the clinical development of RUC-4 and potentially be used to monitor RUC-4's effects in clinical practice.

Impact of morphine use on mortality in STEMI patients treated with primary PCI - preliminary data from a new registry

Funding Acknowledgements Type of funding sources: None. Background Opioids decrease the effect of P2Y12 receptor inhibitors in vitro and observational reports suggest that morphine use is associated with larger infarct size. Our research group presented previously, using a prospective single-center registry, that periprocedural morphine use may have no impact on long-term mortality in STEMI patients treated with primary PCI and clopidogrel. Purpose Our purpose is to check this interaction using a new registry of patients treated according to the current guidelines, including novel antiplatelet agents. Methods From May until November 2020, we collected 196 STEMI patients treated with primary PCI. 88 (44.9%) of them got morphine during the prehospital and periprocedural care. Baseline demographic, anamnestic, procedural, and laboratory data were collected. Survival data were analysed using Kaplan-Meier survival curves and the log-rank test. To adjust for confounding, a 1:1 propensity score-matching analysis was performed using 114 cases. Results An adequate balance on baseline covariates was achieved during propensity score-matching. Kaplan-Meier analysis showed no difference in 30-days mortality of the patients treated with or without morphine neither in the original nor in the propensity score-matched population (p = 0.094 and p = 0.309, respectively). Conclusion Our preliminary data suggest that morphine may have no impact on mortality in STEMI patients treated with primary PCI and medical therapy according to the current guidelines including novel P2Y12 antagonists. Abstract Figure. Kaplan-Meier curves

Lyophilized Human Platelets Restore Hemostasis in the Presence of the P2Y12 Inhibitors Cangrelor, Ticagrelor and Clopidogrel

• Background: P2Y12 inhibitors block platelet ADP receptors thereby reducing clotting capacity. In an emergency, these agents must be reversed or therapeutically overcome to stop bleeding. Additionally, prior to and during elective procedures these inhibitors must be withdrawn, thereby increasing thrombotic risk. Lyophilized human platelets (Thrombosomes®) are stabilized platelet derived hemostatic agents currently under Phase 2 clinical development for thrombocytopenia and have potential as antiplatelet reversal agents. • Aims: The aim of this study was to determine if lyophilized human platelets are resistant to the antiplatelet effect of P2Y12 inhibitors and therefore restore hemostasis in the presence of P2Y12 inhibitors. • Methods: Fresh drawn platelet rich plasma (PRP) was treated with pharmacological concentrations of P2Y12 inhibitors cangrelor or ticagrelor and confirmed to inhibit ADP-stimulated aggregation on the PAP-8E Platelet Aggregometer. Changes in hemostatic properties of the P2Y12 inhibitor treated PRP were also tested under shear force on the Total Thrombus formation Analysis System (T-TAS®). P2Y12 inhibitor treated PRP samples were dosed with lyophilized platelets and tested for return of hemostatic properties by T-TAS. NOD SCID mice were used in a tail snip model to determine hemostatic efficacy of Thrombosomes® after super pharmacological doses of clopidogrel treatment. • Results: Fresh PRP derived platelet aggregation response to ADP was inhibited by cangrelor or ticagrelor whereas lyophilized platelets did not respond to either inhibitor. Normal thrombus formation of PRP as measured by T-TAS occurred at 19.5± 1.5 minutes (n=4) but increases to 28.0±3.0 minutes with cangrelor (n=4) or 28.0±3.0 minutes with ticagrelor (n=5) treatment. The addition of 150k/µL lyophilized platelets to P2Y12-inhibited PRP reduced time to thrombus formation to lower than PRP alone; 15.5 ±0.5 minutes in the presence of cangrelor (n=3) versus 17.5 ±1.5 minutes in the presence of ticagrelor (n=5). In the in vivo tail snip mouse model animals treated with super pharmacological dose of clopidogrel bled for 18.0±10.0 minutes (n=5) unlike those not treated that only bleed for 9.5±2.5 minutes (n=6). Lyophilized platelet treated clopidogrel animals stopped bleeding at 12.5 ±4.5 minutes (n=5) after tail snip. • Conclusion: Lyophilized human platelets, unlike normal platelets were resistant to the antiplatelet effects of P2Y12 inhibitors. In vitro and in vivo studies reveal that lyophilized human platelets can be used to recover the anti-thrombotic effect of P2Y12 antagonists, potentially allowing continued drug compliance prior to elective procedures and as a possible treatment for emergent acute bleeding while on P2Y12 antagonist therapy. Disclosures Dickerson: Cellphire Inc.: Current Employment. Lee:Cellphire, Inc.: Current Employment. Hale:Cellphire: Current Employment. Moskowitz:Cellphire Inc.: Current Employment.

Role of P2Y Receptors in Platelet Extracellular Vesicle Release

Platelet extracellular vesicles (PEVs) are potential new biomarkers of platelet activation which may allow us to predict and/or diagnose developing coronary thrombosis before myocardial necrosis occurs. The P2Y1 and P2Y12 receptors play a key role in platelet activation and aggregation. Whereas the P2Y1 antagonists are at the preclinical stage, at present, the P2Y12 antagonists are the most effective treatment strategy to prevent stent thrombosis after percutaneous coronary intervention. Despite an increasing number of publications on PEVs, the mechanisms underlying their formation, including the role of purinergic receptors in this process, remain an active research field. Here, we outline the clinical relevance of PEVs in cardiovascular disease, summarize the role and downstream signalling of P2Y receptors in platelet activation, and discuss the available evidence regarding their role in PEV formation.

