Method for the evaluation of residual platelet thrombotic potential in patients undergoing antiplatelet therapy with acetylsalicylic acid

Abstract

Disclosed is a method for evaluation of residual platelet thrombotic potential in a patientundergoing antiplatelet therapy with acetylsalicylic acid which comprises determination ofplatelet tissue factor (TF) expression in a patient's blood sample.

Description

[0001] “METHOD FOR THE EVALUATION OF RESIDUAL PLATELET THROMBOTIC POTENTIAL IN PATIENTS UNDERGOING ANTIPLATELET THERAPY WITH ACETYLSALICYLIC ACID”

[0002] The invention relates to a method for evaluating residual platelet thrombotic potential in patients undergoing antiplatelet therapy.

[0003] Background to the invention

[0004] The main therapeutic target in coronary disease treatment is the inhibition of platelet activity. The contribution of platelets to the thrombotic event characteristic of cardiovascular diseases is not limited to their ability to aggregate, but also extends to their role in the coagulation process. Platelets not only supply the surface whereon coagulation factors assemble to give rise to thrombin formation, as historically recognised, but also possess various coagulation factors in the cytosol including tissue factor (TF), a key protein that triggers the coagulation cascade (Camera M. et al., Arterioscler Thromb Vase Biol 2003;23: 1690-1696).

[0005] Among the drugs able to regulate the platelet functions, acetylsalicylic acid is the main therapeutic choice for secondary prevention of coronary disease. Said medicament acts by irreversibly inhibiting cyclooxygenase- 1 (COX-1) in the platelets, thereby reducing the production of thromboxane A2, a potent platelet activator (Patrono C, BMJ. 2002;324:71-86). The benefits of treatment with low doses of aspirin for secondary prevention of new cardiovascular events have been recognised for many years, and corroborated by numerous trials and meta-analyses demonstrating a 20% reduction in the risk of new major cardiovascular events (Baigent C, Lancet, 2009; Antithrombotic Trialists’ Collaboration, BMJ. 2002;324:71- 86).

[0006] However, although aspirin is to date the pharmacological treatment suggested by the guidelines for cardiovascular disease secondary prevention, the risk of new coronary events remains high. This may be attributable not only to an inadequate response to the pharmacological treatment, but also to the presence of platelet activation functions / mechanisms which cannot be adequately controlled by pharmacological treatment, thus contributing to the overall risk of thrombosis.

[0007] On this regard, the procoagulant platelet function, sustained by tissue factor (TF) expression, contributes to the prothrombotic platelet phenotype and is of particular interest. After platelet activation by the classic agonists, functionally active TF is exposed on the surface thereof and, by binding factor Vila, triggers the coagulation process with generation of thrombin (Camera M. et al., Arterioscler Thromb Vase Biol 2003;23: 1690-1696; Brambilla M. et al., Platelets, 2018, 29(4):406-414). The important clinical implications of the presence of TF on the platelet surface involve the direct relationship between the TF levels and the procoagulant / prothrombotic functional activity of the platelets, which are thus able to trigger and amplify the thrombotic process (Brambilla et al, Arterioscler Thromb Vase Biol. 2008;28(5):947-53; Canzano et al, JACC Basic Transl Sci. 2021; 6(3):202-218). Significantly increased levels of TF-positive platelets, compared with those found in healthy volunteers, have been reported in various pathological conditions characterised by a prothrombotic phenotype, especially all cardiovascular diseases, together with thromb ocythaemi a, antiphospholipid syndrome, cancer and viral infections, including SARS-Cov-2 (Brambilla M, et al., Arterioscler Thromb Vase Biol 2008;28:947-953, Falanga A. et al., Exp Hematol. 2007; 35:702-711; Capozzi A et al., Clin Exp Immunol. 2019; 196:59-66; Tilley RE et al., Thromb Res 2008;122:604-609; Mayne E et al., J Acquir Immune Defic Syndr.2012;59:340-346; Canzano P et al., JACC Basic Transl Sci. 2021; 6(3):202-218).

