Pharmacological Study of Antimyeloma Agents on Hemostasis

Abstract Abstract 3175 Introduction: Patients with multiple myeloma (MM) have an increasing risk of developing venous thromboembolism (VTE). The use of thalidomide or lenalidomide, two antimyeloma agents has been associated with an increased risk of VTE especially when associated with dexamethasone or melphalan-prednisone. This increased risk in less pronounced with the proteasome inhibitor bortezomib. The pharmacological effects of thalidomide, lenalidomide and bortezomib have never been studied on platelet aggregation or coagulation. These preliminary data are necessary to understand the differential effects of these drugs on thrombotic events. Aims: The aim of our study is to investigate the pharmacological effects of these drugs on primary hemostasis by light transmission agregometry (LTA) and on secondary hemostasis using thrombin generation test (TGT). Methods: LTA was performed with a Chrono-Log aggregometer (Kordia). Platelet-rich plasma (PRP) of healthy blood donors (n=3 to 10) was adjusted to 300,000 platelets/μl. Platelet-poor plasma (PPP) and PRP were used respectively to adjust the photometric measurement to the minimum and maximum optical density. The platelet reactivity of the healthy donors was previously checked by LTA. Thalidomide (racemic, enantiomer (+) and enantiomer (-)), lenalidomide and bortezomib were spiked at final concentration of 200 μM in PRP except for studying the influence of thalidomide on ADP-induced platelet aggregation where the maximal concentration usable was 100 μM. LTA was performed without (spontaneous aggregation) and with addition of 5μM ADP, 190μg/ml collagen, 600μM arachidonic acid (AA) and 10 μM PAR1-AP (final concentrations). Negative controls (physiological saline and DMSO at concentrations used to dissolve the drugs) were also included. TGT was performed both on PPP (from normal pool plasma (NPP)) and on PRP. We first validated the method on NPP and PRP with two anticoagulants: a thrombin direct inhibitor (i.e. argatroban) and a FXa direct inhibitor (i.e. ZK-807834). Thalidomide ((+/−), (+) and (-)), lenalidomide and bortezomid were tested at the final concentrations of 5, 10, 50, 100 and 200 μM. For the experiments on NPP, PPP reagent (5 pM TF + 4 μM PL in the final assays), PPP reagent low (1 pM TF + 4 μM PL in the final assays) and MP reagent (4 μM PL in the final assays) were used whereas for the experiments on PRP, PRP reagent (1 pM TF in the final assays) was used. Results: LTA Both three drugs did not induce spontaneous aggregation until 200 μM. Lenalidomide and bortezomib showed no effect on induced platelet aggregation until 200 μM, whatever the agonist used. On the contrary, for racemic thalidomide, platelet aggregation was reduced at 50 and 100 μM with 5 μM ADP and at 150 and 200 μM with 600 μM AA. These effects were more pronounced with thalidomide (+) than with thalidomide (-). So, the half maximal inhibitory concentrations (IC50) for platelet aggregation induced by 600 μM AA were 127, 143 and 221 μM for racemic, enantiomer (+) and enantiomer (-), respectively. When 190μg/ml collagen or 10 μM PAR1-AP were used as agonists, no effect was observed on platelet aggregation until 200 μM thalidomide. TGT. With argatroban and ZK-807834, we observed a concentration-dependent decrease and delay of the thrombin activity profiles in PRP and NPP, whatever the inducer used. No significant effect on thrombin activity in NPP or PRP has been observed for antimyeloma drugs at all concentrations tested, whatever the inducer used. Conclusions: Lenalidomide, thalidomide and bortezomid do not induce spontaneous platelet aggregation and do not affect platelet aggregation induced by collagen or PAR1-AP. Conversely to lenalidomide and bortezomib, thalidomide, and especially its enantiomer (+), moderately inhibits platelet aggregation induced by ADP and AA. Moreover, these antimyeloma agents have no effect on thrombin generation. The increased risk of VTE by thalidomide or lenalidomide in patients with multiple myeloma is thus not mediated by a direct impact on primary or secondary hemostasis at therapeutic concentrations. Disclosures: No relevant conflicts of interest to declare.

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A Synthetic Evaluation Of Genetic and Pharmacological Resistance To Clopidogrel and On Treatment Residual Platelet Aggregation In Patients With Atherothrombosis

Blood ◽

10.1182/blood.v122.21.3631.3631 ◽

2013 ◽

Vol 122 (21) ◽

pp. 3631-3631

Author(s):

Grigoris T Gerotziafas ◽

Severin Bouffard ◽

Patrick Van Dreden ◽

Amir Kartechi ◽

Ilias Evmorfiadis ◽

...