Prevention of P2 Receptor-Dependent Thrombocyte Activation by Pore-Forming Bacterial Toxins Improves Outcome in A Murine Model of Urosepsis

Urosepsis is a potentially life-threatening, systemic reaction to uropathogenic bacteria entering the bloodstream of the host. One of the hallmarks of sepsis is early thrombocyte activation with a following fall in circulating thrombocytes as a result of intravascular aggregation and sequestering of thrombocytes in the major organs. Development of a thrombocytopenic state is associated with a poorer outcome of sepsis. Uropathogenic Escherichia coli frequently produce the pore-forming, virulence factor α-haemolysin (HlyA), of which the biological effects are mediated by ATP release and subsequent activation of P2 receptors. Thus, we speculated that inhibition of thrombocyte P2Y1 and P2Y12 receptors might ameliorate the septic response to HlyA-producing E. coli. The study combined in vitro measurements of toxin-induced thrombocyte activation assessed as increased membrane abundance of P-selectin, fibronectin and CD63 and data from in vivo murine model of sepsis-induced by HlyA-producing E. coli under infusion of P2Y1 and P2Y12 antagonists. Our data show that the P2Y1 receptor antagonist almost abolishes thrombocyte activation by pore-forming bacterial toxins. Inhibition of P2Y1, by constant infusion of MRS2500, markedly increased the survival in mice with induced sepsis. Moreover, MRS2500 partially prevented the sepsis-induced depletion of circulating thrombocytes and dampened the sepsis-associated increase in proinflammatory cytokines. In contrast, P2Y12 receptor inhibition had only a marginal effect in vivo and in vitro. Taken together, inhibition of the P2Y1 receptor gives a subtle dampening of the thrombocyte activation and the cytokine response to bacteraemia, which may explain the improved survival observed by P2Y1 receptor antagonists.

Adenosine Receptor Agonists Increase the Inhibition of Platelet Function by P2Y12 Antagonists in a cAMP- and Calcium-Dependent Manner

We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists—cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.

von Willebrand Factor Predicts Mortality in ACS Patients Treated with Potent P2Y12 Antagonists and is Inhibited by Aptamer BT200 Ex Vivo

Background von Willebrand factor (VWF) is crucial for arterial thrombosis and its plasma levels are increased in acute coronary syndromes (ACSs). The effects of conventional platelet inhibitors are compromised by elevated VWF under high shear rates. BT200 is a third-generation aptamer that binds and inhibits the A1 domain of human VWF. This article aims to study whether VWF is a predictor of mortality in ACS patients under potent P2Y12 blocker therapy and to examine the effects of a VWF inhibiting aptamer BT200 and its concentrations required to inhibit VWF in plasma samples of patients with ACS. Methods VWF activity was measured in 320 patients with ACS, and concentration effect curves of BT200 were established in plasma pools containing different VWF concentrations. Results Median VWF activity in patients was 170% (interquartile range % confidence interval [CI]: 85–255) and 44% of patients had elevated (> 180%) VWF activity. Plasma levels of VWF activity predicted 1-year (hazard ratio [HR]: 2.68; 95% CI: 1.14–6.31; p < 0.024) and long-term (HR: 2.59; 95% CI: 1.10–6.09) mortality despite treatment with potent platelet inhibitors (dual-antiplatelet therapy with aspirin and prasugrel or ticagrelor). Although half-maximal concentrations were 0.1 to 0.2 µg/mL irrespective of baseline VWF levels, increasing concentrations (0.42–2.13 µg/mL) of BT200 were needed to lower VWF activity to < 20% of normal in plasma pools containing increasing VWF activity (p < 0.001). Conclusion VWF is a predictor of all-cause mortality in ACS patients under contemporary potent P2Y12 inhibitor therapy. BT200 effectively inhibited VWF activity in a target concentration-dependent manner.

Major Bleeding after Surgical Revascularization with Dual Antiplatelet Therapy

Objective Patients with acute coronary syndrome are treated with dual antiplatelet therapy containing acetylsalicylic acid (ASA) and P2Y12 antagonists. In case of urgent coronary artery bypass grafting this might be associated with increasing risks of bleeding complications. Methods Data from 1200 consecutive urgent operations between 2010 and 2018 were obtained from our institutional patient database. For this study off-pump surgery was excluded. The primary composite end point major bleeding consisted of at least one end point: transfusion ≥ 5 packed red blood cells within 24 hours, rethoracotomy due to bleeding, chest tube output >2000 mL within 24 hours. Demographic data, peri-, and postoperative variables and outcomes were compared between patients treated with mono antiplatelet therapy, ASA + clopidogrel (ASA-C) +ticagrelor (ASA-T) or +prasugrel (ASA-P) < 72 hours before surgery. Furthermore, we compared patients with dual antiplatelet therapy with ASA monotherapy. Results From 1,086 patients, 475 (44%) received dual antiplatelet therapy. Three-hundred seventy-two received ASA-C (77.7%), 72 ASA-T (15%), and 31 ASA-P (6.5%). Major bleeding (44 vs. 23%, p < 0.0001) was more frequently in patients receiving dual therapy with higher rates of massive drainage loss within 24 hours (23 vs. 11%, p < 0.0001) of mass transfusion (34 vs. 16%, p < 0.0001) and rethoracotomy (10 vs. 5%, p = 0.002) when compared with ASA. In this analysis, ASA-T and ASA-P were not associated with higher bleeding complications compared with ASA-C. Conclusion Dual antiplatelet therapy is associated with higher rates of major bleeding. Further studies should examine the difference in the prevalence of major bleeding complications in the different dual antiplatelet therapy regimes in patients requiring urgent surgery.