[0008] However, the evaluation of platelet TF conducted in Brambilla M, et al., Arterioscler Thromb Vase Biol 2008;28:947-953, was not designed to demonstrate residual platelet thrombotic potential, because measurement of TF is not correlated with failure of pharmacological treatment; the authors simply state that aspirin does not reduce TF.

[0009] Pettersen et al (Thrombosis Research Vol 130, no.3, 424-428 2012) analysed soluble markers in patients with stable CAD treated with aspirin, with the aim of identifying the factors responsible for residual platelet reactivity. The results indicate that the plasma TF (not platelet TF) levels, measured with the ELISA technique, are no different in patients with residual platelet reactivity from those who respond to treatment with aspirin.

[0010] Zhu Shengsi et al. (BMC Cardiovascular Disorders, Biomed central, Vol 5, no.l, 15.07.2005, page 22) studied on muscle cells in vitro the effect of sirolimus on TF induction as a possible mechanism for intra-stent thrombosis. TF was measured in cell lysates by the western blot technique and in terms of activity, using the coagulation factor Xa generation assay. The western blot is not a method based on cytofluorometry, although it uses a monoclonal antibody, and it is not applicable to analysis of an individual cell (platelet in whole blood). There is no reference to CAD patients, and above all, no measurements were conducted in vivo on the patients.

[0011] In patent IT 102017000062176, entitled “Method for evaluating residual platelet thrombotic potential in patients undergoing antiplatelet treatment”, the present inventors proposed evaluation of platelet TF in coronary patients undergoing dual antiplatelet therapy (DAPT) with acetylsalicylic acid and clopidogrel for determination of residual platelet thrombotic potential. It was found that 10% of patients undergoing said therapy, despite the good pharmacological response to clopidogrel, exhibited a residual thrombotic risk, as the amount of TF associated with the platelets exceeded the median value measured in the group of patients who failed to respond optimally to the treatment. More recent findings from the same inventors confirm the importance of measuring platelet TF, indicating that high levels of TF-expressing platelets are independent predictors of cardiovascular death in patients who respond correctly to clopidogrel treatment (HR 6.88, 95% CI: 1.0-58.89; p=0.04). However, said prognostic value is not observed in patients undergoing dual antiplatelet therapy who do not respond correctly to clopidogrel, and in whom the antiplatelet action is therefore provided by treatment with acetylsalicylic acid.

[0012] Description of the invention

[0013] It has now been observed that stratifying patients with stable coronary artery disease undergoing chronic therapy with acetylsalicylic acid on the basis of platelet TF measurement, and thereof of platelet thrombotic potential, in a blood sample, significantly improves the prediction of cardiovascular mortality in a 5-year follow-up.

[0014] On the basis of the data already present in the literature, and the data produced by the present inventors, it could not have been expected that TF-expressing platelet levels would improve risk stratification in coronary patients undergoing therapy with acetylsalicylic acid.

[0015] A first object of the invention therefore consists of a method for evaluating residual platelet thrombotic potential in a patient undergoing antiplatelet therapy with acetylsalicylic acid, which comprises determination of platelet TF expression in a patient’s blood sample.

[0016] TF determination can be conducted by known methods or modifications of conventional methods within the reach of one skilled in the art. In particular, flow cytofluorometry is a technique that can conveniently be used to determine platelet TF, as will be described in detail in the experimental part. Other known techniques can be used as an alternative to cytofluorometry, such as ELISA (Enzyme-Linked Immunosorbent Assay), RT-qPCR (Reverse Transcription Quantitative PCR), confocal immunofluorescence microscopy, etc.

[0017] In a second aspect thereof, the invention relates to a kit for evaluation of residual platelet thrombotic potential in a patient undergoing antiplatelet therapy with acetylsalicylic acid which comprises a selective ligand for platelet tissue factor, diluents and fixatives.

[0018] In a preferred form, the ligand is a fluorescent probe-labelled anti-TF mouse monoclonal antibody. The detection system comprises fluorescent probe-labelled anti-IgG mouse antibodies.

