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Background:To date, there is no data on switching to dual pathway inhibition (DPI) patients who have completed a guideline-recommended dual antiplatelet therapy (DAPT) regimen. Objectives: To assess the feasibility of switching from DAPT to DPI and to compare the pharmacodynamic (PD) profiles of these treatments. Methods: This was a prospective, randomized, PD study conducted in 90 patients with chronic coronary syndrome (CCS) on DAPT with aspirin (81mg/qd) plus a P2Y12 inhibitor [clopidogrel (75 mg/qd; n=30), ticagrelor (90mg/bid; n=30), or prasugrel (10mg/qd; n=30)]. Patients in each cohort were randomized to maintain DAPT or switch to DPI (aspirin 81mg/qd plus rivaroxaban 2.5mg/bid). PD assessments included: VerifyNow P2Y12 reaction units; light transmittance aggregometry following stimuli with adenosine diphosphate (ADP), tissue factor (TF), and a combination of collagen, ADP, and TF (CATF, maximum platelet aggregation %); thrombin generation (TG). Assays were performed at baseline and 30 days post-randomization. Results: Switching from DAPT to DPI occurred without major side effects. DAPT was associated with enhanced P2Y12 inhibition, while DPI with reduced TG. Platelet-mediated global thrombogenicity (primary endpoint) showed no differences between DAPT and DPI in the ticagrelor (14.5% [0.0–63.0] vs. 20.0% [0.0–70.0]; p=0.477) and prasugrel (20.0% [0.0–66.0] vs. 4.0% [0.0–70.0]; p=0.482), but not clopidogrel (27.0% [0.0–68.0] vs. 53.0% [0.0–81.0]; p=0.011), cohorts. Conclusion: In patients with CCS, switching from different DAPT regimens to DPI was feasible, showing enhanced P2Y12 inhibition with DAPT and reduced TG with DPI, with no differences in platelet-mediated global thrombogenicity between DPI and ticagrelor- and prasugrel, but not clopidogrel-, based DAPT.

Aims To assess the feasibility of switching from dual antiplatelet therapy (DAPT) to dual pathway inhibition (DPI) and to compare the pharmacodynamic (PD) profiles of these treatment regimens. Methods and results This was a prospective, randomized, PD study conducted in 90 patients with chronic coronary syndrome (CCS) on DAPT with aspirin (81 mg/qd) plus a P2Y12 inhibitor [clopidogrel (75 mg/qd; n = 30), ticagrelor (90 mg/bid; n = 30), or prasugrel (10 mg/qd; n = 30)]. Patients in each cohort were randomized to maintain DAPT or switch to DPI (aspirin 81 mg/qd plus rivaroxaban 2.5 mg/bid). PD assessments included: VerifyNow P2Y12 reaction units; light transmittance aggregometry following stimuli with adenosine diphosphate (ADP), tissue factor, and a combination of collagen, ADP, and TF (CATF, maximum platelet aggregation %); thrombelastograph coagulation analyzer; thrombin generation (TG). Assays were performed at baseline (on DAPT) and 30 days post-randomization (trough and peak). Switching from DAPT to DPI occurred without major side effects. DAPT was associated with enhanced P2Y12 inhibition, while DPI with reduced TG. Platelet-mediated global thrombogenicity (primary endpoint) showed no differences between DAPT and DPI in the ticagrelor (14.5% [0.0–63.0] vs. 20.0% [0.0–70.0]; p = 0.477) and prasugrel (20.0% [0.0–66.0] vs. 4.0% [0.0–70.0]; p = 0.482), but not clopidogrel (27.0% [0.0–68.0] vs. 53.0% [0.0–81.0]; p = 0.011), cohorts. Conclusion In patients with CCS, switching from different DAPT regimens to DPI was feasible, showing enhanced P2Y12 inhibition with DAPT and reduced TG with DPI, with no differences in platelet-mediated global thrombogenicity between DPI and ticagrelor- and prasugrel, but not clopidogrel-, based DAPT.

Aims Dual pathway inhibition (DPI) by adding a vascular dose of rivaroxaban to a single antiplatelet agent has emerged as a promising antithrombotic strategy. However, in most studies the antiplatelet agent of choice used in adjunct to a vascular dose of rivaroxaban was aspirin, and data on a P2Y12 inhibitor and how this DPI regimen compares with standard dual antiplatelet therapy (DAPT) are limited. Methods and results This investigation was a substudy analysis conducted in selected cohorts of patients with stable atherosclerotic disease enrolled from a larger prospective, open-label, parallel-group pharmacodynamic (PD) study. We analysed data from 40 patients treated with either clopidogrel- or ticagrelor-based DAPT first, and clopidogrel- or ticagrelor-based DPI thereafter. PD measures explored key pathways involved in thrombus formation and included markers of (1) P2Y12 reactivity, (2) platelet-mediated global thrombogenicity, (3) cyclooxygenase-1 activity, (4) thrombin receptor-activating peptide (TRAP)-induced platelet aggregation, (5) tissue factor (TF)-induced platelet aggregation, and (6) thrombin generation. Compared with DAPT, on a background of the same P2Y12 inhibitor (clopidogrel or ticagrelor), DPI was associated with reduced thrombin generation, increased markers of cyclooxygenase-1 activity and TRAP-induced platelet aggregation, and no differences in markers of P2Y12 signalling, platelet-mediated global thrombogenicity, and TF-induced platelet aggregation. In an analysis according to P2Y12 inhibitor type, ticagrelor reduced markers of platelet-mediated global thrombogenicity, P2Y12 signalling, and rates of high platelet reactivity compared with clopidogrel. Conclusion Compared with DAPT with aspirin and a P2Y12 inhibitor, the use of a P2Y12 inhibitor in adjunct to a vascular dose of rivaroxaban as part of a DPI strategy is associated with similar effects on platelet-mediated global thrombogenicity but reduced thrombin generation. A DPI strategy with ticagrelor is associated with enhanced antithrombotic efficacy, the clinical implications of which warrant larger scale investigations. Clinical trial registration ClinicalTrials.gov identifier: NCT03718429.

