LAUSR

Introduction: RUC-4 is a novel platelet integrin αIIbβ3 antagonist specifically designed for first point-of-medical contact (ambulance or ER) treatment of patients with ST segment elevation myocardial infarction in combination with aspirin. Ongoing Phase 1 studies indicate that RUC-4 at a dose of 0.075 mg/kg administered subcutaneously can inhibit ADP-induced platelet aggregation by ~80% within 15 minutes, with a return toward baseline within 60-90 minutes. It may be advantageous to monitor the antiplatelet effects of RUC-4 on admission to the hospital, but optical aggregometry (OA) takes too long to guide immediate therapeutic decisions. The automated VerifyNow (VN) assays provide rapid point-of-care assessment of the effect of antiplatelet agents. The VN IIb/IIIa assay uses a thrombin receptor activating peptide (isoTRAP) to activate the platelets and the VN Aspirin assay uses arachidonic acid, whereas the PRU (P2Y12) assay uses isoTRAP in one channel and a mixture of ADP and PGE1 in another. We recently reported that: 1. Aspirin does not affect the IC50 for RUC-4 when measured by OA of platelet-rich plasma (PRP) in response to ADP, and 2. The IC50 is lower in PRP that is prepared from blood anticoagulated with citrate rather than PPACK, consistent with RUC-4's mechanism of action in displacing the Mg2+ ion from the MIDAS domain (Vootukuri et al., J Clin Transl Sci; in press). We now have explored the sensitivity of the VN assays to RUC-4 and the effects of aspirin and different anticoagulants. Methods: Whole blood anticoagulated with either sodium citrate (0.32%) or PPACK (100 µM) from healthy volunteers who were either taking or not taking daily aspirin was treated in vitro with RUC-4 to achieve final whole blood concentrations between 30 and 900 nM. Blood was incubated for 10 minutes at room temperature and then tested with either the IIb/IIIa or PRU cartridge. Results: Whole blood samples were obtained from 13 healthy volunteers who provided consent, of whom 7 were taking aspirin. The antiplatelet effect of aspirin in the latter 7 participants was confirmed with the VN Aspirin assay. 1. PPACK vs citrate anticoagulation in blood from individuals who were not treated with aspirin. The percent inhibition values produced by different concentrations of RUC-4 in whole blood anticoagulated with either PPACK or citrate and activated with isoTRAP were highly correlated (R2=0.95; p<0.01) despite differences in absolute values (IC50s of 141 ± 5 vs 88 ± 7 nM, respectively, p=0.01; n=6). The comparable values using ADP+PGE1 as the activator were also highly correlated (R2=0.95; p<0.01) despite differences in absolute values (IC50s of 128 ± 5 vs 69 ± 8 nM, p=0.01; n=6). Based on these results, we selected citrate-anticoagulated blood for additional studies because vacuum blood collection tubes containing citrate, but not PPACK, are available commercially. 2. Effect of RUC-4 on VN assays: a) Impact of aspirin on results in the PRU cartridge. The IC50s in the isoTRAP channel were similar using blood from individuals who were not taking aspirin (88 ± 7 nM) or taking aspirin (97 ± 20 nM; p=0.35; n=9). Similar results were obtained using the ADP+ PGE1 channel (69 ± 8 vs 70 ± 8 nM; p=0.84; n=9). b) Comparison of the 2 different isoTRAP channels with blood from people on aspirin. RUC-4 showed similar dose-responses in both isoTRAP channels, with an IC50 of 79 ± 26 nM in the IIb/IIIa cartridge vs 97 ± 20 nM in the PRU cartridge (p=0.12; n=5). c) Comparison of the isoTRAP vs ADP+PGE1 channel using blood from people on aspirin. The ADP+PGE1 channel was more sensitive to the lower concentrations of RUC-4 than the isoTRAP channel, showing, for example, ~40% inhibition at 30 nM compared to ~20%, respectively (Figure; p<0.02 based on comparison of IC50s ). Discussion: We conclude that RUC-4 produces dose-dependent inhibition of the VN isoTRAP channels in both the IIb/IIIa and PRU cartridges, as well as the ADP+PGE1 channel in the PRU cartridge. The PRU cartridge ADP+PGE1 channel is more sensitive than the isoTRAP channel to low concentrations of RUC-4. Since P2Y12 antagonists inhibit the ADP+PGE1 channel, but not the isoTRAP channels, the effect of RUC-4 on patients who are not treated with a P2Y12 antagonist may be monitored with the PRU ADP+PGE1 channel or the isoTRAP channel in either the PRU or IIb/IIIa cartridge. The RUC-4 effects in patients treated with a P2Y12 antagonist may be monitored with the isoTRAP channel in either cartridge. Figure Coller: Accumetrics/Instrumentation Laboratory: Patents & Royalties: VerifyNow assay; Scholar Rock: Consultancy, Equity Ownership; CeleCor: Consultancy, Equity Ownership, Research Funding; Centocor/Janssen: Patents & Royalties: abxicimab.