Keyword(s):

Platelet Aggregation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Diagnostic Algorithm ◽

Poor Response ◽

Wild Type ◽

Functional Assays ◽

Pharmacological Response

Abstract Background Clopidogrel is cornerstone treatment for atherothrombotic patients. The incidence of recurrent thrombotic events raises up to 10% of patients on treatment with clopidogrel. Intestinal absorption via the glycoproteine ABCB1, bioactivation by the cytochrome P450 (CYP) system, binding to P2Y12 receptor and inhibition of platelet aggregation are critical steps for the efficiency of clopidogrel treatment. Prospective independent clinical trials showed that the risk of recurrent thrombotic complications is associated with the presence of common polymorphisms of genes encoding ABCB1 (rs1045642C>T) or the CYP2C19 isoenzyme (*2_rs4244285G>A, *3_rs4986893G>A, *4_rs28399504A>G) or the inefficiency of clopidogrel to decrease intracellular VASP or the presence of high residual platelet reactivity (HRPR). Aim To evaluate the available pharmacogenetic, pharmacological and functional assays for the diagnosis of the resistance to clopidogrel treatment on the same population of patients with atherothrombosis. Materials and Methods A cohort of 94 out-patients with atherothrombosis receiving clopidogrel treatment (75 mg o.d) for more than 3 months were included. Polymorphisms ABCB1, CYP *2, *3, *4, *17 were assessed with rPCR method. VASP was measured with flow cytometry assay and poor response (PR) to the treatment was diagnosed if VASP index was > 50. Light Transmission Platelet Aggregation (LTPA) triggered by ADP (5 ìM and 10 ìM) was assessed on citrated platelet rich plasma. Multielectrode aggregometry (WBMEA) was performed on citrated whole blood using Multiplate instrument and ADP reagent (ADP-test®). The cut-off point for the diagnosis of HRPR on clopidogrel treatment was the lower value of the maximum aggregation or the AUC observed in the upper quintile of ADP triggered LTPA or WBMEA measurements. Results The frequency of heterozygous for CYP*2, *3, *4 and ABCB 1 polymorphisms was 28%, 0%, 1%, and 51% respectively. The frequency of the respective homozygocity was 2%, 0%, 0%, and 20%. Patients heterozygous for the CYP*2 G>A polymorphism showed significantly higher VASP index as compared to the wild type but LTPA and WBMEA were not significantly different. VASP index, LTPA and WBMEA were not significantly different in patients heterozygous or homozygous for the ABCB1 C>T polymorphism as compared to the wild type. The VASP index was significantly correlated with the LTPA (r=0.44, p<0.0001) and WBMEA (r=0.376, p=0.001). In addition LTPA was significantly correlated with the WBMEA (r=0.46, p<0.0001). With the VASP assay 42% of patients were diagnosed as PR to clopidogrel. HRPR was found in 12% and 28% of patients tested with LTPA ADP 5 ìM and 10 ìM respectively and in 19% of patients tested with WBMEA. Among patients with HRPR the frequency of PR with the VASP assay was 60% when they were tested with LTPA ADP 10 ìM, 70% when they were tested with LTPA ADP 5 ìM and 76% when they were tested with WBMEA. Less than 50% of patients with HRPR had at least one of the studied polymorphisms. One patient homozygous for CYP*2 and ABCB1 was PR with the VASP assay and had HRPR with the WBMEA. Conclusion The present study performed on a cohort of patients with atherothrombosis on treatment with clopidogrel demonstrates that the genotype of CYP and ABCB1 polymorphisms have limited influence on the pharmacological response to the treatment and on the residual platelet reactivity. Among the studied polymorphisms only the CYP*2G>A was related with increase of VASP index and WBMEA. Pharmacological response to clopidogrel is correlated with the level of residual platelet reactivity. However 25-30% of patients with HRPR showed good pharmacological response to clopidogrel. The present study underlines the need to a synthetic use of bioassays for the evaluation of the risk of failure of clopidogrel treatment to prevent the recurrence of atherothrombosis. The need for the construction of a diagnostic algorithm including the genetic, pharmacological and functional assays is revealed by the present study. Disclosures: No relevant conflicts of interest to declare.

