Comparison of the effects of the GPIIb-IIIa antagonist Zalunfiban and the P2Y12 antagonist Selatogrel on Platelet Aggregation.

Understanding the pharmacodynamic effects of platelet inhibitors is standard for developing more effective antithrombotic therapies. An example is the antithrombotic treatment of acute coronary syndrome (ACS), in particular ST-elevated myocardial infarction (STEMI) patients who are in need for rapid acting strong antithrombotic therapy despite the use of aspirin and oral P2Y12-inhibitors. In this study, we evaluated two injectable platelet inhibitors under clinical development (the P2Y12 antagonist selatogrel and the GPIIb-IIIa antagonist zalunfiban) that may be amenable to pre-hospital treatment of STEMI patients. Platelet reactivity was assessed at inhibitor concentrations that represent clinically relevant levels of platelet inhibition (IC20-50%, 1/2Cmax, and Cmax). Light transmission aggregometry (LTA), was used to evaluate the initial rate of aggregation (primary slope, PS) and maximal aggregation (MA). Both adenosine diphosphate (ADP) and thrombin receptor agonist peptide (TRAP) were used as agonists. Zalunfiban demonstrated similar inhibition of platelet aggregation when blood was collected in PPACK or TSC, whereas selatogrel demonstrated greater inhibition in PPACK. In this study, using PPACK anticoagulant, selatogrel and zalunfiban affected PS in response to ADP equivalently at all drug concentrations tested. In contrast, zalunfiban had significantly greater potency at its Cmax concentration compared to selatogrel using TRAP as agonist. Upon evaluation of MA responses at lower doses, selatogrel had greater inhibition of MA in response to ADP than zalunfiban; however, at concentrations that represent Cmax, the drugs were equivalent. Zalunfiban also had greater inhibition of MA in response to TRAP at the Cmax dose. These data suggest that zalunfiban may provide greater protection in reducing thrombus formation than selatogrel, especially since thrombin is an early, key primary agonist in the pathophysiology of thrombotic events.

Antibody-Based Ticagrelor Reversal Agent in Healthy Volunteers.

BACKGROUND: Ticagrelor is an oral P2Y12 inhibitor that is used with aspirin to reduce the risk of ischemic events among patients with acute coronary syndromes or previous myocardial infarction. Spontaneous major bleeding and bleeding associated with urgent invasive procedures are concerns with ticagrelor, as with other antiplatelet drugs. The antiplatelet effects of ticagrelor cannot be reversed with platelet transfusion. A rapid-acting reversal agent would be useful. METHODS: In 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 P2Y12 platelet-reactivity test, and a vasodilator-stimulated phosphoprotein assay. RESULTS: Of 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 (P<0.001 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. CONCLUSIONS: 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.).

Effects of genetic variation in protease activated receptor 4 after an acute coronary syndrome: Analysis from the TRACER trial.

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 utility of tetraspanin CD9 as a biomarker for metastatic clear cell renal cell carcinoma.

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.

PAR1 antagonists inhibit thrombin-induced platelet activation whilst leaving the PAR4-mediated response intact.

Thrombin-induced platelet activation is initiated by PAR1 and PAR4 receptors. Vorapaxar, a PAR1 antagonist, has been assessed in patients with acute coronary syndromes (ACS) and stable atherosclerotic disease in addition to standard-of-care treatment. In clinical trials, vorapaxar has been observed to reduce the frequency of ischaemic events in some subgroups though in others has increased the frequency of bleeding events. Among patients undergoing CABG surgery, which is associated with excess thrombin generation, bleeding was not increased. The aim of these studies was to investigate the effects of selective PAR1 antagonism on thrombin-induced platelet activation in patients receiving vorapaxar or placebo in the TRACER trial and to explore the roles of PAR1 and PAR4 in thrombin-induced platelet activation in healthy volunteers. ACS patients receiving vorapaxar or placebo in the TRACER trial were studied at baseline and 4 hours, 1 and 4 months during drug administration. Thrombin-induced calcium mobilisation in platelet-rich plasma was assessed by flow cytometry. In vitro studies were performed in healthy volunteers using the PAR1 antagonist SCH79797 or PAR4 receptor desensitisation. Vorapaxar treatment significantly inhibited thrombin-induced calcium mobilisation, leaving a residual, delayed response. These findings were consistent with calcium mobilisation mediated via the PAR4 receptor and were reproduced in vitro using SCH79797. PAR4 receptor desensitization, in combination with SCH79797, completely inhibited thrombin-induced calcium mobilisation confirming that the residual calcium mobilisation was mediated via PAR4. In conclusion vorapaxar selectively antagonises the PAR1-mediated component of thrombin-induced platelet activation, leaving the PAR4-mediated response intact, which may explain why vorapaxar is well tolerated in patients undergoing CABG surgery since higher thrombin levels in this setting may override the effects of PAR1 antagonism through PAR4 activation, thus preserving haemostasis. Further assessment may be warranted.

