Search

Introduction: Direct-acting oral anticoagulants (DOACs) are increasingly evaluated for the secondary prevention of thrombosis-related events; however, DOAC dose-related effects on platelet function, particularly with anti-platelet agents, are unknown. Hypothesis: Factor Xa inhibitors do not have direct anti-platelet activity; however, by their capacity to inhibit thrombin generation (TG), they indirectly regulate thrombin-dependent platelet activation. This indirect mode of action may have additive effects with anti-platelet agents such as aspirin or P2Y12 antagonists. Methods: Platelet-rich plasma (PRP) from healthy individuals (n=6) was incubated with increasing amounts, and combinations of rivaroxaban (Riva), aspirin (ASA) and/or ticagrelor(T). The impact on platelet function was studied using light transmission aggregometry (LTA) and fluorogenic TG assays (TGA). For LTA, maximal aggregation (MA) was measured after initiation of tissue factor (TF)-induced TG. Similarly, for TG assays in platelet-poor plasma (PPP) and PRP low TF concentrations were used to initiate TG. Results and Conclusions: Riva treatment alone decreased % MA by TF-generated thrombin by 24% (15 ng/ml; akin to 2.5 mg bid dosing) and 71% (120 ng/ml; akin to 10 - 20 mg bid dosing). Riva, in combination with both anti-platelet agents, decreased MA substantially by 69% at 15 ng/ml and significantly by 89% at 120 ng/ml (p

Background— Eptifibatide reduces major adverse cardiac events in patients with acute coronary syndromes undergoing percutaneous coronary intervention (PCI). Intracoronary bolus administration of eptifibatide may result in higher levels of platelet glycoprotein IIb/IIIa receptor occupancy in the local coronary bed, disaggregate thrombus in the epicardial artery and microvasculature, and thereby improve coronary flow. Methods and Results— Patients undergoing PCI for an acute coronary syndrome were randomized to either intracoronary or intravenous bolus administration of eptifibatide. The primary end point was the local glycoprotein IIb/IIIa receptor occupancy measured in the coronary sinus. There were no angiographic, electrophysiological, or other adverse findings attributable to intracoronary eptifibatide. Platelet glycoprotein IIb/IIIa receptor occupancy was significantly greater with intracoronary versus intravenous administration: first bolus, 94±9% versus 51±15% ( P P =0.001), respectively. Microvascular perfusion was significantly improved as measured by the corrected thrombolysis in myocardial infarction frame count (cTFC) with intracoronary versus intravenous administration: pre-PCI, 36 (median) (25th and 75th percentiles, 16 and 64) versus 31 (25th and 75th percentiles, 23 and 45; P =0.8); and post-PCI, 18 (25th and 75th percentiles, 10 and 22) versus 25 (25th and 75th percentiles, 22 and 35; P =0.007), respectively. The only multivariate predictor associated with a post-PCI cTFC rank score was the first bolus glycoprotein IIb/IIIa receptor occupancy ( P Conclusions— Intracoronary bolus administration of eptifibatide during PCI in patients with acute coronary syndromes results in higher local platelet glycoprotein IIb/IIIa receptor occupancy, which is associated with improved microvascular perfusion demonstrated by an improved cTFC.