Aim Inhibition of thrombin-mediated signaling processes using a vascular dose of rivaroxaban in adjunct to antiplatelet therapy, known as dual-pathway inhibition (DPI), reduces atherothrombotic events in patients with stable atherosclerotic disease. However, there are limited data on the pharmacodynamic (PD) effects of this strategy and how it compares to standard dual antiplatelet therapy (DAPT). Methods and Results This investigation was conducted in selected cohorts of patients (n = 40) with stable atherosclerotic disease—enrolled within a larger prospective, open-label, parallel-group PD study—who were treated with either aspirin plus clopidogrel (DAPT), aspirin plus rivaroxaban 2.5 mg/bid (DPI), or DAPT plus rivaroxaban 2.5 mg/bid. Multiple PD assays providing a comprehensive assessment of markers of thrombosis were used. PD endpoints included platelet-mediated global thrombogenicity measured by light transmittance aggregometry (LTA) following stimuli with CATF (collagen-related peptide + adenosine diphosphate [ADP] + tissue factor [TF]), markers of P2Y12 reactivity, markers of platelet aggregation using LTA following several stimuli (arachidonic acid, ADP, collagen, TF, and thrombin receptor-activating peptide [TRAP]), thrombin generation, and thrombus formation. There was no difference in platelet-mediated global thrombogenicity between groups. Rivaroxaban significantly reduced thrombin generation and was associated with a trend toward reduced TF-induced platelet aggregation. Clopidogrel-based treatments reduced markers of P2Y12 signaling and TRAP-induced platelet aggregation. There were no differences between groups on markers of cyclooxygenase-1-mediated activity. Conclusion Compared with DAPT, DPI does not result in any differences in platelet-mediated global thrombogenicity, but reduces thrombin generation. These PD observations suggest that modulating thrombin generation—by means of factor Xa inhibition—in adjunct to antiplatelet therapy provides effective antithrombotic effects, supporting the efficacy and safety findings of a DPI strategy observed in clinical trials.

Summary: Vorapaxar reduces thrombotic cardiovascular events at the expense of increased bleeding. However, the differential pharmacodynamic (PD) effects of vorapaxar according to diabetes mellitus (DM) status are unknown. Moreover, although withdrawal of aspirin has emerged as a bleeding reduction strategy, the PD effects of stopping aspirin in patients treated with vorapaxar also are unknown. In this prospective PD investigation, vorapaxar was associated with reduced platelet-mediated thrombogenicity without affecting clot kinetics irrespective of DM status. However, platelet-mediated thrombogenicity increased after aspirin withdrawal, particularly among patients with DM. (Optimizing anti-Platelet Therapy In diabetes MellitUS-5 Study [OPTIMUS-5]; NCT02548650) Key Words: dual antiplatelet therapy, pharmacodynamics, platelets, thrombin, vorapaxar

Ticagrelor is an oral P2YIn this randomized, double-blind, placebo-controlled, phase 1 trial, we evaluated intravenous PB2452, a monoclonal antibody fragment that binds ticagrelor with high affinity, as a ticagrelor reversal agent. We assessed platelet function in healthy volunteers before and after 48 hours of ticagrelor pretreatment and again after the administration of PB2452 or placebo. Platelet function was assessed with the use of light transmission aggregometry, a point-of-care P2YOf the 64 volunteers who underwent randomization, 48 were assigned to receive PB2452 and 16 to receive placebo. After 48 hours of ticagrelor pretreatment, platelet aggregation was suppressed by approximately 80%. PB2452 administered as an initial intravenous bolus followed by a prolonged infusion (8, 12, or 16 hours) was associated with a significantly greater increase in platelet function than placebo, as measured by multiple assays. Ticagrelor reversal occurred within 5 minutes after the initiation of PB2452 and was sustained for more than 20 hours (P0.001 after Bonferroni adjustment across all time points for all assays). There was no evidence of a rebound in platelet activity after drug cessation. Adverse events related to the trial drug were limited mainly to issues involving the infusion site.In healthy volunteers, the administration of PB2452, a specific reversal agent for ticagrelor, provided immediate and sustained reversal of the antiplatelet effects of ticagrelor, as measured by multiple assays. (Funded by PhaseBio Pharmaceuticals; ClinicalTrials.gov number, NCT03492385.).

Direct oral anticoagulants such as apixaban are increasingly being evaluated clinically for the secondary prevention of cardiovascular events; however, their effects on platelet function in combination with dual anti-platelet therapy (DAPT) have not been fully investigated. The purpose of this translational, in vitro study was to determine if apixaban via inhibition of thrombin generation exhibits synergistic activity with DAPT to reduce platelet reactivity. Consented subjects with a prior history (7 days prior to blood collection. Platelet-rich plasma from TSC anticoagulated blood was prepared and treated in vitro with nothing, a carrier control or apixaban (40, 90 and 220 ng/mL). The range of 40 to 220 ng/mL brackets the expected apixaban exposure at steady state with all three approved regimens with the 40 ng/mL treatment corresponding to Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Bristol-Myers Squibb

Background Vorapaxar as an adjunct to dual antiplatelet therapy ( DAPT ) reduces thrombotic events in patients with prior myocardial infarction at the expense of increased bleeding. Withdrawal of aspirin has emerged as a bleeding reduction strategy. The pharmacodynamic effects of vorapaxar with potent P2Y 12 inhibitors as well as the impact of dropping aspirin is unexplored and represented the aim of the VORA‐PRATIC (Vorapaxar Therapy in Patients With Prior Myocardial Infarction Treated With Newer Generation P2Y 12 Receptor Inhibitors Prasugrel and Ticagrelor) study. Methods and Results Post–myocardial infarction patients (n=130) on standard DAPT (aspirin+prasugrel or ticagrelor) were randomized to 1 of 3 arms: (1) triple therapy: aspirin+prasugrel/ticagrelor+vorapaxar; (2) dual therapy (drop aspirin): prasugrel/ticagrelor+vorapaxar; (3) DAPT : aspirin+prasugrel/ticagrelor. Pharmacodynamic assessments were performed at 3 time points (baseline and 7 and 30 days). Vorapaxar reduced CAT (collagen‐ADP‐TRAP)–induced platelet aggregation, a marker of platelet‐mediated global thrombogenicity (triple therapy versus DAPT at 30 days: mean difference=–27; 95% CI ,–35 to –19; P DAPT at 30 days: mean difference=–15; 95% CI ,–23 to –7; P P P 12 reactivity. Markers sensitive to aspirin‐induced effects increased ( P Conclusions In post–myocardial infarction patients treated with potent P2Y 12 inhibitors, vorapaxar reduces platelet‐driven global thrombogenicity, an effect that persisted, albeit attenuated, in the absence of aspirin and without affecting markers of P2Y 12 reactivity or clot kinetics. The clinical implications of these PD observations warrant future investigation. Registration URL : https://www.clini​caltr​ials.gov . Unique identifier: NCT 02545933.

Background Elevated D-dimer concentrations are associated with an increased risk of venous thromboembolism (VTE). However, they may also provide prognostic value. The present analysis sought to study the association of D-dimer levels with VTE event rates and the efficacy of betrixaban versus enoxaparin in the APEX trial. Methods Hospitalized acutely medically ill subjects (n = 7,513) were randomized in a double-dummy double-blind fashion to either extended-duration oral betrixaban (80 mg once daily for 35–42 days) or standard dose subcutaneous enoxaparin (40 mg once daily for 10 ± 4 days) for venous thromboprophylaxis. D-dimer was assessed using a central core laboratory measurement. Results For every 0.25 µg/mL increase in D-dimer concentration, there was a 2% increase in the relative risk of experiencing the primary efficacy endpoint (asymptomatic deep vein thrombosis [DVT], symptomatic DVT, nonfatal pulmonary embolism, or VTE-related death) in both the betrixaban (p Conclusion Extended-duration betrixaban was superior to standard-duration enoxaparin, irrespective of D-dimer level at baseline. To prevent one VTE event, 46 D-dimer-positive patients would need to be treated with betrixaban.

