Aspirin insensitive thrombophilia: transcript profiling of blood identifies platelet abnormalities and HLA restriction

Abstract

Aspirin is the most widely used antiplatelet agent because it is safe, efficient, and inexpensive. However, a significant subset of patients does not exhibit a full inhibition of platelet aggregation, termed 'aspirin resistance' (AR). Several major studies have observed that AR patients have a 4-fold increased risk of myocardial infarction (MI), stroke, and other thrombotic events. Arachidonic acid-stimulated whole blood aggregation was tested in 132 adults at risk for ischemic events, and identified an inadequate response to aspirin therapy in 9 patients (6.8%). Expression profiling of blood RNA by microarray was used to generate new hypotheses about the etiology of AR. Among the differentially expressed genes, there were decreases in several known platelet transcripts, including clusterin (CLU), glycoproteins IIb/IIIa (ITGA2B/3), lipocalin (LCN2), lactoferrin (LTF), and the thrombopoetin receptor (MPL), but with increased mRNA for the T-cell Th1 chemokine CXCL10. There was a strong association of AR with expression of HLA-DRB4 and HLA-DQA1. Similar HLA changes have been linked to autoimmune disorders, particularly antiphospholipid syndrome (APS), in which autoantibodies to phospholipid/protein complexes can trigger platelet activation. Consistent with APS, AR patients exhibited a 30% reduction in platelet counts. Follow-up testing for autoimmune antibodies observed only borderline titers in AR patients. Overall, these results suggest that AR may be related to changes in platelet gene expression creating a hyperreactive platelet, despite antiplatelet therapy. Future studies will focus on determining the protein levels of these differential transcripts in platelets, and the possible involvement of HLA restriction as a contributing factor.

Copyright © 2013 Elsevier B.V. All rights reserved.

Figures

Fig. 1

Distribution of aspirin resistance scores in patient samples. Platelet function was evaluated on a cardiology outpatient population (n = 132), after 1 week on low dose aspirin (81 mg/day). The residual platelet aggregation was measured by VerifyNow ASA using AA-stimulated whole blood aggregometry. The rate of aggregation is reported as aspirin response units (ARU) with higher values indicating increased aggregation despite aspirin treatment. ARUs above 550 are defined as aspirin resistant (AR). Blue bars indicate the frequency of ARUs within the bin range reported on X-axis. Red line indicates a normal distribution based on the mean (453.9 ARU) and standard deviation (52.1) of the sample.

Fig. 2

Heatmap of transcripts used for Support Vector Machine classification of aspirin sensitive versus resistant patients. Microarray analysis was conducted on whole blood RNA from subjects with varying antiplatelet responses to aspirin. DEGs were identified by a combined fold-change (>1.5 folds) and t test cut-off (p

Fig. 3

Scatter plot of transcripts in aspirin-sensitive versus aspirin-resistant groups. Gene expression levels were compared between aspirin-resistant (Y-axis) and aspirin-sensitive patients (X-axis) with absolute expression plotted on a log2 scale color-coded from low expression (below median expression, blue), to high expression (red). Each point reflects one transcript of the 54,000 transcripts quantitated on the Affymetrix U133 + 2 microarray. Transcripts deviating either up or down in aspirin-resistant patients, and with potential relevance to thrombotic pathways, are marked with their gene symbols, and more detailed levels shown in Table 3.

Fig. 4

Expression from HLA-DR and HLA-DQ alleles in patients with varying sensitivity to the antiplatelet effect of aspirin. The expression levels of HLA-DR and HLA-DQ alleles shown in Table 3 were analyzed with respect to which alleles were expressed by the patients as a function of their response to the antiplatelet effect of aspirin. All of the sensitive (AS) subjects were expressing from DQA1 and DQA5 alleles, but none (0%) were expressing from DRB4 alleles, while the high normal (HN) or resistant (AR) had a significantly higher percentage using DRB4 alleles and much lower percentage using both DQA1 and DQA5 alleles, tending to use just one or the other.

Fig. 5

Pathway analysis of transcripts modulated in blood associated with aspirin resistance. The differentially expressed genes (Table 3, and Supplementary Table 2) from patients with aspirin resistance were analyzed by an automated comparison to a database of known gene–gene interactions (Ingenuity Pathway Analysis). A high scoring pathway of platelet adhesion is illustrated using transcript abbreviations and linear connections defining known relationships. The core of the pathway emanates from a reduction in glycoproteins IIb (ITGA2B) and IIIa (ITGB3), which are the principal platelet fibrinogen receptors. Neutrophil elastase (ELANE), which is decreased in AR, has an activating effect upon IIb/IIIa. Transcripts elevated in AR are shown in red, decreased transcripts are shown in green.

Fig. 6

Schematic model of two potential etiologies of aspirin resistance. Using primary antiplatelet syndrome (PAPS) as a model, the general molecular and cellular relationships of the proposed etiology of AR is shown in the upper left. It is commonly believed that autoimmune syndromes such as PAPS or lupus, have an infectious trigger, in which a viral or bacterial antigen is processed through the immunoproteasome in antigen-presenting cells, and presented on HLA to generate a cross-reactive B-cell antibody clone. Certain HLA combinations are more likely to generate auto-reactive antibodies, in this case, reacting against platelet antigens, which leads to platelet activation, possibly via chronic complement reactivity. Alternatively, as shown in the lower right, the primary defect may reside in megakaryocytes, whereby an infectious or inflammatory event may affect the megakaryocyte population and lead to production of platelets with an aberrant repertoire of mRNA, as observed in the present transcript profiling, thereby leading to elevated susceptibility to aggregation.