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity.

Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in two human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9-Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.

Vorapaxar with or without clopidogrel after non-ST-segment elevation acute coronary syndromes: results from the thrombin receptor antagonist for clinical event reduction in acute coronary syndrome trial.

BACKGROUND: Protease-activated receptor 1 antagonism with vorapaxar represents a novel strategy for platelet inhibition. In TRACER, vorapaxar was compared with placebo plus standard of care among 12,944 patients with non-ST-segment elevation acute coronary syndromes. We anticipated that most patients would have received clopidogrel as part of standard care. We investigated the modification of vorapaxar's effect associated with clopidogrel use over time. METHODS: The marginal structural model method was used to estimate causal modification of vorapaxar effect by use of clopidogrel over time. The primary outcomes were the composite of cardiovascular death, myocardial infarction, or stroke and Global Use of Strategies to Open Occluded Coronary Arteries moderate or severe bleeding. The event accrual period excluded the time during which clopidogrel was clinically warranted. RESULTS: Among 12,887 patients who received study medication, 11,117 (86.3%) received clopidogrel before randomization, of whom 38.5% stopped later in the trial (median time to stoppage 200 days with placebo; interquartile range [IQR] 14-367) (186 days with vorapaxar; IQR 17-366). In total, 1,770 (13.7%) patients were not on clopidogrel at randomization, of whom 47.8% started afterward (median time to start 2 days; IQR 2-4). During the period of event accrual, vorapaxar was associated with a 26% reduction in the composite of cardiovascular death, myocardial infarction, or stroke when used with clopidogrel (hazard ratio [HR] 0.74; 95% CI 0.60-0.91) and a 24% reduction when used without clopidogrel (HR 0.76; 95% CI 0.56-1.02) (interaction; P = .89). The hazard of Global Use of Strategies to Open Occluded Coronary Arteries bleeding with vorapaxar was not significantly different without clopidogrel (HR 1.33; 95% CI 0.81-2.20) or with clopidogrel (HR 1.09; 95% CI 0.76-1.56) (interaction; P = .53). CONCLUSIONS: We observed no interaction between vorapaxar and clopidogrel after non-ST-segment elevation acute coronary syndromes on efficacy or safety outcomes, supporting a complementary role of protease-activated receptor 1 and P2Y12 antagonism.

Signaling via P2Y12 may be critical for early stabilization of platelet aggregates.

P2Y(12) receptor antagonism inhibits platelet aggregation by preventing adenosine diphosphate (ADP)-mediated amplification of activation pathways downstream of primary agonists, such as thrombin and collagen. However, the role of ADP signaling in maintaining aggregate stability and the effects of P2Y(12) antagonists on preestablished aggregates in vitro and arterial thrombus in vivo are not well understood. This study evaluated the impact of P2Y(12) signaling on platelet aggregate stability and early thrombotic occlusion using a reversible P2Y(12) antagonist, ticagrelor. There were 2 study objectives: (1) to determine if there was a time-dependent factor on the capacity of a P2Y(12) antagonist to affect human platelet aggregate stability in vitro using light transmission aggregometry and (2) to evaluate the extent of arterial thrombus reversal in a preclinical model upon administration of ticagrelor in vivo. Platelet aggregates were exposed to ticagrelor after ADP or collagen activation, monitored for stability by aggregometry, and visualized by microscopy. Freshly formed ADP- and collagen-induced platelet aggregates were more rapidly dispersed by a P2Y(12) antagonist than drug carrier control at clinically relevant concentrations (P < 0.05). However, stable aggregates were not noticeably affected. A murine arterial thrombosis model was used to evaluate thrombus stability in an in vivo mouse model. Thrombotic occlusion was induced by FeCl(3), followed by a bolus intravenous administration of ticagrelor or vehicle control. Doppler blood flow was monitored before injury and 30 minutes after bolus administration. Arteries were retrieved for inspection for residual thrombus. Early arterial thrombotic occlusion in vivo was partially reversed by ticagrelor administration. Blood flow through the injured artery increased, and thrombus size within the artery decreased (P < 0.05, n = 3). In conclusion, P2Y(12) antagonism disrupts the stability of newly formed platelet aggregates, promoting disaggregation, and reverses thrombotic vascular occlusion. Thus, in addition to activating platelets, signaling via P2Y(12) seems to be required for stabilizing platelet thrombi.