Introduction: GARP is a cell surface protein expressed post activation on regulatory T-cells (Treg), monocytes, B cells, and platelets. GARP binds to latent transforming growth factor beta (TGF-β1) and regulates the release of active TGF-β1. TGF-β1 modulates Treg-mediated immunosuppression and represses effector T cell responses. ABBV-151 is a first-in-class monoclonal antibody that binds to the GARP-TGF-β1 complex and blocks active TGF-β1 release. Here we describe the validation of a TE assay for ABBV-151 using human platelets to measure receptor occupancy (RO) and binding saturation, that will inform dosing decisions during a first-in-human Phase I dose escalation study. Procedure: A competing (LHG10.6 that recognizes the GARP-TGF-β1 complex) and non-competing (1E7 that recognizes total GARP) anti-GARP antibodies were used to measure TE on platelets. Human whole blood was incubated with increasing concentrations of ABBV-151, followed by platelet-rich plasma (PRP) preparation and storage at -80°C. Platelets were thawed, washed, and stained with a fluorescently labeled antibody cocktail (LHGH10.6, 1E7 and CD61) and analyzed on a flow cytometer. The analytical measurement range, inter-operator and inter-assay precision and sample stability at -80°C (up to 12 months) for this TE assay was determined. Results: ABBV-151 TE assay demonstrated a dose response relationship over a range of ABBV-151 concentrations (0.08 to 400 ng/mL) that corresponded to 0-80% occupancy, with an EC50 of 6.2 ± 2.1 ng/mL, and 0-100% saturation with an EC50 of 6.7 ± 1.9 ng/mL. Inter-assay precision demonstrated ≤3% CV at low ABBV-151 doses, and ≤14% in samples at high doses. Inter-operator precision for untreated and treated samples was between 3 to 9% CV, respectively. The stability data indicated frozen platelets could be stored up to 12 months with highly reproducible assay performance. Conclusions: This TE assay for ABBV-151 on platelets demonstrates desirable analytical performance characteristics. The percent saturation (PS) showed higher precision than the RO measurements for this assay and PS was determined to be a reliable metric for measuring TE. Frozen platelets samples were suitable for the TE assay for up to a year allowing for batch processing of samples at a central lab and for minimizing the variability in test results. This novel TE assay overcomes key limitations of implementing TE assays in clinical trials, and quantification of PS using this TE assay in clinical trials is currently ongoing to assess the pharmacodynamic/pharmacokinetic relationship of ABBV-151 therapy. Citation Format: Juan M. Cardenas, Jayaprakash Kotha, Sridevi Kotha, Lisa K. Jennings, Michelle Graham, Stacie Lambert, Kinjal Hew. Development and validation of a flow cytometric assay for measuring glycoprotein A repetitions predominant protein GARP target engagement by ABBV-151 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1308.

Introduction: The anticoagulants rivaroxaban and apixaban inhibit Factor Xa (FXa) activity and are effective therapeutic agents for the prevention and treatment of thromboembolism. Andexanet alfa (andexanet) is the only specific reversal agent approved for anticoagulation reversal in patients presenting with life-threatening bleeding treated with rivaroxaban or apixaban. Anti-FXa assays are functional assays used for measuring direct and indirect FXa inhibitor levels in plasma. Current commercially available anti-FXa assays are not suitable for accurately measuring anti-FXa activity in patient samples in the presence of andexanet due to the high sample dilutions (e.g.,1:44 with STA-Liquid Anti-Xa), which causes dissociation of the inhibitor from andexanet (due to reversible binding). This results in substantial underestimation of the reversal activity of andexanet, and in some cases with erroneously elevated anti-FXa levels in patient samples following andexanet treatment. Therefore, we modified the current anti-FXa assays to measure apixaban and rivaroxaban anti-FXa levels in the presence of andexanet. Methods: The modified anti-FXa assays were performed on STA®-Compact analyzer using reagents from STA-Liquid Anti-Xa assay kits (Stago). Andexanet was provided as a 10 mg/mL frozen stock. Plasma samples with FXa inhibitor in the presence or absence of andexanet were prepared using Stago Calibrator 1 and pooled normal human plasma (Precision Biologic). Previous anti-FXa assays (calibrator range: 0-500 ng/mL) employed 1:4 dilution of test plasma in Owren-Koller buffer followed by addition of FXa substrate and bovine FXa with an overall 1:44 sample dilution. In the modified assays, calibration curves for the anti-FXa assay were generated using apixaban or rivaroxaban calibrators in the range of 0 to 80 ng/mL; test samples were analyzed neat with an overall 1:2.6 sample dilution. Rivaroxaban and apixaban concentrations in the test samples were interpolated from the respective assay specific calibration curves. Validation of the modified anti-FXa assays was performed by assessing a) linearity and precision of the calibration curve and controls, b) lower limit of quantitation (LLOQ), c) inter-assay precision and d) sample stability. Results: The calibration curves for both apixaban and rivaroxaban assays demonstrated good correlation with a mean "r" value of 0.997 and 0.998, respectively (n=6). The percent recovery and precision of the modified anti-FXa assay are summarized in Table 1. The LLOQ for apixaban and rivaroxaban using the modified assays were Conclusions: The modified anti-FXa assays intended for measuring FXa inhibitor levels in the presence of andexanet produced acceptable analytical performance characteristics. Application of the modified anti-FXa assays in plasma samples from andexanet-treated human subjects has yet to be studied. Table 1 Disclosures Cardenas: CirQuest Labs/MLM Medical Labs: Current Employment. Kotha:CirQuest Labs/MLM Medical Labs: Current Employment. Lu:Portola Pharmaceuticals, Inc.: Current Employment. Conley:Portola Pharmaceuticals, Inc.: Current Employment. Bourdin:Diagnostica Stago: Current Employment. Herve:Diagnostica Stago: Current Employment. Jennings:CirQuest Labs/MLM Medical Labs: Current Employment, Current equity holder in private company.