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An Ex Vivo and In Silico Study Providing Insights into the Interplay of Circulating miRNAs Level, Platelet Reactivity and Thrombin Generation: Looking beyond Traditional Pharmacogenetics

Journal of Personalized Medicine ◽

10.3390/jpm11050323 ◽

2021 ◽

Vol 11 (5) ◽

pp. 323

Author(s):

Alix Garcia ◽

Sylvie Dunoyer-Geindre ◽

Séverine Nolli ◽

Jean-Luc Reny ◽

Pierre Fontana

Keyword(s):

Platelet Aggregation ◽

Platelet Function ◽

In Silico ◽

Thrombin Generation ◽

Light Transmission ◽

Platelet Reactivity ◽

Circulating Mirnas ◽

Candidate Mirnas ◽

The Impact

Platelet reactivity (PR), a key pharmacodynamic (PD) component of the action of antiplatelet drugs in cardiovascular disease (CVD) patients, is highly variable. PR is associated with occurrence or recurrence of thrombotic and bleeding events, but this association is modulated by several factors. Conventional pharmacogenetics explains a minor part of this PR variability, and among determinants of PR, circulating microRNAs (miRNAs) have been the focus of attention during these last years as biomarkers to predict PR and clinical outcomes in CVD. This being said, the impact of miRNAs on platelet function and the mechanisms behind it are largely unknown. The level of a set of candidate miRNAs including miR-126-3p, miR-150-5p, miR-204-5p and miR-223-3p was quantified in plasma samples of stable CVD patients and correlated with PR as assessed by light-transmission aggregometry and in vivo thrombin generation markers. Finally, miRNA target networks were built based on genes involved in platelet function. We show that all candidate miRNAs were associated with platelet aggregation, while only miR-126-3p and miR-223-3p were positively correlated with in vivo thrombin generation markers. In silico analysis identified putative miRNA targets involved in platelet function regulation. Circulating miRNAs were associated with different aspects of platelet reactivity, including platelet aggregation and platelet-supported thrombin generation. This paves the way to a personalized antithrombotic treatment according to miRNA profile in CVD patients.

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Comparison of methods to evaluate clopidogrel-mediated platelet inhibition after percutaneous intervention with stent implantation

Thrombosis and Haemostasis ◽

10.1160/th08-09-0577 ◽

2009 ◽

Vol 101 (02) ◽

pp. 333-339 ◽

Cited By ~ 83

Author(s):

Sabine Steiner ◽

Daniela Seidinger ◽

Renate Koppensteiner ◽

Thomas Gremmel ◽

Simon Panzer ◽

...

Keyword(s):

Stent Implantation ◽

Light Transmission ◽

Platelet Reactivity ◽

Platelet Inhibition ◽

Percutaneous Intervention ◽

Adverse Outcomes ◽

Gold Standard Method ◽

Platelet Aggregometry ◽

Increased Risk ◽

The Impact

SummaryA high on-treatment residual ADP-inducible platelet reactivity in light transmission aggregometry (LTA) has been associated with an increased risk of adverse outcomes after percutaneous coronary intervention (PCI). However, LTA is weakly standardized, and results obtained in one laboratory may not be comparable to those obtained in another one. We therefore sought to determine the test correlating best with LTA to estimate clopidogrel-mediated platelet inhibition in 80 patients on dual antiplatelet therapy after elective percutaneous intervention with stent implantation. We selected the VerifyNow P2Y12 assay, the vasodilator-stimulated phosphoprotein phosphorylation assay, multiple electrode platelet aggregometry and the Impact-R for comparisons with LTA. Cut-off values for residual ADP-inducible platelet reactivity were defined according to quartiles of each assay. Sensitivities and specificities of the different platelet function tests were based on the results from LTA. The results from all four assays correlated significantly with those from LTA. The VerifyNow P2Y12 assay revealed the strongest correlation (r = 0.61, p < 0.001). Sensitivities and specificities ranged from 35% to 55%, and from 78.3% to 85%, respectively. In conclusion, although all assays correlated significantly with LTA, they need to be improved to become clinically used diagnostic tests. Further, it may be too early to define the gold standard method for assessing residual ADP-inducible platelet reactivity and generally acceptable cut-off values.

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Fucosyltransferase VI Induces Platelet Activation: A Novel Property of a Plasma Glycosyltransferase.