Background— Infants and young children with certain types of heart disease are at increased risk for thromboses. Clopidogrel 75 mg/d is used in adults to prevent thrombotic events. The dose to achieve similar platelet inhibition in children is unknown. The objectives of the present study were (1) to determine the dose of clopidogrel needed in infants and young children to achieve a mean 30% to 50% inhibition of 5-μmol/L ADP–induced platelet aggregation (ie, inhibition similar to that observed with 75 mg in adults) and (2) to assess the safety and tolerability of clopidogrel in infants and young children. Methods and Results— We performed a prospective, multicenter, randomized, placebo-controlled trial evaluating the pharmacodynamics of clopidogrel in children (0 to 24 months) with a cardiac condition at risk for arterial thrombosis. Patients were randomized to clopidogrel versus placebo in a 3:1 ratio in 4 sequential groups (0.01, 0.10, 0.20, and 0.15 mg/kg) for ≥7 and ≤28 days. Platelet aggregation was assessed at baseline and steady state by light-transmission aggregometry. Of 116 patients enrolled, 92 (50% neonates, 50% infants/toddlers) were randomized, and 73 completed the study. A total of 79% of the randomized and treated patients were taking aspirin. Compared with placebo, clopidogrel 0.20 mg · kg −1 · d −1 resulted in a mean 49.3% (95% confidence interval 25.7% to 72.8%) inhibition of the maximum extent of platelet aggregation and a mean 43.9% (95% confidence interval 18.6% to 69.2%) inhibition of the rate of platelet aggregation. There was marked interpatient variability in the degree of platelet aggregation inhibition within each treatment-dose group and age group. No serious bleeding events occurred. Conclusions— Clopidogrel 0.20 mg · kg −1 · d −1 in children 0 to 24 months of age achieves a platelet inhibition level similar to that in adults taking 75 mg/d. Clopidogrel is well tolerated in infants and young children at this dose.

Background— Paradoxically, fibrinolytic agents may systemically activate platelets, which in turn secrete plasminogen activator inhibitor (PAI-1), an antagonist of the fibrinolytic process in proportion to total body platelet mass. We hypothesized that improved epicardial patency, myocardial perfusion, and ST-segment resolution would be associated with higher levels of platelet receptor occupancy (RO) by a glycoprotein IIb/IIIa antagonist in ST-elevation MI (STEMI). Methods and Results— Patients were drawn from the low-dose tenecteplase plus eptifibatide arm of the INTEGRITI study. Angiographic and platelet RO data were analyzed at 2 independent core laboratories. To take into account the absolute platelet count and receptors available for cross-linking, absolute platelet count was multiplied by percent of available receptors to obtain the index of the absolute number of receptors available (IANRA). Percent RO was higher among patients with a patent artery (TIMI flow grade 2/3; 78.2±9.2, n=63 versus 63.9±29.7, n=7; P =0.005), those with TIMI myocardial perfusion grade 2/3 (79.6±9.5, n=40 versus 73.0±16.2, n=30; P =0.036), and those with complete (≥70%) ST-segment resolution at 60 minutes (81.3±8.3%, n=27 versus 73.1±17.4%, n=24; P =0.034). The absolute number of glycoprotein IIb/IIIa receptors available for cross-linking was reduced (ie, the IANRA was lower) among patients with a patent artery ( P =0.0015), patients with TIMI myocardial perfusion grade 2/3 ( P =0.026), and patients with ≥70% ST-segment resolution ( P =0.029). Conclusions— This study links restoration of epicardial flow, normal myocardial perfusion, and complete ST-segment resolution with higher levels of platelet glycoprotein IIb/IIIa receptor occupancy after therapy with eptifibatide administered with tenecteplase.