Self-compassion and life satisfaction in gay men.

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.

Switching from Dual Antiplatelet Therapy with Aspirin Plus a P2Y12 Inhibitor to Dual Pathway Inhibition with Aspirin Plus Vascular-Dose Rivaroxaban: The Switching Anti-Platelet and Anti-Coagulant Therapy (SWAP-AC) Study.

BACKGROUND: To date, there are 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 (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 (TF), and a combination of collagen, ADP, and TF (maximum platelet aggregation %); thrombin generation (TG). Assays were performed at baseline and 30 days postrandomization. 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. CLINICAL TRIAL REGISTRATION: http://www. CLINICALTRIALS: gov Unique Identifier: NCT04006288.

Pro-MMP-9 upregulation in HT1080 cells expressing CD9 is regulated by epidermal growth factor receptor.

Degradation of the surrounding extracellular matrix (ECM) by matrix metalloproteinases (MMPs) drives invasion and metastasis of cancer cells. We previously demonstrated that tetraspanin CD9 expression upregulates pro-MMP-9 expression and release and promotes cellular invasion in a human fibrosarcoma cell line (HT1080). These events were dependent upon the highly functional second extracellular loop of CD9. We report here that the epidermal growth factor receptor (EGFR) tyrosine kinase expression and activity are involved in the CD9-mediated increase in pro-MMP-9 release and cellular invasion. Pro-MMP-9 expression was significantly decreased in a dose-dependent manner using first a broad spectrum receptor tyrosine kinase inhibitor and multiple specific EGFR inhibitors in CD9-HT1080 cells. Furthermore, gefitinib treatment of CD9-HT1080 cells reduced invasion through matrigel. EGFR knockdown using short interfering RNA resulted in decreased pro-MMP-9 expression and release into the media and subsequent cellular invasion without affecting CD9 expression or localization. Conclusively, this study points to EGFR as a key mediator between CD9-mediated pro-MMP-9 release and cellular invasion of HT1080 cells.

Elevating local concentrations of GPIIb-IIIa antagonists counteracts platelet thrombus stability.

Glycoprotein IIb-IIIa (GPIIb-IIIa) antagonists have the capacity to destabilize coronary thrombi and restore vessel patency. Antagonist concentration and residence time, which can be increased by local intracoronary (LIC) administration, and thrombus age may be key factors that influence thrombus stability. Light transmission aggregometry was used to examine the effects of exposing human platelet aggregates to extremely high local levels of GPIIb-IIIa antagonists versus conventional therapeutic levels in vitro. Freshly-formed or aged platelet aggregates were subjected to GPIIb-IIIa antagonists (abciximab, eptifibatide) or direct thrombin inhibitor bivalirudin at concentrations simulating either conventional intravenous (IV) or LIC administration. The degree of antagonist-induced disaggregation was significantly higher using elevated (LIC) doses versus conventional (IV) doses (60.1 % vs. 7.4 % for abciximab, 41.6 % or 45.3 % vs. 17.6 % for eptifibatide, p < 0.01). Bivalirudin did not promote disaggregation. Microscopy confirmed noticeably smaller, more dispersed aggregates for antagonist LIC treatments. Dosing at LIC levels also induced more disaggregation than IV levels when aggregates were aged for 30 min prior to exposure. An in vitro perfusion model was used to simulate the fluid dynamics of IV or LIC administration of abciximab using a microporous local drug delivery balloon catheter such as the Atrium ClearWay™ RX. The perfusion model resulted in more rapid thrombus clearance with LIC dosing levels compared to IV. In summary, boosting the concentration of GPIIb-IIIa antagonists enhances dispersal of human platelet aggregates in vitro. These data provide a foundation for investigating increased local concentrations of GPIIb-IIIa antagonists in patients, as with LIC administration.

The Utility of Platelet and Coagulation Testing of Antithrombotics: Fusing Science with Patient Care.

[Table: see text] 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.

No differences in the pharmacodynamics and pharmacokinetics of the thrombin receptor antagonist vorapaxar between healthy Japanese and Caucasian subjects.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSION: There were no substantial differences in the safety, pharmacokinetics or pharmacodynamics of vorapaxar between Japanese and Caucasian subjects.