Background Ticagrelor is a P2Y12 antagonist used in combination with aspirin to reduce the risk of recurrent thrombotic cardiovascular events in patients with acute coronary syndrome. Ticagrelor is associated with a risk for spontaneous major bleeding or bleeding associated with invasive procedures, particularly cardiac surgery. A rapid-acting reversal agent for ticagrelor would be advantageous. The pharmacodynamic effects of ticagrelor and a ticagrelor reversal agent are best evaluated using a panel of platelet function tests that have different sensitivities and methodologies and using a variety of agonists at different concentrations. Purpose In a first-in-human randomized, double-blind, placebo-controlled, healthy volunteer Phase 1 study, intravenous (IV) PB2452, a monoclonal antibody fragment that binds ticagrelor with high affinity, was evaluated as a ticagrelor reversal agent. Methods Platelet function was assessed using light transmission aggregometry (LTA) and 5 and 20 μM adenosine diphosphate (ADP), 1.6 mM arachidonic acid (AA), and 15 μM thrombin receptor agonist peptide (TRAP-6) as agonists. The VerifyNow P2Y12 cartridges, which assess whole blood platelet function, were evaluated as a point-of-care test. A modified vasodilator-stimulated phosphoprotein (VASP) ELISA was used to assess the extent of P2Y12 signaling. These assessments were performed 48 h prior to ticagrelor administration and at multiple time points up to 48 h after administration of PB2452 or placebo. Results 64 subjects were randomized; 48 received PB2452 and 16 received placebo. After ticagrelor administration, LTA response to 20 μM ADP was inhibited by 87% compared to the pre-ticagrelor values. Platelet function as measured by the VerifyNow P2Y12 cartridge was completely inhibited (80% and >90% of baseline as measured by LTA using ADP and TRAP-6, respectively, to >180 PRU using VerifyNow, and to >90% PRI as measured by VASP. The VASP assay enabled batch analyses in a central laboratory, eliminating the need for special equipment on-site and reducing operator variability. When platelet function was assessed by VASP, PB2452 administration produced rapid ticagrelor reversal within 5 min consistent with restoration of P2Y12 signaling. Onset of reversal by all measurements occurred within 15 min of PB2452 administration and was sustained for 20–24 h. Conclusions PB2452 is a specific reversal agent for ticagrelor and produces a rapid and sustained reversal of ticagrelor inhibition of platelets. Utilizing multiple platelet function assays provided a broader understanding of the PB2452 pharmacodynamics in this first-in-human Phase 1 study. Acknowledgement/Funding PhaseBio Pharmaceuticals

The use of tetraspanin CD9 as a biomarker for renal cell carcinomas (RCC) has been explored with minor conclusions. Identification of a biomarker that not only distinguishes between the different types of renal cell carcinomas, but also predicts the metastatic potential of these tumors would significantly advance diagnosis and prognosis of kidney cancers. We utilized established cell lines to better understand the contribution of CD9 to the metastatic potential of clear cell renal cell carcinomas, and then applied our findings to the TCGA database and immunohistochemical analysis of human samples based on tumor grading to determine the utility of CD9 as a biomarker for RCC. Clear cell renal cell carcinoma (ccRCC) cell expression of tetraspanin CD9 was compared to normal kidney cells and found to be elevated. Upon knockdown of CD9, ccRCC cells obtained a more metastatic phenotype. We found E-cadherin expression to be repressed and the endothelial to mesenchymal transition markers Snail, Twist1, and Zeb1 to be elevated upon CD9 knockdown. Upon observing these gene expression changes in the TCGA database and in 10 cases, we found that CD9 and E-cadherin expression was lowered in higher grade ccRCC tumors. There was a significant correlation between CD9 and either E-cadherin, Snail, or Zeb1 in these tumors. Collectively, using tetraspanin CD9 in tandem with E-cadherin as a biomarker in renal cell carcinoma will help to not only distinguish between types, but also predict the metastatic potential of RCC.

Introduction: High rates of recanalization of Emergent Large Vessel Occlusion (ELVO) provide an opportunity to examine retrieved thrombi. We studied device and endothelial (ET) interactions to evaluate ET injury and the relationship between thrombi and Acute Ischemic Stroke (AIS) mechanism. Methods: Prospective registry examining immunohistochemical characteristics of retrieved thrombi in ELVO patients. Thrombi were categorized histopathologically and ET injury quantified by CD34 staining and immunohistochemistry, ELISA and reverse transcribed quantitative PCR to detect ET components. ET mRNA expression of CD31 was quantified from thrombi RNA. Plasma at the thrombectomy site was tested for ET derived biomarkers: microparticles (MPs), soluble Intercellular Cell Adhesion Molecule-1 (sICAM1) and soluble Thrombomodulin (sTM). Statistical analysis was performed with unpaired student’s t-tests and chi-square tests. Results: Twenty-five patients (age 68±3 , NIHSS 16±2, 48% female) were enrolled. The most common sites of occlusion were the MCA (9) and ICA (7). Eighty eight % of patients required three or less attempts at recanalization and 71 % achieved TICI 2b or 3. Histopathology demonstrated fibrin rich (5), erythrocytic (2), layered (7), and serpentine (11) thrombi and four were CD 34 positive. Levels of plasma biomarkers were: sTM 2.2±0.4 ( 3 revascularization attempts had higher levels of sTM (5.5±0.4 vs 1.7±0.2, p Conclusions: ET markers can be detected in the retrieved thrombi and plasma milieu of ELVO patients. ET markers relate to increasing number of attempts at thrombectomy indicating ET disruption. sTM and sICAM are more common in patients with cardioembolic stroke and may serve as thrombus associated markers of AIS mechanism.

Variation in platelet response to thrombin may affect the safety and efficacy of PAR antagonism. The Thr120 variant of the common single nucleotide polymorphism (SNP) rs773902 in the protease-activated receptor (PAR) 4 gene is associated with higher platelet aggregation compared to the Ala120 variant. We investigated the relationship between the rs773902 SNP with major bleeding and ischemic events, safety, and efficacy of PAR1 inhibition in 6177 NSTE ACS patients in the TRACER trial. There was a lower rate of GUSTO moderate/severe bleeding in patients with the Thr120 variant. The difference was driven by a lower rate in the smaller homozygous group (recessive model, HR 0.13 [0.02–0.92] P = 0.042). No significant differences were observed in the ischemic outcomes. The excess in bleeding observed with PAR1 inhibition was attenuated in patients with the Thr120 variant, but the interactions were not statistically significant. In summary, lower major bleeding rates were observed in the overall TRACER cohort with the hyperreactive PAR4 Thr120 variant. The increase in bleeding with vorapaxar was attenuated with the Thr120 variant, but we could not demonstrate an interaction with PAR1 inhibition. These findings warrant further exploration, including those of African ancestry where the A allele (Thr120) frequency is ~65%.

The role of thrombin in vascular physiology and pathophysiology continues to impact our understanding of many cellular processes and systems including the function of platelets, endothelial cells, smooth muscle cells, leukocytes and the regulation of the coagulation cascade. Recent acute coronary syndrome clinical trial results that have compared the use of parenteral or oral anticoagulants versus or in combination with anti-platelet agents have forced a reexamination of the importance of thrombin activity in influencing patient outcomes, particularly in the area of secondary prevention. The debate of the need to include oral anticoagulation as a concomitant or replacement therapy to an antiplatelet regimen as a means to improve patient outcomes requires further examination and larger prospective clinical trials.

Studies have shown that gay men are at increased risk for anxiety and depression due to social oppression; research suggests that self-compassion is positively associated with life-satisfaction and emotional resilience. In a sample of 68 gay men ( M age = 39.7 yr., SD = 16.3), the influence of self-compassion on satisfaction with life was examined while controlling for age, income, and openness about sexual orientation. Analysis of the data revealed that self-compassion was a significant predictor of satisfaction with life. Implications of this finding were discussed.