New oral fXa inhibitors have been increasingly adopted for VTE or AF treatment in the outpatient setting instead of warfarin. Andexanet alfa (AnXa) is a modified, recombinant human fXa molecule developed as a specific antidote to reverse anticoagulant activity of fXa inhibitors during episodes of serious bleeding or before urgent surgery. PER977, a small molecule under development by Perosphere, Inc., is reported to reverse the effect of a broad range of anticoagulants (fXa and thrombin inhibitors, LMWH). In order to compare AnXa and PER977 mechanisms of action, we studied the in vitro activity of both agents in the presence or absence of fXa inhibitors rivaroxaban, apixaban, edoxaban or enoxaparin. In a buffer system containing purified human fXa with physiologic Ca 2+ (5 mM), AnXa dose-dependently reversed oral fXa inhibitor activity. Reversal activity was not observed for any fXa inhibitor with PER977 over a wide concentration range (≤2 mM). In human plasma, AnXa reversed both direct and ATIII-dependent fXa inhibitors, whereas no reversal effect by PER977 was detected. In the absence of a fXa inhibitor, PER977 potentiated fX activation by fIXa in a buffer system (purified human fX, fIXa and 5mM Ca 2+ ). Similar cofactor activity was observed with polylysine, indicating that PER977 may have properties similar to poly-cationic molecules. Procoagulant activity was observed in human plasma with PER977 at low concentrations (≤100 μM) as measured by clotting (aPTT) or thrombin generation, but inhibition in these assays was seen at higher concentrations ( ~1 mM). PER977 also potentiated human platelet activation as determined by P-selectin expression induced by 10 μM ADP. Platelet aggregation, whole blood hemolysis, or complement activation testing showed no effect with either AnXa or PER977. These data indicate that PER977 does not reverse the anticoagulant activity of a direct or indirect fXa inhibitor in vitro. Its observed in vivo effect on blood loss in animals may not be mediated by direct interaction of PER977 with the inhibitor, but may be in part attributed to an off-target effect, suggested by its potential procoagulant activity. In contrast, AnXa acts as a specific antidote to fXa inhibitors by sequestering them with a defined stoichiometry (1:1 molar ratio).

Background Pharmacodynamics of eptifibatide, a cyclic heptapeptide antagonist of platelet glycoprotein IIb/IIIa, are substantially altered by anticoagulants that chelate calcium, resulting in overestimation ex vivo of the in vivo effects of this agent. We conducted a dose-ranging study to characterize the pharmacodynamics and pharmacokinetics of eptifibatide under physiological conditions. Methods and Results Patients (n=39) undergoing elective percutaneous coronary intervention were randomly assigned to an eptifibatide bolus followed by an infusion (180-μg/kg bolus followed by 2 μg/kg per minute or 250-μg/kg bolus followed by 3 μg/kg per minute) for 18 to 24 hours. In a 2:1 ratio, these patients received either a second bolus of eptifibatide (90 μg/kg or 125 μg/kg for the initial 180-μg/kg or 250-μg/kg groups, respectively) or placebo 30 minutes after the initial bolus. Bleeding times, ex vivo platelet aggregation, receptor occupancy, and plasma eptifibatide levels at baseline and at 1, 2, 3, 4, 6, and 8 hours were evaluated. Platelet inhibition was dose dependent and >80% in all groups by steady state. The single-bolus regimens had a transient loss of inhibition at 1 hour, consistent with rapid distribution and drug elimination. Pharmacokinetic modeling suggested that optimal dosing of eptifibatide would be obtained with a 180-μg/kg bolus and a 2-μg/kg per minute infusion followed by a second 180-μg/kg bolus 10 minutes later. Conclusions A novel higher-dose, double-bolus regimen of eptifibatide in coronary intervention attains and maintains >90% inhibition of platelet aggregation in >90% of patients, providing the pharmacodynamic construct for the design of the Enhanced Suppression of the Platelet IIb/IIIa Receptor with Integrilin Therapy (ESPRIT) trial of adjunctive eptifibatide in coronary stent implantation.

We thank Caruthers and Dorsch for their interest in the Platelet Inhibition in Children On cLOpidogrel (PICOLO) trial.1 The variability in inhibition of ADP-induced platelet aggregation by clopidogrel reported for pediatric patients in PICOLO (Figure 2 of our article1) is similar to the well-described variability in inhibition of ADP-induced platelet aggregation by clopidogrel observed in adult patients.2 Caruthers and Dorsch’s main concern is that because clopidogrel undergoes metabolism through CYP3A4, lower CYP3A4 activity …

