Séverine Nolli

Biological effects of aspirin and clopidogrel in a randomized cross‐over study in 96 healthy volunteers

Summary.  Background: Some data suggest that biological ‘resistance’ to aspirin or clopidogrel may influence clinical outcome. Objective: The aim of this study was to evaluate the relationship between aspirin and clopidogrel responsiveness in healthy subjects. Methods: Ninety‐six healthy subjects were randomly assigned to receive a 1‐week course of aspirin 100 mg day−1 followed by a 1‐week course of clopidogrel (300 mg on day 1, then 75 mg day−1), or the reverse sequence, separated by a 2‐week wash‐out period. The drug effects were assessed by means of serum TxB2 assay, platelet aggregation tests, and the PFA ‐100® and Ultegra RPFA ‐Verify Now® methods. Results: Only one subject had true aspirin resistance, defined as a serum TxB2 level > 80 pg μL−1 at the end of aspirin administration and confirmed by platelet incubation with aspirin. PFA‐100® values were normal in 29% of the subjects after aspirin intake, despite a drastic reduction in TxB2 production; these subjects were considered to have aspirin pseudo‐resistance. Clopidogrel responsiveness was not related to aspirin pseudo‐resistance. Selected polymorphisms of platelet receptor genes were not associated with either aspirin or clopidogrel responsiveness. Conclusions: In healthy subjects, true aspirin resistance is rare and aspirin pseudo‐resistance is not related to clopidogrel responsiveness.

Antiplatelet Drug Response Status Does Not Predict Recurrent Ischemic Events in Stable Cardiovascular Patients Results of the Antiplatelet Drug Resistances and Ischemic Events Study

Background— The biological response to antiplatelet drugs has repeatedly been shown to predict the recurrence of major adverse cardiovascular events (MACEs). However, most studies involved coronary artery disease patients with recent vessel injury shortly after the initiation of antiplatelet therapy. Data on stable cardiovascular patients are scarce, and the added predictive value of specific assays (the vasodilator phosphoprotein assay for the clopidogrel response and serum thromboxane B2 for the aspirin response) and aggregation-based assays relative to common predictors has rarely been addressed. Methods and Results— Stable cardiovascular outpatients participating in the Antiplatelet Drug Resistances and Ischemic Events (ADRIE) study (n=771) were tested twice, at 2 separate visits, with specific and aggregation-based assays. Follow-up lasted 3 years, and <1% of patients were lost to follow-up. MACEs were adjudicated by an independent committee. Multivariate survival analyses included relevant variables identified in univariate analysis and platelet function test results. The C-index was used to express the prognostic value of various multivariate models. MACEs, the primary end point, occurred in 16% of patients. Hypertension, smoking, older age, and elevated low-density lipoprotein cholesterol were predictive of MACE recurrence, with a C-index of 0.63 (P<0.001). Neither the specific nor the aggregation-based assays added significant predictive value for the primary end point. Conclusions— Biological antiplatelet drug responsiveness, measured with specific or aggregation-based assays, has no incremental predictive value over common cardiovascular risk factors for MACE recurrence in stable cardiovascular outpatients. These results do not support platelet function testing for MACE risk evaluation in stable cardiovascular patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00501423.

Antiplatelet Drug Response Status Does Not Predict Recurrent Ischemic Events in Stable Cardiovascular Patients: Results of the ADRIE Study

Background— The biological response to antiplatelet drugs has repeatedly been shown to predict the recurrence of major adverse cardiovascular events (MACEs). However, most studies involved coronary artery disease patients with recent vessel injury shortly after the initiation of antiplatelet therapy. Data on stable cardiovascular patients are scarce, and the added predictive value of specific assays (the vasodilator phosphoprotein assay for the clopidogrel response and serum thromboxane B2 for the aspirin response) and aggregation-based assays relative to common predictors has rarely been addressed. Methods and Results— Stable cardiovascular outpatients participating in the Antiplatelet Drug Resistances and Ischemic Events (ADRIE) study (n=771) were tested twice, at 2 separate visits, with specific and aggregation-based assays. Follow-up lasted 3 years, and <1% of patients were lost to follow-up. MACEs were adjudicated by an independent committee. Multivariate survival analyses included relevant variables identified in univariate analysis and platelet function test results. The C-index was used to express the prognostic value of various multivariate models. MACEs, the primary end point, occurred in 16% of patients. Hypertension, smoking, older age, and elevated low-density lipoprotein cholesterol were predictive of MACE recurrence, with a C-index of 0.63 (P<0.001). Neither the specific nor the aggregation-based assays added significant predictive value for the primary end point. Conclusions— Biological antiplatelet drug responsiveness, measured with specific or aggregation-based assays, has no incremental predictive value over common cardiovascular risk factors for MACE recurrence in stable cardiovascular outpatients. These results do not support platelet function testing for MACE risk evaluation in stable cardiovascular patients. Clinical Trial Registration— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00501423.

