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 + PGE 1 , 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 + PGE 1 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.

A 19 mM ticagrelor stock solution was made by dissolving ticagrelor (Cayman Chemical Company, Ann Arbor, MI) in DMSO and then working stock solutions were prepared in sterile 0.9% saline at concentrations between 40 and 120 µM. To achieve final whole blood concentrations between 0.5 and 3 µM, 50 µL was added to 2 mL whole blood. The negative control was prepared by adding 50 µL of DMSO in sterile 0.9% saline to whole blood (final DMSO concentration 0.01%). Samples were incubated for 30 minutes at 37°C.

Flow Cytometry with PAC1
PPACK-anticoagulated whole blood (3,952 µL) was treated with 28 µL aspirin [made by dissolving acetyl salicylic acid (Sigma Aldrich, Saint Louis, MO) in sterile water and adjusting the pH to 8.1; final concentration 0.3 mM], or control sterile water. Working stock dilutions of RUC-4 were made up in 0.9% saline at concentrations between 2.5 and 125 µM to achieve final whole blood concentrations between 0.01 and 0.63 µM when 20 µL was added to achieve a final volume of 4 mL.
Whole blood was incubated with aspirin and RUC-4, or buffer controls, for 20 minutes at room temperature, after which 2 mL was transferred to a VN tube. A 13.75 µL aliquot was set aside for flow cytometry of whole blood, and the remaining $2 mL was used to prepare platelet-rich plasma (PRP) for flow cytometry and LTA. PRP was prepared by centrifuging the whole blood at 200 Â g for 6 minutes at room temperature. After removing the PRP, the remaining blood was centrifuged at 1,200 Â g for 8 minutes at room tempera-ture to prepare platelet-poor plasma (PPP). Platelet counts were obtained with the automated counter and samples were adjusted to 3 Â 10 5 platelets/μL with PPP if the PRP count was higher.
Flow cytometry with PAC1 was performed as previously described (reference 24 in manuscript) by adding 13.75 µL of either whole blood or PRP to 6.25 μL of FITC-labeled PAC1 (6.25 µg/mL final concentration; BD Biosciences, San Jose, CA) and either 5 μL of PGE 1 (final concentration 1 µM; Santa Cruz Biotechnology, Dallas, TX) or ADP (final concentration 20 µM; CHRONO-LOG, Havertown, PA) in HEPES-buffered modified Tyrode's solution (HBMT) with 1 mM Mg 2þ . After incubation for 30 minutes at room temperature, 250 µL of HBMT with 1 mM Mg 2þ was added to dilute the samples, and flow cytometry was conducted immediately at room temperature (FACSCalibur; BD Biosciences, San Jose, CA). Forward and side light scatter was displayed on logarithmic scales and used to gate the platelet population. Samples were analyzed with the acquisition of 25,000 events. Flow cytometry data were analyzed using BD CellQuest Pro Analysis software. Data are expressed as the percentage PAC1 geometric mean fluorescence intensity observed in the absence of RUC-4.

Statistical Analysis
All continuous variables are reported as mean AE SD. Continuous variables were plotted on scatter plots and a Pearson correlation coefficient was computed to assess the linear relationship. The multivariate R 2 was computed and a mixed effects logistic regression model was implemented to account for repeated measurements on each participant. Differences in means between 2 dependent or independent groups were analyzed using the student's t-test and a 2tailed p value <0.05 was considered significant. The half maximal inhibitory concentration (IC50) for RUC-4 was determined by fitting a 4-parameter logistic curve to the scatter plot of log [RUC-4 concentration] vs percent inhibition of platelet function. For Receiver Operating Characteristic (ROC) curves, the optimal cut-off points for the VN assays for determining platelet inhibition compared with LTA were identified by calculating the Younden index [J ¼ max(sensitivity þ specificity -1)] by maximizing the distance between the chance diagonal and a point on the ROC curve, which represents the maximum potential of effectiveness for the diagnostic test. ROC analysis included a mixed effects logistic regression model with a random effects for subjects to account for repeated measurements in the same donors (SAS Studio 3.8).
TH Open © 2021. The Author(s). Supplementary Fig. S1 Comparison of the primary slope and the maximal and final platelet aggregation values of all samples tested in the in vitro studies (n ¼ 6, 30 measurements). The blue dotted line denotes linear regression of maximal aggregation (R 2 ¼ 0.92, p < 0.001) and the red dotted line denotes the linear regression for final aggregation at 10 minutes (R 2 ¼ 0.80, p < 0.001).
Supplementary Fig. S3 Concentration-response curve of RUC-4 measured by the VN assays in citrate-anticoagulated blood. (n ¼ 7 without aspirin and n ¼ 6 with aspirin, data are mean AE SD). VN ¼ VerifyNow.