Blood ◽

10.1182/blood.v114.22.4016.4016 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4016-4016

Author(s):

José-Tomás Navarro ◽

Shwan Tawfiq ◽

Roland Wohlgemuth ◽

Karin M. Hoffmeister ◽

Robert Sackstein

Keyword(s):

Flow Cytometry ◽

Platelet Aggregation ◽

Platelet Activation ◽

Light Transmission ◽

Conflicts Of Interest ◽

Platelet Rich Plasma ◽

Surface Protein ◽

Surface Flow ◽

Platelet Surface ◽

Biochemical Studies

Abstract Abstract 4016 Poster Board III-952 A number of glycosyltransferases are present in human plasma with the α(1→3) fucosyltransferase, Fucosyltransferase VI (FTVI), having the highest plasma concentration. Notably, elevated plasma levels of FTVI are associated with a variety of cancers and correlate with tumor load/progression. The well-known association of neoplasia with thromboembolic complications prompted us to examine whether FTVI has direct effect(s) on platelet function. We obtained human platelets from blood of healthy donors and separated from platelet-rich plasma by differential centrifugation. Freshly isolated platelets (x108/ml) were stirred and exposed at 37°C to varying concentrations (20, 40, 60 and 80 mU/mL) of glycosyltransferases FTVI, β-1-4-galactosyltransferase-I (βGalT-I), or α,2-3-N-sialyltransferase (α2,3-N-ST), or to 1 U/mL thrombin. Platelet aggregation and activation was assessed by aggregometry (light transmission) or by flow cytometry of FSC/SSC characteristics and of surface expression of P-Selectin, respectively. FT-VI reproducibly induced platelet aggregation and activation, whereas other glycosyltransferases (β4GalT-I and α2,3-N-ST) had no effect on platelets. FTVI activation of platelets was concentration-dependent, and the aggregation curve for FTVI was one wave, similar to that for thrombin. FTVI-induced platelet activation was independent of catalytic conversion of surface glycans, but was inhibited by FTVI denaturation, indicating that FTVI-induced platelet activation is a lectin-mediated process. To determine the membrane target(s) mediating FTVI-induced platelet activation, biochemical studies were performed after catalytic exofucosylation of the platelet surface. Flow cytometry after platelet exofucosylation showed formation of the carbohydrate structure sLex, detected by the mAb Heca452, but no formation of Lex (CD15). Western blot showed that enforced fucosylation induced sLex on a single platelet surface protein, and further biochemical studies revealed that this protein is GPIbα. These findings unveil a previously unrecognized property of FTVI as an activator of platelets, mediated via a specific lectin/carbohydrate interaction on GP1ba, and offer novel perspectives on the pathobiology of tumor-associated thrombogenesis. Disclosures: No relevant conflicts of interest to declare.

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Military Service in Vietnam/Korea and Serum Dioxin Levels Do Not Affect the Outcomes of Patients Diagnosed with Plasma Cell Dyscrasias

Blood ◽

10.1182/blood.v118.21.5098.5098 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5098-5098

Author(s):

Prasanth Ganesan ◽

Emil Kuriakose ◽

Carla Smith ◽

Robert T Harris ◽

Jonathan E Dowell ◽

...

Keyword(s):