[0019] DESCRIPTION OF FIGURE

[0020] Figure 1: Kaplan-Meier survival curves analysed in the population of patients with stable coronary artery disease treated with acetylsalicylic acid only, stratified according to the percentage of TF-expressing platelets. Curve with continuous line: platelet TF <4%; curve with broken line: platelet TF >4%. Panel A shows the all-cause mortality curves, while panel B shows the cardiovascular mortality curves.

[0021] The invention will now be described in detail in the following experimental part, provided by way of example.

[0022] MATERIALS AND METHODS

[0023] Recruitment of patients

[0024] 527 patients with a diagnosis of chronic coronary syndrome (CCS) and acute coronary syndrome (ACS), hospitalised at the Centro Cardiologico Monzino IRCCS in Milan between 2012 and 2018, were recruited to this prospective observational study. In particular, 149 ACS patients without persistent ST-segment elevation who presented within 24 hours of the start of the symptoms, and 378 CCS patients admitted to the hospital for coronary angiography, were included in the study. In all patients, the presence of coronary artery disease was confirmed by coronary angiography. The exclusion criteria were the presence of severe kidney failure (stage 4-5), haemodynamic instability (acute pulmonary oedema, cardiogenic shock) and / or electrical instability (ventricular arrhythmia, high-grade conduction disturbances) or other major clinical complications at the time of admission to hospital. Patients with angina associated with anaemia or other factors such as type 2 acute myocardial infarction, and those with serious heart valve disease, malignant tumours, systemic inflammatory diseases, recent trauma or major surgery, were also excluded. The study, conducted in compliance with the Declaration of Helsinki, was approved by the institutional Ethics Committee. On recruitment, each patient supplied the informed consent form for participation in the study.

[0025] Study protocol

[0026] For each patient included in the study, demographic, clinical, biochemical and echocardiographic data were recorded. The glomerular filtration rate was calculated according to the Modification of Diet in Renal Disease (MDRD) formula. The left ventricular ejection fraction (LVEF) was measured in all patients within 24 hours of admission. The choice of pharmacological treatment and surgical strategy for management of CCS and ACS was left to the doctor’s discretion, on the basis of the current standards of treatment recommended by the guidelines.

[0027] After being discharged from hospital all patients were monitored for a five-year followup period, mainly conducted by means of regularly scheduled out-patient appointments or, in a minority of cases, telephone contacts conducted by dedicated medical personnel. The primary and secondary endpoints of the study were all-cause mortality and cardiovascular mortality after 5 years respectively.

[0028] Blood drawing and platelet isolation

[0029] Whole blood samples were taken during the hospitalisation period with a 19 gauge needle without venous stasis in tubes containing sodium citrate (0.129 M, 1 / 10 volume / volume) (Vacutainer, Becton Dickinson), discarding the first 4 ml. The blood samples were then treated within 15 minutes of sample-drawing.

[0030] Flow cytometry analysis

[0031] Surface expression of platelet activation markers was analysed by flow cytometry as previously described (Brambilla M, et al., Arterioscler Thromb Vase Biol. 2008;28(5):947- 953). Briefly, the samples were incubated with saturating concentrations of mouse monoclonal antibodies recognising human tissue factor (HTF1; Thermo Fisher), P-selectin (APC; Becton Dickinson), PAC1 (FITC; Becton Dickinson). Labelling with an aspecific antibody (Becton Dickinson isotype), and / or with the secondary antibody only, was used to quantify the aspecific labelling signal in all the experiments (AlexaFluor ® 633 labelled IgG; Thermo Fisher). Before use, all antibodies were centrifuged at 17,000 rpm for 5 min at 4°C to remove any aggregate. A total of 10,000 platelets per sample were acquired with a Gallios flow cytofluorimeter (Beckman Coulter), equipped with four solid-state lasers at 488 nm, 638 nm, 405 nm and 561 nm. Flow-check Pro Fluorospheres (Beckman Coulter) were used daily in accordance with the manufacturer’ s instructions to monitor the stability of the flow cytofluorimeter. All the data were analysed with Kaluza analysis software vl.5 (Beckman Coulter), and reported as percentage of cells positive for the activation marker.

