Pierre Sié

Antiphosphatidylethanolamine antibodies are associated with an increased odds ratio for thrombosis - A multicenter study with the participation of the European Forum on antiphospholipid antibodies

A multicenter study was set up to evaluate the prevalence, clinical and biological significance of antiphosphatidylethanolamine antibodies (aPE) in thrombotic patients with or without the main known clinical and biological risk factors for thrombosis. APE and antibodies, defined as the laboratory criteria of antiphospholipid syndrome (APS) -lupus anticoagulant, anticardiolipin and anti-beta(2)-GPI antibodies were measured in 270 patients with thrombosis (234 venous and 37 arterial) and 236 matched controls. APE were found in 15% of thrombotic patients compared to 3% of controls (p < 0.001) with no predominant isotype, no association with the main known clinical or biological risk factors for thrombosis neither with a type of thrombosis, arterial or venous. In a multivariate logistic regression analysis of antibodies, aPE showed the highest association with thrombosis (odds ratio [OR]: 4.2, p < 0.001). Moreover, using a multivariate analysis in a case-control subgroup study on 158 patients, IgGaPE were found to be significantly associated with venous thrombosis (OR:6;p = 0.005). Interestingly, 25 of the 40 aPE-positive patients (63%) were negative for the APS laboratory criteria. Most of them (21/25) had venous thrombosis, recurrent in ten of them. Four patients also suffered from early or late miscarriages. Our results underline the strength of the association between the presence of aPE and thrombosis and suggest their measurement in thrombotic patients, especially when lupus anticoagulant, anticardiolipin or anti-beta(2)-GPI antibodies are absent.

Internalization of microparticles by endothelial cells promotes platelet/endothelial cell interaction under flow

Summary.u2002 Background: Microparticles (MPs) released by activated or apoptotic cells increase in number in the blood of subjects with vascular or metabolic diseases and may contribute to thrombotic complications. Objectives: In this study, we investigated whether MPs promoted platelet recruitment to endothelial cells in flow conditions, and by which mechanism. Methods: Human umbilical vein endothelial cells (HUVECs) grown in microslide perfusion chambers were exposed to MPs prepared in vitro from HUVECs, monocytes or platelets. Results: Videomicroscopy of DIOC‐labelled blood perfused at arterial rate on human umbilical vein ECs demonstrated that, irrespective of their cell origin, MPs promoted the formation of platelet strings at the surface of HUVECs. This platelet/endothelial cell interaction was dependent on von Willebrand factor (VWF) expression at the HUVEC surface and involved Glycoprotein Ib and P‐selectin. Interestingly, HUVECs internalized MPs within a few hours through a process involving anionic phospholipids, lactadherin and αvβ3 integrin. This uptake generated the production of reactive oxygen species via the xanthine/xanthine oxidase system (inhibited by allopurinol and the ROCK inhibitor Y‐27632) and the NADPH oxidase (inhibited by SOD). Reactive oxygen species appeared essential for VWF expression at the endothelial cell surface and subsequent platelet/endothelial cell interaction under flow. The pathophysiological relevance of this process is underlined by the fact that circulating MPs from Type I diabetic patients induced platelet/endothelial cell interaction under flow, with an intensity correlated with the severity of the vasculopathy.

Expanding the Mutation Spectrum Affecting αIIbβ3 Integrin in Glanzmann Thrombasthenia: Screening of the ITGA2B and ITGB3 Genes in a Large International Cohort.

We report the largest international study on Glanzmann thrombasthenia (GT), an inherited bleeding disorder where defects of the ITGA2B and ITGB3 genes cause quantitative or qualitative defects of the αIIbβ3 integrin, a key mediator of platelet aggregation. Sequencing of the coding regions and splice sites of both genes in members of 76 affected families identified 78 genetic variants (55 novel) suspected to cause GT. Four large deletions or duplications were found by quantitative real‐time PCR. Families with mutations in either gene were indistinguishable in terms of bleeding severity that varied even among siblings. Families were grouped into type I and the rarer type II or variant forms with residual αIIbβ3 expression. Variant forms helped identify genes encoding proteins mediating integrin activation. Splicing defects and stop codons were common for both ITGA2B and ITGB3 and essentially led to a reduced or absent αIIbβ3 expression; included was a heterozygous c.1440‐13_c.1440‐1del in intron 14 of ITGA2B causing exon skipping in seven unrelated families. Molecular modeling revealed how many missense mutations induced subtle changes in αIIb and β3 domain structure across both subunits, thereby interfering with integrin maturation and/or function. Our study extends knowledge of GT and the pathophysiology of an integrin.