Multiple Myeloma ◽

Plasma Cell ◽

Military Service ◽

Conflicts Of Interest ◽

Military Veterans ◽

Cell Transplant ◽

Increased Risk ◽

Plasma Cell Dyscrasias ◽

Chlorinated Dioxins ◽

The Impact

Abstract Abstract 5098 Military Service in Vietnam/Korea and Serum Dioxin Levels Do Not Affect the Outcomes of Patients Diagnosed with Plasma Cell Dyscrasias. Background: Exposure to dioxin, a contaminant found in herbicides has been associated with increased risk of cancers including multiple myeloma and postulated to cause poorer survival in the exposed population. Military personnel, especially those who had served in Vietnam and Korea have an increased risk of dioxin (which contaminated the herbicide Agent Orange which was sprayed during these wars) exposure. We looked at the impact of dioxin exposure and blood levels of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) which is the most toxic of the poly-chlorinated dioxins on the survival outcomes of military veterans diagnosed with plasma cell dyscrasias (PCD). Methods: A prospective analysis of newly diagnosed and existing myeloma patients was done. Information regarding the patient and disease characteristics, the military record, and outcomes were collected. Approximately 60 ml of heparinised peripheral blood was collected and immediately frozen at −20 degrees. These samples were shipped to Eurofins Laboratory, Hamburg, Germany for dioxin level measurement. Patients' blood lipid levels were also measured and the dioxin toxic equivalent (Teq) was calculated. Overall survival (OS) was calculated from the date of diagnosis till death (Kaplan Meier method). Cox regression and log rank analysis were used to look for prognostic variables. Results: Fifty two (52) patients of PCD were available for analysis. Majority had a diagnosis of multiple myeloma. Forty one underwent treatment including stem cell transplant in 16 (Table 1 shows the patient characteristics, laboratory results and treatment outcomes). During a median follow up of 54 months (2–348), 21 patients died (progressive myeloma: 12(23%), cardiac failure: 3 (5.7%), infections: 1 (1.9%), acute myeloid leukemia: 1 (1.9%), pulmonary embolism: 1 (1.9%) and unknown: 3 (5.7%). The median OS was 111 mos (95% CI 56–155) and the estimated survival at 5 yrs was 69.5% (+/− SE 0.067). The 5 yr OS was negatively impacted by abnormal cytogenetics (40.3 % vs. 75.5%; p=0.012), and service in the army (non-army vs. army: 83% vs. 40%; p=0.032). Patients who had served in Vietnam had outcomes similar to others; Korean War veterans had a poorer OS, but this was not statistically significant (5 yr OS 68% vs. 48%; p=0.1). There was no association between TCDD levels or the Teq with OS. Abnormal cytogenetics was the only significant factor on multivariate analysis. Conclusions: We did not find an association between military service in Korea/Vietnam or serum dioxin levels and poor survival in military veterans diagnosed with Plasma cell dyscrasias. However, a study of a larger sample of myeloma patients with similar service and exposure histories maybe warranted. Disclosures: No relevant conflicts of interest to declare.

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Implications of TP53 alterations for Therapy Response in Multiple Myeloma

Blood ◽

10.1182/blood-2018-99-118658 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 3189-3189 ◽

Cited By ~ 1

Author(s):

Umair Munawar ◽

Santiago Barrio ◽

Markus Roth ◽

Hermann Einsele ◽

Ralf C Bargou ◽

...

Keyword(s):

Multiple Myeloma ◽

High Risk ◽

Cell Viability ◽

Conflicts Of Interest ◽

Clonal Selection ◽

Proteasome Inhibition ◽

Sleeping Beauty ◽

Targeted Sequencing ◽

Fish Analysis ◽

Increased Risk

Abstract Deletion of the tumor suppressor gene TP53is significantly associated with an unfavorable clinical course of Multiple Myeloma (MM). In addition, point mutations that abrogate p53 function similarly shorten survival. Most recently 'double hit', bi-allelic TP53inactivated MM was identified as an ultimate high risk feature of MM, affecting 3.7% of newly diagnosed MM patients (NDMM, Walker et al., Leukemia 2018). For TP53genetic analysis we combined data from two targeted sequencing M3P cohorts with targeted sequencing and FISH data, one of NDMM (n=142, Kortüm et al. Blood Cancer J. 2016), and one of multirefractory patients (rMM, n=40; Kortüm et al. Blood 2016). We also included two independent cohorts with paired NDMM and rMM, one with mutational data (n=43; Corre J et al 2017) and one with paired FISH analysis (Merz M et al. Haematologica 2017). We confirmed an increase of mutations in TP53 (8% NDMM / 16.9% rrMM), as well as del17p (12.6% / 22%). Similarly, mono- (12.7% / 22.5%) and bi-allelic events (5.6% / 17.5%) both demonstrated a significant increase over time (Figure, top). Importantly we observed deletion after mutation and vice versa in our cohorts. Next we established an AMO-1 MM cell line model (TP53+/+) mimicking mono and bi-allelic TP53-inativation. After CRISPR/Cas9-mediated TP53destruction, we introduced a modified Sleeping Beauty (SB) vector with two separate expression cassettes (p53 wt / wt and mutant p53 (R282W or R175H). Functionality of the p53 system was confirmed using nutlin-3, as inhibitor of MDM2-p53 interaction, as described elsewhere. Results: Doxorubicin and Melphalan are commonly used compounds in the treatment of MM. The IC50 of Melphalan in AMO1 naïve cells was 5µM and 5.5 µM after the reintroduction of 2 copies of wt TP53 in the KO model, with cell viability at 10µM of 30% and 31% respectively, measured by AlamarBlue Assay. Strikingly, we observed significant resistance induction in our hemizygous systems (del/wt p53; cell viability at 10µM 60% and mut/wt (62%). This furthermore increased within our homozygous models (TP53 del/del (85%); TP53 mut/del 80%) (Figure, bottom). Similar results were observed under doxorubicin treatment. Remarkably, this effect was absent against proteasome inhibition. Conclusions Here we present first evidence of TP53 inactivation impacting drug response to Melphalan and Doxorubicin, which might lead to the clonal selection of MM subclones harboring increased risk. The fact, that response to proteasome inhibition was not affected in our model might, at least in part, might explain their ability to confine high risk in MM. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