[0032] Statistical analysis

[0033] The continuous variables were reported as mean ± standard deviation (SD) if distributed normally, or if not, as median and interquartile range (IQR). The categorical variables were expressed as frequencies and percentages. The comparisons between the continuous variables were conducted with ANOVA, Student’ s “t” test or the Wilcoxon-Mann-Whitney test, while the comparisons between the categorical variables were conducted with the test or Fisher’s exact test. The variables with non-normal distribution were analysed after log transformation of the values. The best cut-off value relating to the percentage of TF-expressing platelets, required for evaluation of the primary endpoint of the study, was calculated by the Euclidean distance method and confirmed by cross-validation analysis conducted as described below. The study sample was divided in half randomly 200 times to create a training set and a test set, and the best cut-off values, and the sensitivity and specificity thereof, calculated in the 200 training set groups, were validated in the patients belonging to the corresponding 200 test set groups. The mean value of each cut-off was considered as the final value. The 5-year survival analysis was conducted with the Cox regression model. A multivariate model was implemented to investigate the possible predictors of the primary and / or secondary endpoint. The survival analysis in groups, reported with the Kaplan-Meier method, was analysed with the log-rank test. The analyses were also conducted after adjustment for age, CAD diagnosis (CCD vs ACS), antiplatelet therapy (single vs dual antiplatelet therapy - DAPT) and kidney function. A value was considered statistically significant when p was <0.05. The statistical analyses were conducted with SAS software, version 9.4 (SAS Institute, Cary, NC, USA). RESULTS

[0034] 1~ Characteristics of population included in the study

[0035] The characteristics of the population included in the study are reported in Table 1. Almost all the patients (92.4%) were undergoing therapy with low-dose acetylsalicylic acid. In particular, 48.9% were undergoing single antiplatelet therapy with acetylsalicylic acid, while 46.8% were undergoing dual antiplatelet therapy (DAPT) with acetylsalicylic acid and P2Y12 receptor antagonists. On recruitment, 71.7% (•: 378) of patients were suffering from chronic coronary syndrome , while 28.3% (n=149) of patients suffered from ACS.

[0036] Table 1. Case history characteristics of population included in the study.

[0037] 2-The percentage of TFposplatelets is an independent predictor of all-cause mortality.

[0038] Multivariate Cox Regression Analysis conducted on the entire population recruited indicated that of the platelet activation markers analysed (TF expression, activated GpIIblla and P-selectin, and percentage of platelet-monocyte aggregates), only TF was an independent predictor of all-cause mortality (HR=2.021, 95% CI 1.026-3.982; Table 2).

[0039] Table 2. Multivariate Cox Regression Analysis.

[0040] Hazard Ratio CI 95% P value

[0041] TFposPLT 2.021 1.026 3.982 0.0419

[0042] GPIIbIIIaposPLT 2.461 0.318 19.033 0.3883

[0043] P-selectinp0SPLT 0.814 0.154 4.321 0.8095

[0044] Platelet-Monocyte j503 0.445 5.07 0.5115 aggregates

[0045] TF, tissue factor; GPIIbllla, glycoprotein Ilbllla. In the ROC curve analysis, the best cut-off value regarding the percentage of TF- expressing platelets for forecasting the primary endpoint was 3.93%; said value was confirmed by cross-validation analysis (as described in detail in the Methods section). The patients stratified above and below said threshold amounted to 34.3% (181) and 65.6% (346) respectively of the population included in the study. In particular, in the first group, the median percentage of TF -expressing platelets was 8.97% [IQR 6.33-15.12] while in the second group it was 0.81% [0.27-1.80] (p<0.0001); the latter levels were comparable with the levels measured in healthy volunteers (Canzano P et al., JACC BTS 2021; 6(3):202-218). The expression of the classic platelet activation markers fell within the reference range for the healthy volunteers (Canzano P et al., JACC BTS 2021; 6(3):202-218), but a slight difference was observed between the two groups (Table 3).

[0046] Table 3. Percentage of platelets expressing activation markers in the total study population (All) and after stratification on the basis of TFposplatelet levels.