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

Thrombin Receptor Antagonist for Clinical Event Reduction in Acute Coronary Syndrome (TRACER) trial compared vorapaxar and placebo in 12,944 high-risk patients with non-ST-segment elevation acute coronary syndrome. We explored aspirin (ASA) use and its association with outcomes. Kaplan-Meier event rates were compared in groups defined by ASA dose (low, medium, and high). Landmark analyses with covariate adjustment were performed for 0 to 30, 31 to 180, and 181 to 365 days. Of 12,515 participants, 7,523, 1,049, and 3,943 participants were treated with low-, medium-, and high-dose ASA at baseline, respectively. Participants enrolled in North America versus elsewhere were more often treated with a high dose at baseline (66% vs 19%) and discharge (60% vs 3%). Unadjusted cardiovascular death, myocardial infarction, stroke, hospitalization for ischemia, or urgent revascularization event rates tended to be higher with higher baseline ASA (18.45% low, 19.13% medium, and 20.27% high; p for trend = 0.15573). Unadjusted and adjusted hazard ratios (95% confidence intervals) for effect of vorapaxar on cardiovascular (unadjusted p for interaction = 0.065; adjusted p for interaction = 0.140) and bleeding (unadjusted p for interaction = 0.915; adjusted p for interaction = 0.954) outcomes were similar across groups. Landmark analyses showed similar safety and efficacy outcomes with vorapaxar and placebo by ASA dose at each time point except for 0 to 30 days, when vorapaxar tended to be worse for efficacy (hazard ratio 1.13, 95% confidence interval 0.89 to 1.44, p for interaction = 0.0157). In conclusion, most TRACER participants were treated with low-dose ASA, although a high dose was common in North America. High-dose participants tended to have higher rates of ischemic and bleeding outcomes. Although formal statistical testing did not reveal heterogeneity in vorapaxar's effect across dose subgroups, consistent trends support use of low-dose ASA with other antiplatelet therapies.

Strategy, Management and Health PolicyEnabling Technology, Genomics, ProteomicsPreclinical ResearchPreclinical Development Toxicology, Formulation Drug Delivery, PharmacokineticsClinical Development Phases I-III Regulatory, Quality, ManufacturingPostmarketing Phase IV There is an increasing need for the standardization of platelet function and coagulation testing for the assessment of antithrombotic therapies. Investigators continue to strive to identify ideal laboratory testing and monitoring procedures for acquired and inherited platelet function defects as well as for evaluating patient status when treated with existing or emerging antithrombotics. These therapies are used primarily in the treatment of ischemic complications. In patients receiving antithrombotic therapy, the balance between hemostasis and thrombosis is a challenge as there is an ongoing risk for bleeding when patients are receiving antiplatelet agents or anticoagulants to lessen their risk for secondary thrombotic events. There are several diverse tests for monitoring anticoagulant therapy; however, as new agents are developed, more specific tests will be required to directly assess these agents in relationship to overall coagulation status. Research in the platelet biology field is ongoing to provide point-of-care methodologies for the assessment of platelet reactivity in terms of both bleeding and thrombosis risk. Currently there are no instruments that reliably assess the risk of bleeding. The challenges that routinely faced are the complexity of physiology, the need for standardization of platelet testing methodology, and the necessity for appropriate interpretation of the test results.

Background— Elinogrel is the only selective, competitive and reversible platelet P2Y 12 inhibitor available in both intravenous (IV) and oral formulations. Methods and Results— This substudy of the Intravenous and Oral Administration of Elinogrel to Evaluate Tolerability and Efficacy in Nonurgent Percutaneous Coronary Intervention patients (INNOVATE-PCI) trial evaluated the pharmacokinetic and pharmacodynamic effects of two dosing regimens of IV followed by oral elinogrel (120 mg IV plus 100 mg oral twice daily; 120 mg IV plus 150 mg oral twice daily) versus standard clopidogrel therapy (300–600 mg oral loading dose plus 75 mg oral maintenance dose) in 56 patients undergoing nonurgent PCI. At time of randomization, 71.4% (40/56) of patients were using maintenance clopidogrel therapy. In the acute phase, an IV bolus of elinogrel achieved more rapid and potent antiplatelet effects compared with clopidogrel, which were sustained during the transition from the IV to the oral formulation in the first 24 hours of the peri-PCI period. During chronic therapy, elinogrel achieved similar levels of platelet reactivity compared with clopidogrel before the next oral dose and, although platelet reactivity was lower with elinogrel up to 6 hours after daily oral maintenance dosing, these differences were not statistically significant. These pharmacodynamic effects matched the pharmacokinetic profile of elinogrel. There were no differences in pharmacodynamic and pharmacokinetic effects between the two elinogrel dosing regimens. Conclusions— Compared with clopidogrel, the combination of IV and oral elinogrel achieves more rapid and enhanced antiplatelet effects that were sustained through the transition to oral elinogrel in the peri-PCI period, but these were not significant during chronic dosing in this pilot investigation. Clinical Trial Registration— URL: http://www.clinicaltrials.gov . Unique identifier: NCT00751231.

A clinical need continues for consistent bone remodeling within problematic sites such as those of fracture nonunion, avascular necrosis, or irregular bone formations. In attempt to address such needs, a biomaterial system is proposed to induce early inflammatory responses after implantation and to provide later osteoconductive scaffolding for bone regeneration. Biomaterial-induced inflammation would parallel the early stage of hematoma-induced fracture repair and allow scaffold-promoted remodeling of osseous tissue to a healthy state. Initiation of the wound healing cascade by two human concentrated platelet releasate-containing alginate/β-tricalcium phosphate biocomposites has been studied in vitro using the TIB-71™ RAW264.7 mouse monocyte cell line. Inflammatory responses inherent to the base material were found and could be modulated through incorporation of platelet releasate. Differences in hydrogel wt% (2 vs. 8 %) and/or calcium phosphate granule vol.% (20 vs. 10 %) allowed for tuning the response associated with platelet releasate-associated growth factor elution. Tunability from completely suppressing the inflammatory response to augmenting the response was observed through varied elution profiles of both releasate-derived bioagents and impurities inherent to alginate. A 2.5-fold upregulation of inducible-nitric oxide synthase gene expression followed by a tenfold increase in nitrite media levels was induced by inclusion of releasate within the 8 wt%/10 vol.% formulation and was comparable to an endotoxin positive control. Whereas, near complete elimination of inflammation was seen when releasate was included within the 2 wt%/20 vol.% formulation. These in vitro results suggested tunable interactions between the multiple platelet releasate-derived bioagents and the biocomposites for enhancing hematoma-like fracture repair. Additionally, minimally invasive delivery for in situ curing of the implant system via injection was demonstrated in rat tail vertebrae using microcomputed tomography.