Connexin 37 Limits Thrombus Propensity by Downregulating Platelet Reactivity

Background— Formation of platelet plug initiates hemostasis after vascular injury and triggers thrombosis in ischemic disease. However, the mechanisms leading to the formation of a stable thrombus are poorly understood. Connexins comprise a family of proteins that form gap junctions enabling intercellular coordination of tissue activity, a process termed gap junctional intercellular communication. Methods and Results— In the present study, we show that megakaryocytes and platelets express connexin 37 (Cx37). Deletion of the Cx37 gene in mice shortens bleeding time and increases thrombus propensity. Aggregation is increased in murine Cx37−/− platelets or in murine Cx37+/+ and human platelets treated with gap junction blockers. Intracellular microinjection of neurobiotin, a Cx37-permeant tracer, revealed gap junctional intercellular communication in platelet aggregates, which was impaired in Cx37−/− platelets and in human platelets exposed to gap junction blockers. Finally, healthy subjects homozygous for Cx37–1019C, a prognostic marker for atherosclerosis, display increased platelet responses compared with subjects carrying the Cx37–1019T allele. Expression of these polymorphic channels in communication-deficient cells revealed a decreased permeability of Cx37–1019C channels for neurobiotin. Conclusions— We propose that the establishment of gap junctional communication between Cx37-expressing platelets provides a mechanism to limit thrombus propensity. To our knowledge, these data provide the first evidence incriminating gap junctions in the pathogenesis of thrombosis.

Characterisation of the influences of aspirin-acetylation and glycation on human plasma proteins.

UNLABELLED The competition effect between aspirin-mediated acetylation and protein glycation has been a matter of concern for decades. However, the exact interactions between these two post-translational modifications are still not well understood. Several efforts have been made to explain how aspirin prevents glycation, but the influence of prior protein glycation on the action of aspirin has never been investigated. This study involved qualitative and quantitative analyses to: 1) identify acetylated and glycated proteins; 2) quantify rates of acetylation and glycation; and 3) elucidate the common modification sites. Human plasma was incubated with 30mM glucose and then 500μM aspirin. A label-free mass spectrometry approach indicated an increase in the acetylation level after this sequential glucose-then-aspirin incubation; these results were also confirmed by Western blot. Interestingly, for several proteins, decreases in glycation levels were evidenced after aspirin incubation. The common modification sites, where both acetylation and glycation took place, were also identified. The influence that glycation and acetylation processes have on each other could reflect conformational changes induced by glucose and aspirin. In future studies, in order to better understand the interactions between these two PTMs, we intend to apply this strategy to other blood compartments and to diabetic patients. BIOLOGICAL SIGNIFICANCE Non-enzymatic glycation represents an early stage in the development of the long-lasting complications that are associated with diabetes. Aspirin has been shown to prevent this process in a few reference proteins, but how the two post-translational modifications (PTMs) of aspirin-mediated acetylation and protein glycation interact with each other remains poorly investigated. This study used a label-free quantitative proteomic approach to characterise the extent of aspirin-induced acetylation and protein glycation in human plasma. The results clearly supported a mutual influence between these PTMs, which lead us to propose a potential model based on structural conformational changes.

Turbidimetry on Human Washed Platelets: The Effect of the Pannexin1-inhibitor Brilliant Blue FCF on Collagen-induced Aggregation

Turbidimetry is a laboratory technique that is applied to measure the aggregation of platelets suspended in either plasma (platelet-rich plasma, PRP) or in buffer (washed platelets), by the use of one or a combination of agonists. The use of washed platelets separated from their plasma environment and in the absence of anticoagulants allows for studying intrinsic platelet properties. Among the large panel of agonists, arachidonic acid (AA), adenosine di-phosphate (ADP), thrombin and collagen are the most frequently used. The aggregation response is quantified by measuring the relative optical density (OD) over time of platelet suspension under continuous stirring. Platelets in homogeneous suspension limit the passage of light after the addition of an agonist, platelet shape change occurs producing a small transitory increase in OD. Following this initial activation step, platelet clots form gradually, allowing the passage of light through the suspension as a result of decreased OD. The aggregation process is ultimately expressed as a percentage, compared to the OD of platelet-poor plasma or buffer. Rigorous calibration is thus essential at the beginning of each experiment. As a general rule: calibration to 0% is set by measuring the OD of a non-stimulated platelet suspension while measuring the OD of the suspension medium containing no platelets represents a value of 100%. Platelet aggregation is generally visualized as a real-time aggregation curve. Turbidimetry is one of the most commonly used laboratory techniques for the investigation of platelet function and is considered as the historical gold standard and used for the development of new pharmaceutical agents aimed at inhibiting platelet aggregation. Here, we describe detailed protocols for 1) preparation of human washed platelets and 2) turbidimetric analysis of collagen-induced aggregation of human washed platelets pretreated with the food dye Brilliant Blue FCF that was recently identified as an inhibitor of Pannexin1 (Panx1) channels.