Background: TRA-PCI was a multicenter, randomized, double-blind, placebo-controlled study demonstrating the safety of SCH 530348, a potent thrombin receptor antagonist (TRA), in patients undergoing non-urgent percutaneous coronary intervention (PCI). A secondary endpoint in a sub-study within the primary evaluable patient cohort was inhibition of platelet aggregation (IPA) induced by appropriate agonists relative to baseline. We hypothesized that TRA therapy would selectively inhibit platelet aggregation induced by the thrombin receptor agonist peptide (TRAP) and that sustained inhibition would be observed. Pharmacokinetic (PK) studies were also carried out. Methods: Platelet aggregation responses to TRAP, adenosine diphosphate (ADP), collagen and arachidonic acid (AA) were measured using light transmission aggregometry (LTA) at baseline, 30, 60, 90, 120 min following a loading dose (10, 20 or 40 mg vs placebo) and after a maintenance dose (0.5, 1.0 or 2.5 mg/day) at 30 and 60 days. PK was assessed at 30 and 60 min and 2 hr after loading dose administration. Results: TRA was active in inhibiting 15 μM TRAP-induced platelet aggregation with onset of inhibition directly related to dose. Loading doses of 10, 20 or 40 mg achieved >90% inhibition of platelet aggregation (IPA) 60 –120 min post dose with the 40 mg dose achieving >90% inhibition between 60 and 90 min. Patients receiving maintenance doses of 0.5, 1.0 or 2.5 mg exhibited >90% IPA at 30 and 60 days. Placebo treated patients had on average ≥10% IPA to TRAP. TRA had no significant effects on ADP, collagen or AA -induced platelet aggregation compared with placebo controls. TRA PK was characterized by fast distribution and slow elimination (t1/2 = 165–311 hr) with clear evidence of hysteresis. Conclusion: Among PCI patients treated with TRA, there was a specific, dose-related, sustained inhibition of TRAP-induced platelet aggregation, without an impact on other aggregation related pathways. These data suggest that TRA is a potent, selective inhibitor of PAR-1 activity induced by thrombin activation of platelets, and, in view of this, is a promising therapeutic utility for treatment of thrombosis.

Background Platelet deposition and aggregation are central to the pathogenesis of ischemic complications of acute coronary syndromes (ACS). Pharmacodynamic effects of the platelet glycoprotein IIb/IIIa antagonist eptifibatide have been delineated in healthy subjects but not in patients with ACS. We assessed effects of eptifibatide on ex vivo platelet aggregation in patients enrolled in the Platelet glycoprotein IIb/IIIa in Unstable angina: Receptor Suppression Using Integrilin (eptifibatide) Therapy (PURSUIT) trial of ACS. Methods and Results Patients were randomly assigned to an intravenous bolus (180 μg/kg) and 72-hour infusion of eptifibatide (2.0 μg/kg per minute, n=48) or placebo (n=50). We assessed correlations of plasma eptifibatide levels with receptor occupancy and inhibition of ex vivo platelet aggregation at 5 minutes and 1, 4, 24, 48, and 72 hours during treatment and 4 and 8 hours after termination of infusion. Blood was collected in buffered citrate and d -phenylalanyl- l -prolyl- l -arginine chloromethylketone anticoagulants. Although eptifibatide produced profound, prolonged inhibition of platelet aggregation during therapy, aggregation appeared to recover partially by 4 hours after the bolus. The aggregation response was greater with thrombin receptor agonist peptide versus ADP stimulation; inhibition of platelet aggregation was greater in blood samples anticoagulated with citrate versus d -phenylalanyl- l -prolyl- l -arginine chloromethylketone (PPACK). Plasma eptifibatide levels correlated significantly with receptor occupancy but not with inhibition of platelet aggregation. Conclusions A bolus and infusion of eptifibatide inhibits platelet aggregation profoundly in patients with ACS and is followed by brief, partial recovery. These results enhance our understanding of the relation between pharmacodynamic and clinical effects of eptifibatide in such patients and may have important implications for its use in percutaneous interventions.

Background Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-IIIa, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca 2+ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug. Methods and Results Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean±SD IC 50 =140±40 nmol/L, n=6; Ca 2+ =40 to 50 μmol/L) than with PPACK (IC 50 =570±70 nmol/L, P 2+ ≈1 mmol/L). Chelation of Ca 2+ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca 2+ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa. Conclusions These data suggest that citrate anticoagulation removes Ca 2+ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.