A significant gap exists in our understanding of subchondral and cancellous bone changes that may regulate osteoarthritis progression. Herein, we complement our prior osteochondrogenesis work with growth factor elution and monocyte and endothelial cell activation using two biomaterial formulations. The design of these biomaterials was inspired by the roles of a fracture hematoma, more specifically, the potential of significant cross-talk among cells and cellular factors that affect bone remodeling. Biomaterials, referred to herein as F1+ and F2+, are human concentrated platelet releasate-containing alginate/beta-tricalcium phosphate composites. F1+ has a higher calcium phosphate volume percentage and lower alginate polymer weight percent hydrogel versus F2+. The majority of releasate-derived platelet-derived growth factor eluted over 24 h for F1+ and 48 h for F2+, suggesting sustained release with an increase in alginate weight percentage. Simple monocyte and endothelial cell migration studies demonstrated 650% and 900% increases with F1+ eluate over medium alone, respectively. Induction of endothelial cell invasion over supplemented medium positive control was also shown for F2+ eluate (p = 0.03) with F1+ eluate being similar to the control. Monocyte transendothelial migration was increased over 300% and 400% for F1+ and F2+ eluates compared with medium alone, respectively. In addition, F1+ and F2+ eluates induced spontaneous endothelial tube formations similar to supplemented medium, demonstrating a well-formed network of capillary-like structures. This work demonstrated our biomaterial formulations ability to induce characteristics in vitro that parallel the in vivo behavior of fracture hematomas and potential to induce bone remodeling for the early treatment of osteoarthritic joints.

Little data on pediatric percent platelet aggregation (%PA) exist in the literature, particularly in cardiac patients and in response to clopidogrel. The objectives were to estimate the %PA range expected in pediatric patients and to measure the clopidogrel effect on %PA in the PICOLO (Platelet Inhibition in Children on Clopidogrel) trial. To estimate a neonatal/infant %PA response range, %PA induced by 5 µM adenosine diphosphate (ADP) was assessed using light transmission aggregometry in 16 cord and 11 normal adult blood samples and prior to clopidogrel therapy in 49 neonatal and 49 infant/toddler cardiac patients enrolled in PICOLO. The %PA induced by 5 µM thrombin receptor-activating peptide (TRAP) was also assessed for 10 neonates and 21 infants/toddlers enrolled in PICOLO and compared with 11 adult samples. Percent inhibition of platelet aggregation (%IPA) induced by 5 µM ADP at steady-state clopidogrel levels was assessed in 33 neonates and 39 infants/toddlers. ADP-induced %PA was lowest in cord blood samples, intermediate in study neonates and infants/toddlers, and highest in adults. Similarly, TRAP-induced platelet aggregation was lower in neonates and infants/toddlers than adults. For all groups, %PA and %IPA were highly variable, with 11% of neonates and 13% of infants/toddlers showing

Underlying changes in subchondral and cancellous bone precede joint degeneration in spinal discs, knees, hips, and other joints. We hypothesize that an early bone remodeling intervention therapy may alleviate or postpone the later manifestation of joint failure. Utilization of a novel biomaterial for injection deliverability to the underlying subchondral and cancellous bone during the onset of osseous changes, before severe cartilaginous damage, is proposed. In this preliminary study, we introduced novel platelet releasate-containing alginate/calcium phosphate composites for this intervention therapy and evaluated them in vitro through mesenchymal stem cell biocompatibility and induction of osteochondral differentiation. DNA quantification and gene expression profiles suggested mesenchymal stem cells were directed along an osteochondral differentiation pathway, more specifically, to the immature nonhypertrophic chondrocyte phenotype. These conclusions were based on reduced mitogenic activity, a rapid upregulation followed by suppression of Runx2 transcription factor mRNA, sustained upregulation of Sox9 transcription factor mRNA and the absence of late marker expressions for both mature articular chondrocytes and osteoblasts over the course of 14 days. Initial findings in regard to the use of these materials for bone remodeling were positive and support the execution of future studies evaluating the inflammatory and angiogenic aspects of the materials. In this study, we have considered one of the many aspects of the endochondral ossification process; neovascularization and resorption of bone with an irregular physiology need to be evaluated. © 2011 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2012.

Vorapaxar, a novel antiplatelet agent in advanced clinical development for the prevention and treatment of atherothrombotic disease, is a potent, orally bioavailable thrombin receptor antagonist selective for the protease-activated receptor 1 (PAR-1).Since race/ethnicity may affect the safety, efficacy and dosage of drugs, this study was conducted to evaluate potential differences in the pharmacodynamics, pharmacokinetics and safety of vorapaxar after single (5, 10, 20, or 40 mg) or multiple (0.5, 1, or 2.5 mg once daily) doses in healthy Japanese and matched (gender, age, height, and weight) Caucasian volunteers.Vorapaxar was well tolerated in both Japanese and Caucasian subjects. Pharmacodynamic and pharmacokinetic profiles of vorapaxar in the two racial/ethnic groups were similar. In both racial groups, complete inhibition of platelet aggregation was achieved most rapidly with vorapaxar 40 mg and was consistently achieved and maintained with a 2.5 mg daily maintenance dose.There were no substantial differences in the safety, pharmacokinetics or pharmacodynamics of vorapaxar between Japanese and Caucasian subjects.

Context. In adults with acute coronary syndrome, decreased platelet inhibition associated with concomitant use of clopidogrel and proton pump inhibitors (PPI) has been reported. Objective. To evaluate platelet activity associated with PPI + clopidogrel vs. clopidogrel alone in children enrolled in the Platelet Inhibition in Children On cLOpidogrel (PICOLO) trial of clopidogrel in children with a cardiac condition at risk for arterial thrombosis. Design. Patients 0–24 m randomized to active therapy in the PICOLO trial were included in the present analysis. Platelet aggregation inhibition at baseline and steady state were evaluated in patients taking clopidogrel + PPI vs. clopidogrel only in the overall cohort and sub-group of clopidogrel responders. Results. A total of 49 patients were included (44 clopidogrel only, five clopidogrel + PPI); median age 38 days (interquartile range [IQR] 17–157 days). The majority of patients in each group had undergone systemic-to-pulmonary artery shunt. Compared with the clopidogrel group, patients in the clopidogrel + PPI group had a trend toward lower percent inhibition of maximum extent of platelet aggregation overall (median 6%, IQR 0–44% vs. 49%, IQR 19–63%, P= 0.09), and a significant reduction in the clopidogrel responders sub-group (median 25%, IQR 3–45% vs. 53%, IQR 38–65%, P= 0.04). There was no difference in percent inhibition of rate of platelet aggregation. Conclusions. Concomitant use of PPI + clopidogrel may be associated with decreased platelet inhibition in children with cardiac disease. Further study in a larger population and assessment of associated clinical outcomes is warranted.

The positive effects of religiousness on coping with transitions and other challenges in life are well documented. However, little is known about specific roles that religious beliefs and activities provide for residents who are becoming integrated into assisted living (AL) settings. The purpose of this study was to explore the experiences and meanings associated with religiousness among AL residents. Qualitative data were collected via semi-structured interviews with 29 residents in four AL settings in a Southern state. Findings revealed four key themes: the opportunity for interactions, coping mechanisms, sense of self, and privacy. Practice implications for the AL community were suggested.

