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Mahaffey K.W.,Stanford University | Huang Z.,Duke Clinical Research Institute | Wallentin L.,Uppsala Clinical Research Center | Storey R.F.,University of Sheffield | And 14 more authors.
American Journal of Cardiology | Year: 2014

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. © 2014 Elsevier Inc. All rights reserved.


Storey R.F.,University of Sheffield | Kotha J.,CirQuest Labs LLC | Smyth S.S.,University of Kentucky | Moliterno D.J.,University of Kentucky | And 18 more authors.
Thrombosis and Haemostasis | Year: 2014

Vorapaxar is an antagonist of the protease activated receptor-1 (PAR-1), the principal platelet thrombin receptor. The Thrombin Receptor Antagonist for Clinical Event Reduction (TRACER) trial evaluated vorapaxar compared to placebo in non-ST-elevation (NSTE)-acute coronary syndrome (ACS) patients. It was the study's objective to assess the pharmacodynamic effects of vorapaxar versus placebo that included aspirin or a thienopyridine or, frequently, a combination of both agents in NSTE-ACS patients. In a substudy involving 249 patients, platelet aggregation was assessed by light transmittance aggregometry (LTA) in 85 subjects (41 placebo, 44 vorapaxar) using the agonists thrombin receptor activating peptide (TRAP, 15 μM), adenosine diphosphate (ADP, 20 μM), and the combination of collagen-related peptide (2.5 μg/ml) + ADP (5 μM) + TRAP (15 μM) (CAT). VerifyNow® IIb/IIIa and vasodilator-stimulated phosphoprotein (VASP) phosphorylation assays were performed, and platelet PAR-1 expression, plasma platelet/endothelial and inflammatory biomarkers were determined before and during treatment. LTA responses to TRAP and CAT and VerifyNow results were markedly inhibited by vorapaxar. Maximal LTA response to TRAP (median, interquartile range) 2 hours post loading dose: placebo 68% (53-75%) and vorapaxar 3% (2-6%), p<0.0001. ADP inhibition was greater in the vorapaxar group at 4 hours and one month (p<0.01). In contrast to the placebo group, PAR-1 receptor number in the vorapaxar group at one month was significantly lower than the baseline (179 vs 225; p=0.004). There were significant changes in selected biomarker levels between the two treatment groups. In conclusion, vorapaxar caused a potent inhibition of PAR-1-mediated platelet aggregation. Further studies are needed to explore vorapaxar effect on P2Y12 inhibition, PAR-1 expression and biomarkers and its contribution to clinical outcomes. © Schattauer 2014.


Judge H.M.,University of Sheffield | Jennings L.K.,University of Tennessee Health Science Center | Jennings L.K.,CirQuest Labs LLC | Moliterno D.J.,University of Kentucky | And 6 more authors.
Platelets | Year: 2015

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. © 2014 Informa UK Ltd.


Herr M.J.,University of Tennessee Health Science Center | Kotha J.,CirQuest Labs LLC | Hagedorn N.,University of Tennessee Health Science Center | Smith B.,University of Tennessee Health Science Center | And 2 more authors.
PLoS ONE | Year: 2013

Tumor cell metastasis, a process which increases the morbidity and mortality of cancer patients, is highly dependent upon matrix metalloproteinase (MMP) production. Small molecule inhibitors of MMPs have proven unsuccessful at reducing tumor cell invasion in vivo. Therefore, finding an alternative approach to regulate MMP is an important endeavor. Tetraspanins, a family of cell surface organizers, play a major role in cell signaling events and have been implicated in regulating metastasis in numerous cancer cell lines. We stably expressed tetraspanin CD9 in an invasive and metastatic human fibrosarcoma cell line (CD9-HT1080) to investigate its role in regulating tumor cell invasiveness. CD9-HT1080 cells displayed a highly invasive phenotype as demonstrated by matrigel invasion assays. Statistically significant increases in MMP-9 production and activity were attributed to CD9 expression and were not due to any changes in other key tetraspanin complex members or MMP regulators. Increased invasion of CD9-HT1080 cells was reversed upon silencing of MMP-9 using a MMP-9 specific siRNA. Furthermore, we determined that the second extracellular loop of CD9 was responsible for the upregulation of MMP-9 production and subsequent cell invasion. We demonstrated for the first time that tetraspanin CD9 controls HT1080 cell invasion via upregulation of an integral member of the MMP family, MMP-9. Collectively, our studies provide mounting evidence that altered expression of CD9 may be a novel approach to regulate tumor cell progression. © 2013 Herr et al.


Herr M.J.,University of Tennessee Health Science Center | Herr M.J.,CirQuest Labs. LLC | Mabry S.E.,University of Tennessee Health Science Center | Jennings L.K.,University of Tennessee Health Science Center | Jennings L.K.,CirQuest Labs. LLC
PLoS ONE | Year: 2014

The most prevalent cardiovascular diseases arise from alterations in vascular smooth muscle cell (VSMC) morphology and function. Tetraspanin CD9 has been previously implicated in regulating vascular pathologies; however, insight into how CD9 may regulate adverse VSMC phenotypes has not been provided. We utilized a human model of aortic smooth muscle cells to understand the consequences of CD9 deficiency on VSMC phenotypes. Upon knocking down CD9, the cells developed an abnormally small and rounded morphology. We determined that this morphological change was due to a lack of typical parallel actin arrangement. We also found similar total RhoA but decreased GTP-bound (active) RhoA levels in CD9 deficient cells. As a result, cells lacking a full complement of CD9 were less contractile than their control treated counterparts. Upon restoration of RhoA activity in the CD9 deficient cells, the phenotype was reversed and cell contraction was restored. Conversely, inhibition of RhoA activity in the control cells mimicked the CD9-deficient cell phenotype. Thus, alteration in CD9 expression was sufficient to profoundly disrupt cellular actin arrangement and endogenous cell contraction by interfering with RhoA signaling. This study provides insight into how CD9 may regulate previously described vascular smooth muscle cell pathophysiology. © 2014 Herr et al.


Garner J.M.,University of Tennessee Health Science Center | Herr M.J.,University of Tennessee Health Science Center | Herr M.J.,CirQuest Labs LLC | Hodges K.B.,University of Houston | And 2 more authors.
Biochemical and Biophysical Research Communications | Year: 2016

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. © 2016 Elsevier Inc. All rights reserved.


Speich H.E.,University of Tennessee Health Science Center | Bhal V.,CirQuest Labs LLC | Houser K.H.,University of Tennessee Health Science Center | Caughran A.T.,University of Tennessee Health Science Center | And 6 more authors.
Journal of Cardiovascular Pharmacology | Year: 2014

P2Y12 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 P2Y12 antagonists on preestablished aggregates in vitro and arterial thrombus in vivo are not well understood. This study evaluated the impact of P2Y12 signaling on platelet aggregate stability and early thrombotic occlusion using a reversible P2Y12 antagonist, ticagrelor. There were 2 study objectives: (1) to determine if there was a time-dependent factor on the capacity of a P2Y12 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 P2Y12 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 FeCl3, 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, P2Y12 antagonism disrupts the stability of newly formed platelet aggregates, promoting disaggregation, and reverses thrombotic vascular occlusion. Thus, in addition to activating platelets, signaling via P2Y12 seems to be required for stabilizing platelet thrombi. Copyright © 2014 by Lippincott Williams & Wilkins.

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