[0047] The data are reported as median [IQR]. GPIIbllla, glycoprotein Ilbllla.

[0048] Table 4 shows the clinical and case-history characteristics of the patients recruited, broken down by TFposplatelet levels. In the groups compared, the percentage of patients treated with acetylsalicylic acid alone, and the percentage of patients undergoing DAPT, was similar (45.1% vs 45.8%; and 45.2% vs 49.7%, in the group with TFposplatelets >4% and <4% respectively). The group of patients with TFposplatelets >4% was significantly older (p=0.03), with greater electrical instability (p=0.03) than the group with TFposplatelets <4%. No statistically significant differences emerged between the two groups for any of the other parameters evaluated. Table 4. Case history characteristics of the population included in the study, stratified on the basis of TFposplatelet levels. 3- The percentage of TFposplatelets predicts all-cause mortality and cardiovascular mortality in patients undergoing therapy with aspirin.

[0049] In the cohort of subjects recruited, the incidence of all-cause mortality was 30.6 and 16.1 per 1000 person-years (p=0.0192) in patients with a percentage of TFposplatelets above and below the cut-off respectively. The annual CV mortality rate was significantly higher in the patients with a TFposplatelet value >4% (23.3 and 9.3 per 1000 person-years above and below the cut-off respectively, p=0.0058) with HR=2.51 (95% CI: 1.278-4.95; p=0.0057). Said associations persisted after adjustment for age, diagnosis (CCD or ACS), estimated glomerular filtration rate and pharmacological antiplatelet therapy (HR=2.45, 95% CI: 1.23-4.87; p<0.0001).

[0050] The association of TFposplatelet levels with all-cause mortality and CV mortality on the basis of pharmaceutical antiplatelet therapy was evaluated in sub-group analyses.

[0051] The patients treated with DAPT (n=246, 46.8%), broken down on the basis of their therapeutic response to P2Y 12 antagonists (n=136 responders, n=l 10 non-responders according to the VASP test), demonstrate that the responders with TF1®5-platelet levels >4% were those with the greatest risk of all-cause mortality (HR: 4.11, 95% CI: 1.11-15.17; p=0.021) and CV mortality (HR 6.88, 95% CI: 1.0-58.89; p=0.041) with a sensitivity and specificity of the cutoff value for CV mortality of 83% and 60% respectively. Conversely, TFposplatelet levels >4% were not predictive of events in the ACS patients who were non-responders to pharmacological therapy with clopidogrel, in whom the only antiplatelet action was therefore provided by the treatment with acetylsalicylic acid.

[0052] Conversely, the survival analysis conducted over a 5-year follow-up period on patients with stable chronic coronary artery disease undergoing therapy with acetylsalicylic acid only (n=239, 45.3%), described in Figure 1 by Kaplan-Meier curves, demonstrated that the percentage of circulating platelets expressing TF was predictive of both all-cause mortality (HR=3.03, 95% CI: 1.29-7.08) and cardiovascular mortality (HR=3.56, 95% CI: 1.40-9.05; Figure 1), with a sensitivity and specificity for the latter of 67% and 68% respectively.

Claims

CLAIMS1. A method for the evaluation of residual platelet thrombotic potential in a patient with stable coronary artery disease undergoing antiplatelet therapy with acetylsalicylic acid, said method comprising the determination of platelet tissue factor (TF) expression in a patient’s blood sample.

2. A method according to claim 1 wherein the determination of TF is performed by flow cytometry.

3. A kit for evaluating residual platelet thrombotic potential in a patient undergoing antiplatelet therapy with acetylsalicylic acid comprising a selective ligand for platelet tissue factor, a detection system for said ligand, diluents and fixatives.

4. A kit according to claim 3, wherein the ligands are anti-TF mouse monoclonal antibodies.

5. A kit according to claim 4, wherein the detection system comprises fluorescent probe-labelled anti-TF mouse monoclonal antibodies.

6. A kit according to claim 4, wherein the detection system comprises fluorescent probe-labelled anti-IgG mouse antibodies.