The addition of clopidogrel to aspirin treatment reduces ischemic events in a wide range of patients with cardiovascular disease. However, recurrent ischemic event occurrence during dual antiplatelet therapy, including stent thrombosis, remains a major concern. Platelet function measurements during clopidogrel treatment demonstrated a variable and overall modest level of P2Y(12) inhibition. High on-treatment platelet reactivity to adenosine diphosphate (ADP) was observed in selected patients. Multiple studies have now demonstrated a clear association between high on-treatment platelet reactivity to ADP measured by multiple methods and adverse clinical event occurrence. However, the routine measurement of platelet reactivity has not been widely implemented and recommended in the guidelines. Reasons for the latter include: 1) a lack of consensus on the optimal method to quantify high on-treatment platelet reactivity and the cutoff value associated with clinical risk; and 2) limited data to support that alteration of therapy based on platelet function measurements actually improves outcomes. This review provides a consensus opinion on the definition of high on-treatment platelet reactivity to ADP based on various methods reported in the literature and proposes how this measurement may be used in the future care of patients.

Very low–dose (VLD) factor Xa (FXa) inhibition, in combination with acetylsalicylic acid (ASA) and clopidogrel, is associated with improved outcomes in patients with acute coronary syndrome (ACS) with a tolerable bleeding risk profile. To date, there are no data documenting platelet inhibition and the anticoagulatory effects of VLD FXa inhibition on top of guideline-adherent dual-antiplatelet therapy (DAPT) in patients with ACS. Patients with non–ST-elevation myocardial infarction (NSTEMI) receiving oral DAPT (ASA + clopidogrel, n = 20; or ASA + ticagrelor, n = 20) were prospectively enrolled in a nonrandomized study. Coagulation- and platelet-dependent thrombin generation (TG), measured by means of the calibrated automated thrombogram, were significantly decreased after in vitro and in vivo addition of rivaroxaban. As shown by a total thrombus-formation analysis approach, rivaroxaban treatment led to a significantly decreased coagulation-dependent (AR-chip) thrombus formation in patients treated with ASA plus P2Y 12 inhibitor (clopidogrel/ticagrelor), whereas the pure platelet-dependent (PL-chip) thrombus formation was not affected at all. Adjunctive rivaroxaban therapy was not associated with significant differences in platelet aggregation assessed by light-transmission aggregometry (LTA). Nevertheless, according to fluorescence-activated cell sorter analysis, VLD rivaroxaban treatment resulted in a significantly reduced expression of platelet HMGB-1, whereas P-selectin exposure was not affected. Furthermore, an enhanced effect of rivaroxaban on total thrombus formation and TG was observed in particular in clopidogrel nonresponder patients defined as adenosine 5′-diphosphate-induced LTA ≥40%. VLD rivaroxaban reduces thrombus formation and platelet-dependent TG in patients with ACS receiving DAPT, which can be of potential ischemic benefit. This trial was registered at www.clinicaltrials.gov as #NCT01417884.

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/Aims: Eptifibatide is a parenteral glycoprotein IIb-IIIa inhibitor that prevents platelet aggregation. Although contraindicated in dialysis patients due to limited safety and dialysis data, eptifibatide is prescribed in this population and is associated with bleeding complications. This study was done to determine dialysis clearance (CLD) of eptifibatide using an in vitro system. Methods:Three common dialyzers were tested. In vitro dialysis was performed at a dialysate flow rate of 500 ml/min, ‘blood’ flow rate (QB) of 200 and 400 ml/min, and the minimal ultrafiltration rate. Eptifibatide CLD and fraction removed were calculated for each condition. Results: CLD ranged from 122 to 225 ml/min and was not significantly different among the dialyzers tested. CLD was flow dependent with higher clearances observed at higher QB. The estimated fraction of eptifibatide removed was 73–83%. Conclusions: These data suggest that hemodialysis is an effective method to decrease the effects of eptifibatide in patients with impaired kidney function.

Summary. Background: Utilization of glycoprotein IIb–IIIa (GPIIb–IIIa) inhibitors improves outcomes of patients with acute coronary syndromes (ACS), including those undergoing percutaneous coronary intervention (PCI). These results may be related to the ability of the inhibitors to destabilize coronary thrombi, reduce microembolization, and restore vessel patency. Objective: To evaluate in vitro the ability of GPIIb–IIIa antagonists, abciximab and eptifibatide, to promote the disaggregation of platelet-rich thrombus. Methods: Antagonist-induced disaggregation was assayed in plasma by aggregometry, as well as in whole blood by point of care and capillary perfusion systems. Fibrinogen dissociation from the platelet surface was quantified by flow cytometry. Results: Significant disaggregation of 5 μm ADP-induced aggregates was observed after addition of either agent. The maximum extent and rate of disaggregation were significantly higher with eptifibatide than with abciximab. Both antagonists also dispersed 2 μg mL−1 collagen-induced aggregates, again with eptifibatide having a greater effect. Eptifibatide, but not abciximab (up to 10 μg mL−1), was efficient at dissociating aggregates to single platelets in whole blood and dispersing aggregates that had been aged for 30 min before treatment. Eptifibatide also reduced existing thrombus burden in the perfusion model under arterial flow conditions. A key mechanism of aggregate dispersal was antagonist-induced displacement of platelet-bound fibrinogen, which was greater with eptifibatide, a competitive inhibitor of fibrinogen binding, than with the noncompetitive inhibitor, abciximab. Conclusions: These results suggest that drug concentration and residence time, along with thrombus extent and age, may be critical determinants in promoting timely recanalization.

Summary Background An antithrombotic drug is needed that safely reduces cardiovascular events in patients undergoing percutaneous coronary intervention (PCI). We therefore assessed the tolerability and safety of SCH 530348—an oral platelet protease-activated receptor-1 antagonist. Methods We randomly assigned patients aged 45 years or older and undergoing non-urgent PCI or coronary angiography with planned PCI to an oral loading dose of SCH 530348 (10 mg, 20 mg, or 40 mg) or matching placebo in a 3:1 ratio in a multicentre international study. Those in the SCH 530348 group who subsequently underwent PCI (primary PCI cohort) continued taking an oral maintenance dose (0·5 mg, 1·0 mg, or 2·5 mg per day), and patients in the placebo group continued placebo for 60 days. The primary endpoint was the incidence of clinically significant major or minor bleeding according to the thrombolysis in myocardial infarction (TIMI) scale. Both investigators and patients were unaware of treatment allocation. Analysis was by intention to treat. This study is registered with ClinicalTrials.gov, number NCT00132912. Findings 257 patients were assigned to placebo and 773 to SCH 530348. The primary endpoint occurred in 2 (2%) of 129, 3 (3%) of 120, and 7 (4%) of 173 patients, respectively, in the SCH 530348 10 mg, 20 mg, and 40 mg groups compared with 5 (3%) of 151 patients in the placebo group (p=0·5786). TIMI major plus minor bleeding occurred in 3 (2%) of 136, 5 (4%) of 139, and 4 (3%) of 138 patients given SCH 530348 0·5 mg, 1·0 mg, and 2·5 mg once per day, respectively (p=0·7561). Interpretation Oral SCH 530348 was generally well tolerated and did not cause increased TIMI bleeding, even when administered concomitantly with aspirin and clopidogrel. Further testing in phase III trials to accurately define the safety and efficacy of SCH 530348 is warranted. Funding Schering-Plough Research Institute.