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Use of increased concentrations of adenosinediphosphate for high residual platelet reactivity in patients with coronary heart disease

10.18411/lj-02-2021-33 ◽

2021 ◽

Vol 70 (1) ◽

pp. 129-132

Author(s):

O.A. Trubacheva ◽

S.N. Belyaeva ◽

T.E. Suslova ◽

I.V. Petrova

Keyword(s):

Coronary Heart Disease ◽

Heart Disease ◽

Platelet Aggregation ◽

Antiplatelet Therapy ◽

Light Transmission ◽

Platelet Reactivity ◽

Platelet Rich Plasma ◽

Adenosine Diphosphate ◽

Transmission Curve ◽

Platelet Suspension

Detection of a tendency to increased thrombosis in patients with coronary heart disease (CHD) is of important prognostic value in the selection of drugs aimed at achieving a persistent antithrombotic effect. The aim of the study was to evaluate the use of elevated ADP inducer concentrations to improve the accuracy of ADP-induced platelet aggregation in patients with coronary heart disease. Material and method. Material and method. We studied 48 patients with CHD who were on continuous double antiplatelet therapy for 6 months (aspirin 75mg and clopidogrel 75mg per day). The aggregation activity of the platelet suspension was studied using the Born method G. in the modification of Gabbasov Z. A. Platelet activity was evaluated by the degree of aggregation of platelet-rich plasma along the light transmission curve under the influence of the inducer adenosine diphosphate (ADP) at a concentration of 2 mmol/l and by its own patented method against the background of additional ADP application. Results. In patients, platelet aggregation decreased to 5-35% (p<0.005) compared to the standard values, which are 50-60%. The values of platelet aggregation with the additional introduction of the inducer of aggregation ADP in a ratio of 2:1 to 2 µmol/l for 1, 2, 3, and 4-minute registration of platelet aggregation, resulted in increased aggregation from 55% to 75% (p<0.001), indicating high residual platelet reactivity on the background of double antiplatelet therapy. Correlations of the degree of aggregation for elevated ADP concentrations with multivessel arterial lesion and dyslipidemia were also found, r=0.86 and r=0.92, respectively. Conclusion. The use of elevated concentrations of adenosine diphosphate in platelet aggregation in patients with ischemic heart disease increases the accuracy of assessing ADP-induced platelet aggregation against the background of dual antiplatelet therapy and contributes to the detection of high residual platelet reactivity.

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Mean Corpuscular Volume Predicts Adverse Outcomes Following Peripheral Angioplasty With Stenting and Is Associated With On-Treatment Platelet Reactivity

Angiology ◽

10.1177/0003319720943816 ◽

2020 ◽

Vol 72 (1) ◽

pp. 16-23

Author(s):

Maximilian Tscharre ◽

Silvia Lee ◽

Christoph W. Kopp ◽

Simon Panzer ◽

Thomas Gremmel

Keyword(s):

Mean Corpuscular Volume ◽

Cox Regression ◽

Light Transmission ◽

Platelet Reactivity ◽

Adenosine Diphosphate ◽

Adverse Outcomes ◽

Corpuscular Volume ◽

Characteristic Analysis ◽

Cox Regression Analysis ◽

Increased Risk

Structural aspects of red blood cells have been associated with cardiovascular disease. No data linking mean corpuscular volume (MCV) to clinical outcomes and on-treatment platelet reactivity in patients with peripheral artery disease (PAD) are available. We investigated a composite of atherothrombotic events and target vessel restenosis or reocclusion following infrainguinal stenting for stable PAD. Residual platelet reactivity was measured by light transmission aggregometry (LTA) and the VerifyNow assays. We included 104 patients receiving dual antiplatelet therapy (DAPT) with aspirin and clopidogrel. In receiver-operating characteristic analysis, MCV effectively discriminated between patients with and without adverse outcomes and identified a MCV ≤90.8 fL as optimal cutoff. Adverse outcomes occurred significantly more often in patients with low MCV (log-rank P = .002). In univariable Cox regression analysis, low MCV was associated with an increased risk of future adverse outcomes (hazard ratio [HR]: 2.662 [95%CI: 1.304-5.434]; P = .007) and remained significantly associated after adjustment (HR: 2.591 [95%CI: 1.242-5.403]; P = .011). Mean corpuscular volume was inversely correlated with arachidonic acid (AA)- and adenosine diphosphate (ADP)-inducible platelet reactivity by LTA and with the VerifyNow aspirin assay. Low MCV is associated with adverse outcomes over 2 years following infrainguinal stenting. Mean corpuscular volume correlates inversely with AA- and ADP-inducible platelet reactivity during DAPT.