Pharmacokinetics and pharmacodynamics of the novel PAR-1 antagonist vorapaxar in patients with end-stage renal disease.

PURPOSE: To determine whether impaired renal function alters the pharmacokinetics (PK) of vorapaxar or its ability to inhibit thrombin receptor agonist peptide (TRAP)-induced platelet aggregation. METHODS: This was an open-label study in which 8 patients with end-stage renal disease (ESRD) on hemodialysis and 7 matched (based on age, gender, weight, and height) healthy controls were administered a single 10-mg oral dose of vorapaxar. Blood samples for vorapaxar PK and pharmacodynamic analysis were collected predose and at frequent intervals up to 6 weeks postdose. RESULTS: Mean vorapaxar bioavailability (based on area under the curve of plasma vorapaxar concentration over time) was identical in the two subject groups; the ESRD/healthy geometric mean ratio (GMR, expressed in percent) was 98. Mean maximum observed plasma concentration (77.4-98.2 ng/mL) was numerically lower in patients with ESRD compared with matched controls (GMR=76; 90% confidence interval=48 to 118). Median time of maximum observed plasma concentration was 2 h in both subject groups. The observed means for elimination half-life were 186 and 231 h in the ESRD and control groups, respectively. Inhibition of platelet aggregation was similar in the two groups. Four out of 15 (27%) subjects reported adverse events, all of which were characterized by the investigator as mild and unrelated to treatment. CONCLUSIONS: ESRD had no clinically relevant effect on the PK profile of vorapaxar or its ability to inhibit TRAP-induced platelet aggregation.

Pharmacodynamic effects of clopidogrel in pediatric cardiac patients: a comparative study of platelet aggregation response.

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 <10% IPA. In conclusion, ADP- and TRAP-induced %PA is lower in pediatric cardiac patients than normal adults, but highly variable in both. The lower baseline %PA may explain why the pediatric clopidogrel dose providing 30-50% IPA (0.20 mg/kg/day) is lower than a simple weight-based extrapolation of the adult dose (75 mg/day) providing similar inhibition.

Hematoma-inspired alginate/platelet releasate/CaPO4 composite: initiation of the inflammatory-mediated response associated with fracture repair in vitro and ex vivo injection delivery.

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/ß-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.

Pharmacodynamic Profiles of Dual-Pathway Inhibition with or without Clopidogrel versus Dual Antiplatelet Therapy in Patients with Atherosclerotic Disease.

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.

Platelet P2Y12 inhibiting therapy in adjunct to vascular dose of rivaroxaban or aspirin: a pharmacodynamic study of dual pathway inhibition vs. dual antiplatelet therapy.

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.

Bentracimab for Ticagrelor Reversal in Patients Undergoing Urgent Surgery.

BACKGROUND: Ticagrelor is a reversible oral P2Y12 platelet inhibitor used to treat patients with acute coronary syndromes, prior myocardial infarction, high-risk coronary artery disease, transient ischemic attack, or ischemic stroke. A healthy volunteer study showed that the intravenous monoclonal antibody bentracimab rapidly reverses ticagrelor, but the effect in patients was unknown. METHODS: In a prespecified interim analysis of a single-arm, prospective study, bentracimab was evaluated in ticagrelor-treated patients who required urgent surgery or had major hemorrhage. The extent of reversal was determined using the VerifyNow P2Y12 assay. Clinical hemostasis was assessed by central adjudication using validated criteria. Treatment-emergent safety events were evaluated. The trial is ongoing and will enroll approximately 200 patients with evaluable data. RESULTS: Of 150 enrolled patients, 142 required urgent surgery and 8 had major hemorrhage. For the end-point analysis, 129 patients had analyzable platelet data; 122 had data on adjudicated hemostasis. Bentracimab provided a rapid reversal of ticagrelor's antiplatelet effects within 5 to 10 minutes. The reversal was sustained for more than 24 hours, as measured with the VerifyNow P2Y12 and vasodilator-stimulated phosphoprotein phosphorylation assays (P<0.001, with both assays and in all subgroups). Adjudicated hemostasis was achieved for more than 90% of patients (P<0.001); approximately 5% of patients had thrombotic events. No allergic or infusion-related reactions were reported. CONCLUSIONS: Bentracimab provided immediate and sustained reversal of the antiplatelet effects of ticagrelor in patients undergoing surgical procedures. (Funded by PhaseBio Pharmaceuticals, Inc.; ClinicalTrials.gov number, NCT04286438.)