Are P2Y12 reaction unit (PRU) and % inhibition index equivalent for the expression of P2Y12 inhibition by the VerifyNow® assay? Role of haematocrit and haemoglobin levels

The results of the whole blood VerifyNow P2Y12 assay can be expressed as platelet reaction units (PRU) or % inhibition index (%inh), but an optimal cut-off for the assessment of high on-treatment platelet reactivity (HPR) predictive of clinical events has been validated only for PRU. The aim of the study was to study the influence of haematological variables, such as platelet and leukocyte counts or haematocrit / haemoglobin, within the limits indicated by the manufacturer for assay validity, on the results of the test. We performed a comparison of PRU and %inh in a series 186 samples obtained from a clinical trial on patients under dual antiplatelet therapy. The results show that PRU significantly decreases with increasing haematocrit / haemoglobin, whereas %inh does not, due to a parallel change in PRU and iso-TRAP baseline value. PRU and % inhibition index are not equivalent for the definition of HPR, because of their different sensitivities to haematocrit / haemoglobin.

Platelet activation and arterial peripheral serotonin turnover in cardiac remodeling associated to aortic stenosis.

Peripheral serotonin (5‐HT) has been involved in adverse cardiac remodeling and valve fibrosis. The peripheral levels of 5‐HT mainly depend on its release from activated platelets and degradation by monoamine oxidase A (MAO‐A). The SERAOPI study investigated the relationship between arterial serotoninergic system, degree of platelet activation and cardiac remodeling, in patients with aortic valve stenosis (AS). Thirty patients with severe AS and 15 control subjects underwent transthoracic echocardiography, radial, and aortic arterial blood sampling. Measurements of 5‐HT and its MAO‐A‐dependent degradation product, 5‐HIAA, were performed by HPLC. Arterial platelet activation was assessed by flow cytometry analysis of platelet surface expression of P‐selectin and activated integrin GPIIb/IIIa. Activated platelets and arterial plasma 5‐HT increased in AS patients as compared to control subjects (P‐selectin 1.08u2009±u20090.2MFI vs. 0.49u2009±u20090.1MFI, Pu2009=u20090.04; GPIIb/IIIa 0.71u2009±u20090.1MFI vs. 0.35u2009±u20090.1MFI; Pu2009=u20090.0015 and arterial plasma 5‐HT 11.55u2009±u20091.6 nM vs. 6.18u2009±u20090.7 nM, Pu2009=u20090.028, respectively). Moreover, 5‐HT was strongly correlated to left ventricular hypertrophy assessed by echocardiography. The correlation was independent of cardiovascular risk comorbidities and others echocardiographic AS parameters. Finally, plasma 5‐HIAA increased in AS patients (74.64u2009±u20099.7 nM vs. 37.16u2009±u20094.1 nM; Pu2009=u20090.0002) indicating a higher 5‐HT degradation rate by MAO‐A. Platelet activation, arterial circulating serotonin, and serotonin degradation increased in patients with AS. These observations suggest that the serotoninergic system may contribute to the pathogenesis of AS including valve fibrosis and adverse ventricular remodeling. Am. J. Hematol. 90:15–19, 2015.

Assessment of platelet response to clopidogrel through measurement of ADP-induced Akt phosphorylation.

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Alterations of the Platelet Procoagulant or Fibrinolytic Functions

Discovered in the 1980s, the procoagulant function of platelets is due to their inherent ability to provide, following activation, a critical platform at their plasma membrane surface for the activation of blood clotting enzyme complexes to generate thrombin. Upon platelet activation, the asymmetric orientation of membrane phospholipids rapidly collapses resulting in a calcium-dependent exposure of the anionic phospholipid, phosphatidylserine (PS), at the outer platelet surface. Binding of blood clotting enzyme complexes to this procoagulant membrane surface allows a dramatic increase in the rate of conversion of zymogens to active serine proteases and in turn the production of a burst of thrombin leading to fibrin clot formation and to further platelet activation. The energy-independent, calcium-dependent, platelet scramblase activity, which governs the bidirectional exchange of phospholipids between the two leaflets of the bilayer, is essential for PS exposure during platelet activation. The platelet scramblase protein has remained elusive for years until a significant advance was recently made with the identification of TMEM16F, a membrane protein essential for calcium-dependent PS exposure with loss-of-function mutations in Scott syndrome, a bleeding disorder due to an impaired platelet procoagulant activity.