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Is the New Point-of-Care Test VerifyNow® Aspirin Able To Identify Aspirin Resistance Using the Recommended Cut-Off?.

Blood ◽

10.1182/blood.v110.11.3896.3896 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3896-3896

Author(s):

Helle L. Nielsen ◽

Steen D. Kristensen ◽

Anne-Mette Hvas

Keyword(s):

Platelet Aggregation ◽

Healthy Volunteers ◽

Low Dose ◽

Light Transmission ◽

Point Of Care ◽

Reference Method ◽

Platelet Inhibition ◽

Aspirin Resistance ◽

Inadequate Response ◽

Increased Risk

Abstract Low dose aspirin inhibits platelet aggregation and is widely used in the treatment of cardiovascular disease. However, a considerable interindividuel variation in response to aspirin has been reported. Some studies suggest that patients with inadequate platelet inhibition by aspirin are at increased risk of suffering future cardiovascular events. Identification of these patients is of great interest. So far there is no consensus regarding the choice of method and definition for identifying patients with inadequate response to aspirin treatment, also referred to as aspirin resistance (AR). The VerifyNow® Aspirin system is a new point-of-care method for measurement of platelet aggregation response to aspirin. The main objectives were: to determine the repeatability of VerifyNow® Aspirin to determine the agreement between VerifyNow® and light transmission aggregometry (LTA) - the considered reference method to determine the frequency of AR defined by the two methods. We included 21 healthy volunteers and 40 patients with documented coronary artery disease. Duplicate measurements of platelet aggregation were performed with VerifyNow® Aspirin and LTA (agonist arachidonic acid 1.0 mM) in healthy volunteers before onset of aspirin treatment. After treatment of healthy volunteers with plain aspirin 75 mg daily for 7 days steady state was reached, and duplicate platelet aggregation measurements were performed once a day for four consecutive days in both healthy subjects and patients during continued treatment with plain aspirin 75 mg daily. Test results were expressed in Aspirin Reaction Units (ARU) using VerifyNow® Aspirin and in percent of max aggregation with LTA. The cut-off for determining AR was ≥ 550 ARU for VerifyNow® and ≥ 20% for LTA. To confirm compliance we measured serum-thromboxaneB2 (se-TXB2) levels. All participants were compliant to aspirin treatment as measured by se-TXB2; < 4,5 ng/mL 99% suppression. All duplicate analyses with VerifyNow® showed a high degree of repeatability as the coefficient of variance prior to aspirin treatment was 0.5% and 3.0% during aspirin treatment. Correlation of measurements of platelet aggregation during aspirin therapy with VerifyNow® and LTA respectively was moderate, Spearmans ρ=0.50. The agreement between the two methods regarding identification of individuals with AR was poor: According to VerifyNow® Aspirin none of the participants had AR, whereas LTA identified 7 (12%) subjects with AR. We performed ROC analysis using LTA as reference method. We found poor sensitivity and good specificity when using 550 ARU as a cut-off, table. ROC statistics on VerifyNow AR cut-off with LTA as reference VerifyNow cut-off Sensitivity, % Specificity, % ≥ 460 ARU 100 72 ≥ 480 ARU 43 80 ≥ 500 ARU 30 91 In conclusion we found that VerifyNow® Aspirin showed a very high repeatability, but correlated only moderately well with LTA. In 100% compliant participants treated with a low dose of aspirin 75 mg daily we found that none of the study participants were classified as AR with VerifyNow® Aspirin, but 12% were identified as AR by LTA. Our results imply that the recommended cut-off level at 550 ARU for identifying AR with VerifyNow® Aspirin is too high. Further studies are needed to investigate the relevance of a cut-off level lower than 550 ARU for determining AR and its clinical implications.

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Evaluation of Residual Platelet Reactivity in Patients Treated with Aspirin and or Clopidogrel: Comparison of Results Obtained On Multiplate®, PFA-100TM and Classical Light Transmission Aggregometry.