Platelet reactivity (eg, platelet adhesion, activation, aggregation) is the underlying pathology for atherothrombotic processes and subsequent ischemic complications. Antiplatelet drugs, including aspirin, dipyridamole, thienopyridines (clopidogrel and ticlopidine), and glycoprotein IIb/IIIa antagonists, have proven efficacy in atherothrombotic event prevention. However, variability of platelet response measured in the laboratory has been reported and is a subject of keen interest.It is unclear to what extent variability of platelet response to antiplatelet agents is associated with clinical outcomes. A better understanding of this issue requires a general consensus for a standard, preferably point-of-care, ex vivo or in vitro assay to determine the effects of antiplatelet agents on key platelet functions. Currently, results using various methods have not yielded an obvious answer. Small-scale studies have examined the correlation between ex vivo inhibition of platelet aggregation or residual platelet activity and clinical endpoints, and although evidence shows that such correlations may exist, results have not been consistent or definitive. Data from large-scale prospective trials are needed to expand our current understanding of the benefits and limitations of utilizing platelet function tests to effectively manage the balance between protection and risks associated with the antiplatelet therapies, aspirin, and clopidogrel.

Platelets play a pivotal role in atherothrombosis and therefore are primary targets of antithrombotic therapy. They release an array of agonists, such as adenosine diphosphate (ADP); adhesive molecules, such as P-selectin, thrombospondin, fibrinogen, and von Willebrand factor; coagulation factors; and growth factors. In turn, they present transmembrane receptors for a plethora of agonists and ligands. Heterodimeric glycoproteins of the integrin family bind extracellular matrix and plasma proteins; mediate adhesion, activation, spreading, and aggregation; and facilitate intercellular bidirectional signal transduction. Glycoprotein IIb/IIIa is the most abundant platelet integrin and membrane surface glycoprotein. Glycolipids, heparins, proteoglycans, tetraspanins, and a multitude of other molecules, such as tumor necrosis factor-alpha, CD40L, growth arrest-specific 6, Eph receptor tyrosine kinases, and signaling lymphocytic activation molecule receptors, have been implicated in atherothrombosis. ADP promotes platelet aggregation by binding to platelet surface receptors P2Y(1) and P2Y(12); the thienopyridines inhibit aggregation by binding covalently to P2Y(12). Thrombin, a potent initiator of platelet aggregation, activates platelets by cleaving protease-activated receptors (PARs) PAR-1 and PAR-4 and further propagates its effect by activating nearby platelets. A number of pharmacologic agents with antiplatelet actions have been developed, but the search continues for agents that strike an optimal balance between control of thrombosis and serious bleeding.

Antiplatelet agents are the cornerstone of treatment for patients with acute coronary syndromes and patients undergoing percutaneous coronary intervention. The current "gold standard" consists of a combination of aspirin and clopidogrel administered orally shortly before invasive procedures and then continued in the form of maintenance doses. Not all patients respond optimally to standard therapy. Resistance to the antiplatelet activity of both drugs when used either singly or in combination has been observed and may lead to treatment failure, including further atherothrombotic events. Potential limitations associated with the combined use of aspirin and clopidogrel have inspired clinical investigation into several promising new antiplatelet agents as potential additions or alternatives to standard therapy. The candidates include prasugrel, which has a mechanism similar to that of clopidogrel but with superior pharmacokinetics; ticagrelor, a nonthienopyridine that binds reversibly to the platelet P2Y(12) receptor; cangrelor, an intravenously administered analogue of ticagrelor; and various thrombin receptor antagonists.

Platelet activation and aggregation are important pathophysiologic elements of both non-ST-elevation acute coronary syndromes and the ischemic complications of percutaneous coronary intervention, making antiplatelet agents necessary components of the pharmacotherapeutic treatment paradigm for these patients. This review evaluates and interprets the role of oral antiplatelet agents, glycoprotein IIb-IIIa inhibitors, and bivalirudin in the context of current clinical evidence and practice.The current standard of care for patients with non-ST-elevation acute coronary syndromes--aspirin, clopidogrel, and glycoprotein IIb-IIIa inhibitors for the majority of patients--is being challenged by recent clinical trials (Intracoronary Stenting and Antithrombotic Regimen: Rapid Early Action for Coronary Treatment, Intracoronary Stenting and Antithrombotic Regimen: Rapid Early Action for Coronary Treatment 2, Randomized Evaluation of Percutaneous coronary intervention Linking Angiomax to Reduced Clinical Events-2, Acute Catheterization and Urgent Intervention Triage StrategY), raising important questions regarding the value of glycoprotein IIb-IIIa inhibitors as accompaniments of high-dose clopidogrel pretreatment and increased use of the anticoagulant bivalirudin.Current data indicate that antiplatelet regimens consisting of aspirin, clopidogrel, and a glycoprotein IIb-IIIa inhibitor provide substantial benefit among patients who undergo percutaneous coronary intervention. Optimized antiplatelet and anticoagulant therapy--including aspirin, clopidogrel, a glycoprotein IIb-IIIa inhibitor, and an anticoagulant--may reduce the incidence of subclinical and clinical events.

Tetraspanin CD9 regulates cell motility and other adhesive processes in a variety of tissue types. Using transfected Chinese Hamster Ovary cells as our model system, we examined the cellular pathways critical for CD9 promoted cell migration. alpha 5 beta 1 integrin was directly involved as CD9 enhanced migration was abolished by the alpha 5 beta 1 blocking antibody PB1. Furthermore, the ligand mimetic peptide RGDS, significantly upregulated the expression of a beta1 ligand induced binding site (LIBS) demonstrating for the first time that CD9 expression potentiates beta1 integrin high affinity conformation states. CD9 promoted cell motility was significantly blocked by phosphatidylinositol-3 kinase (PI-3K) inhibitors, wortmannin and LY294002, whereas inhibitors targeting protein kinase C or mitogen-activated protein kinase had no effect. PI-3K dominant/negative cDNA transfections confirmed that PI-3K was an essential component. CD9 enhanced the phosphorylation of the PI-3K substrate, Akt, in response to cell adhesion on FN. CD9 expression also upregulated p130Cas phosphorylation and total protein levels; however, p130Cas siRNA knockdown did not alter the motile phenotype. CD9 enhanced migration was also unaffected by serum deprivation suggesting that growth factors were not critical. Our studies demonstrate that CD9 upregulates beta1 LIBS, and in concert with alpha 5 beta 1, enhances cell motility to FN via a PI-3K dependent mechanism.

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.