035 Switching patients from clopidogrel to prasugrel at the early phase of an acute coronary syndrome: impact of prasugrel reloading

with minor effect in *2/*2 carriers. After 900mg LD, the effect of the CYP2C19*2 variant on platelet inhibition was fully compensated in wt/*2 carriers but not in *2/*2 carriers (–83.6±25.8% in wt/wt vs. –77.2±26.9% in wt/ *2 vs. –29.5±26.8% in *2/*2; overall p-value=0.0003, p=0.20 for wt/wt versus wt/*2, p<0.001 for wt/*2 versus *2/*2). A similar pattern was observed for the active metabolite AUC0-6 and there was a significant correlation between PK and PD responses irrespective of the LD.

029 Dual antiplatelet responsiveness detected by point-of-care assay VerifyNow® in elderly patients (≥75 years) receiving percutaneous intervention

Background Dual antiplatelet therapy with aspirin and clopidogrel is the cornerstone of treatment after percutaneous coronary intervention (PCI). Platelet responsiveness to these two agents is not well known in elderly patients. We sought to evaluate aspirin and clopidogrel response in elderly patients soon after PCI and on chronic maintenance treatment. Methods and Results We prospectively included 93 elderly patients (≥75 years) who underwent PCI from January 2008 to April 2009. All patients were treated with aspirin and clopidogrel. We used a standardized point of care assay VerifyNow ® aspirin and VerifyNow ® clopidogrel P2Y12 to measure aspirin and clopidogrel responsiveness with cut-offs previously validated in clinical trials. Measurements were performed in-hospital after PCI (T1) and 5–6 weeks later (T2) when the patients had been on maintenance therapy (75xa0mg aspirin plus 75xa0mg clopidogrel once a day) for at least 1 week. Aspirin non/poor responders were found in 10% of patients at T1 and in 14% at T2. Clopidogrel non/poor response was noted in 32% of patients at T1 and in 68% at T2. Changes in platelet reactivity to clopidogrel was significant from T1 to T2 ( p -value p -value = 0.042 for Ht p -value = 0.033 for Ht Conclusions The rate of biological non/poor response to dual antiplatelet therapy in elderly patients soon after PCI was close to that reported in younger patients. In contrast, there was high residual platelet reactivity under chronic treatment with 75xa0mg of clopidogrel (almost two-thirds).

044 Natural History Of Dual Anti-Platelet Responsiveness After Angioplasty In Elderly Patients

Background Dual antiplatelet therapy with aspirin (Asa) and clopidogrel (Clopi) is the cornerstone of treatment for patients (pts) after angioplasty (PCI). The state of dual antiplatelet responsiveness is not well known especially in elderly pts. Objectives The aim of this monocentric prospective observational study is to evaluate Asa and Clopi activity after PCI in elderly pts and to asses the evolution of this responsiveness at 5 weeks. Methods We enrolled 81 elderly pts (≥75 years) from Jan to Dec 2008 on Asa (75 mg/day ≥ 7 days) and Clopi (after loading dose 300 mg before PCI and 150mg/day for 4 weeks fellowed by 75 mg/day for 1 year). We used the VerifyNow Asa assay and the VerifyNow Clopi P2Y12 assay (Accumetrics Inc, USA) to determinate respectively Asa and Clopi responsiveness in-hospital at 11.30 am (T1) and during a medical consultation at 5 weeks (T2). Results Asa non responders were noted in 10% and in 14% pts at respectively T1 and T2, Clopi non responders in 37% and in 70% pts at respectively T1 and T2. Significant changes were observed in Clopi response from responder to non responder (pxa0 Conclusions In elderly, after PCI, there are changes in dual antiplatelet responsiveness between tests in-hospital and at 5 weeks marked for Clopi activity. Low hematocrit remains an independent predictor of Clopi non responsiveness.