Blood ◽

10.1182/blood.v114.22.3129.3129 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3129-3129

Author(s):

Annick Ankri ◽

Isabelle Martin-Toutain ◽

Anne Baranger ◽

Marie-Claude Couty ◽

Jean Philippe Collet ◽

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Keyword(s):

Arachidonic Acid ◽

Light Transmission ◽

Platelet Reactivity ◽

Biological Activities ◽

Impedance Aggregometry ◽

Increased Risk ◽

Light Transmission Aggregometry ◽

Group A ◽

Platelet Functions ◽

Good Agreement

Abstract Abstract 3129 Poster Board III-66 Residual platelet reactivity (RPR), despite antiplatelet therapy (AT), is currently associated with an increased risk of recurrent ischemic events and is linked to a biological resistance to AT. We determined whether whole blood impedance aggregometry using the Multiplate® (Dynabyte and IL France) detects the effects of AT as reliably as does classical light transmission aggregometry (LTA) (PAP-8, Biodis). We compared also results with those obtained on PFA-100TM (Siemens). Patients and Methods Ninety-three controls, healthy volunteers or patients without intake of AT or other drugs or pathology impairing platelet functions and 182 consecutive patients on AT were studied. Among patients, 61 received Aspirin 100mg (group A), 36 Clopidogrel 75mg (group C) and 85 the association of the two drugs. Among these 85 patients, 58 received continuously Aspirin 75mg and Clopidogrel 75mg (group AC) and 27 received a loading dose for both drugs before coronarography (group LAC). Venous blood samples were obtained on Becton Dickinson vacutainer containing citrate 0.129M. Multiplate® measures change in electrical resistance as arbitrary aggregation units (AU) over time. Aggregation is quantified as AU and area under the curve (AUC) of AU.min. On LTA, aggregation was quantified according to manufacturer's recommendations as AUC. “Resistance” to Aspirin or Aspirin RPR was determined on Multiplate® (M) using arachidonic acid at 0.5 mM (ASPITEST), in LTA with arachidonic acid at 1 mM (AA-LTA) and on PFA-100TM using the cartridge with membrane coated with epinephrine (PFA-EPI). In the same way, “resistance” to Clopidogrel was determined on M using ADP at 6.4 μM (ADPTEST), in LTA with the presence of ADP at 10 μM (ADP-LTA) and occlusion time on PFA-100TM using cartridge with membrane coated with ADP (PFA-ADP). Results All patients were tested on M, 169 on PFA-100TM and 37 with LTA. Significant correlations (p<.0001) were observed between ASPITEST and AA-LTA (r = .771), ASPITEST and PFA-EPI (r = -.42), AA-LTA and PFA-EPI (r = -.47), ADPTEST and ADP-LTA (r = .6) ADPTEST and PFA-ADP (r = -.39) and ADP-LTA and PFA-ADP (r = -.56). To define RPR on M and LTA, cut-off values (5th percentile) were determined from controls for each inducer. Treated patients presenting reactivity higher than the threshold value were considered as ‘resistant’. For PFA-100TM, results of patients under cut-off point defined by the manufacturer were considered as ‘resistant’. The frequency of “resistant” patients according to the different platelet functions tests was: a) for Aspirin: 10% on M, 4.5% with LTA and 32.8% with PFA-EPI. b) for Clopidogrel 23.1% on M 16.1% with LTA and 54.5% with PFA-ADP. A good agreement was found between ASPITEST and AA-LTA and between ADPTEST and ADP-LTA (respectively 92% and 88.7%). On the other hand, results on comparison between ASPITEST and PFA-EPI and LTA and PFA-EPI were respectively 70% and 72.6%. Results for ADPTEST and PFA-ADP were 69% and 72.3% for ADP-LTA and PFA-ADP. A significant gradation of mean AUC was observed using ASPITEST on M between all groups of subjects: controls (mean = 494±176); group A (mean = 214±186); group C (mean = 310±198); group AC (mean = 118±109); group LAC (mean = 74±45). Similar results are obtained with LTA and on PFA-100TM (except for the group C, Clopidogrel alone). Thus, a potentiating effect of Aspirin by the association with Clopidogrel may be postulated in this study. Using ADPTEST to assess the treatment response by clopidogrel, mean AUC were significantly lower for all group of patients with Clopidogrel than for controls. No gradient has been observed. Mean AUC of patients with Aspirin alone was similar to controls. In conclusion, our results confirm that Multiplate® is more effective than PFA-100TM in monitoring patients on AT. The good agreement between Multiplate® and LTA could lead to use the Multiplate® in first intention to detect RPR in these patients. Furthermore the easiness of Multiplate® use may contribute to development of new studies on biological activities of AT. Disclosures No relevant conflicts of interest to declare.

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