The main findings in this study are as follows. (i) Aspirin ‘resistance’ (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. (ii) The platelet count contributes to pre-aspirin platelet reactivity as measured by serum TXB2 and the VerifyNow Aspirin Assay, but does not contribute to post-aspirin platelet reactivity. (iii) Aspirin resistance defined by serum TXB2 shows a poor correlation with aspirin resistance defined by five other widely used assays: urinary 11-dehydro TXB2, arachidonic acid-induced platelet aggregation, ADP-induced platelet aggregation, VerifyNow Aspirin Assay, and the TEG PlateletMapping System.
Aspirin ‘resistance’: role of pre-existent platelet reactivity
We have previously demonstrated, in a study of 700 aspirin-treated patients with coronary artery disease, that residual platelet activation after the in vitro addition of arachidonic acid occurred in direct proportion to the degree of baseline platelet activation (i.e. no in vitro addition of arachidonic acid) . This finding raised the question as to whether measurements of ‘aspirin resistance’ reflect, at least in part, baseline heterogeneity in platelet response . Therefore, a major goal of the present study was to determine whether the variability in platelet response after aspirin is partly explained by a pre-existent variability in platelet response. To enable pre-aspirin samples to be drawn, this study was performed in normal subjects rather than in patients, because virtually all clinically relevant patients are already taking aspirin. An additional advantage of the use of normal subjects rather than patients is that the platelet response to stimuli of normal subjects is not influenced (with resultant increased scatter of the data) by an underlying disease (e.g. coronary artery disease), which is well known to cause platelet hyperreactivity [1,20,21]. Thus, the use of normal volunteers in this study rather than patients enabled us to distinguish true aspirin resistance from underlying platelet hyperreactivity, an important point of confusion in the literature . For example, two recent meta-analyses of published studies provided evidence for an association of laboratory-defined aspirin resistance with a higher risk of recurrent cardiovascular events [10,11]. However, these meta-analyses and their 20 included studies did not distinguish between (i) underlying platelet hyperreactivity with resultant high residual platelet reactivity and (ii) true aspirin resistance. In the present study, by analyzing blood samples pre- and post-aspirin, we demonstrate that the phenomenon of ‘aspirin resistance’ is, in part, the result of underlying platelet hyperreactivity, and therefore unrelated to aspirin. The present study does not address the question as to whether or not a higher dose of aspirin, or other additional antiplatelet therapy, would be clinically beneficial in patients with high residual platelet reactivity after aspirin therapy. Definitive evidence in this regard will require a randomized trial that guides antiplatelet therapy based on the results of a platelet function test .
The proportion of post-aspirin platelet function predicted by pre-aspirin platelet function is 28.3 ± 7.5% (mean ± asymptotic standard error) for serum TXB2, 39.3 ± 6.8% for urinary 11-dehydro TXB2, 4.4 ± 7.7% for arachidonic acid-induced platelet aggregation, 40.4 ± 7.1% for ADP-induced platelet aggregation, 26.3 ± 9.2% for the VerifyNow Aspirin Assay, and 45.0 ± 10.9% for the TEG PlateletMapping System with arachidonic acid as the agonist (Table 1). (The explanation for the low correlation coefficient with arachidonic acid-induced platelet aggregation is that the post-aspirin samples were so inhibited that there is little variation in these values.) The factors that contribute to this pre-existent variability in platelet function are unclear. In this study, platelet count was found to contribute to pre-existent variability in two of the assays (serum TXB2 and the VerifyNow Aspirin Assay) but not to contribute to post-aspirin variability in any of the assays. A number of other factors have been shown to contribute to variations in platelet reactivity, including diet , platelet turnover , and single nucleotide polymorphisms (especially in platelet signaling molecules) . However, further studies are needed to identify the specific factors that contribute to both pre-aspirin and post-aspirin platelet response variability.
Aspirin ‘resistance’: correlation between platelet function tests
As recently summarized by Cattaneo , the term ‘resistance’ to a drug should be used when a drug is unable to hit its pharmacological target, as a result of inability to reach it (as a consequence of reduced bioavailability, in vivo inactivation, or negative interactions with other substances) or alterations of the target. Based on this definition, the term ‘aspirin resistance’ should be limited to situations in which aspirin is unable to inhibit COX-1-dependent TXA2 production [7,8]. Even when arachidonic acid, the precursor of TXA2, is used as a platelet agonist, the results may overestimate the prevalence of aspirin resistance [7,8]. The concentration of 11-dehydro TXB2, a urinary metabolite of TXB2, may be partly dependent on extraplatelet sources of TXA2 . Therefore, the most specific test for ‘aspirin resistance’ is serum TXB2, which directly reflects the capacity of platelets to synthesize TXA2, of which TXB2 is a stable metabolite [7,8]. For this reason, in the present study we prospectively considered serum TXB2 to be the most definitive marker of aspirin ‘resistance’, because aspirin specifically inhibits COX-1 and therefore directly inhibits the generation of TXA2, and its stable metabolite TXB2 . However, there is no standard definition in the literature for the specific cutoff of serum TXB2 concentration to define ‘aspirin resistance’. In this study, we therefore used two different methods to define the TXB2 cutoff. First, we used an ROC curve-generated post-aspirin TXB2 cutoff of >12 ng mL−1, which has the advantage that it specifically applies to our study’s subject population and aspirin dose. Secondly, we used a post-aspirin TXB2 cutoff of >2.2 ng mL−1, based on the experimental data of Maree et al. . In patients taking aspirin 75 mg daily, these investigators  demonstrated that when serum TXB2 levels exceed 2.2 ng mL−1, platelets continue to generate TX from exogenous arachidonic acid, indicating the presence of uninhibited COX-1 and therefore an incomplete response to aspirin.
Irrespective of the serum TXB2 cutoff (12 ng mL−1 or 2.2 ng mL−1), we found that those subjects defined as aspirin-resistant by serum TXB2 were not the same subpopulation of subjects who were identified as aspirin-resistant by other widely used assays: urinary 11-dehydro TXB2, arachidonic acid-induced platelet aggregation, ADP-induced platelet aggregation, VerifyNow Aspirin Assay, and the TEG PlateletMapping Assay [see Figs 2 and 3, in which the black squares in each panel indicate subjects who are aspirin-resistant defined by a serum TXB2 of either >12 ng mL−1 (Fig. 2) or >2.2 ng mL−1 (Fig. 3)]. Even when residual post-aspirin platelet function was assessed by correlation of continuous results rather than categorical analysis using a cutoff, none of the platelet function assays showed a significant correlation with the gold standard serum TXB2 (Table 4B). A number of previous studies [17,24,30–35] have shown a poor correlation between different assays for aspirin resistance but, unlike the present study, most of these studies did not use serum TXB2 as a point of comparison and, also unlike the present study, the dose of aspirin varied. Of these studies, only the small study of Gonzalez-Conejero et al.  and the large study of Becker et al.  examined whether the variability in platelet response after aspirin is partly explained by the pre-existent variability in platelet response; both found that pre-aspirin platelet function contributed to the variance in platelet reactivity after aspirin therapy. However, unlike the present study, neither the study by Gonzalez-Conejero et al.  nor the study by Becker et al.  measured serum TXB2.
Clinically relevant definitions of aspirin resistance can only be based on data linking laboratory tests to poor clinical outcomes in patients [4,10,11]. There is evidence from small clinical studies of an association between subsequent poor clinical outcomes in aspirin-treated patients and urinary 11-dehydro TXB2 [14,36], arachidonic acid-induced platelet aggregation [15,37], ADP-induced platelet aggregation [15,37], VerifyNow Aspirin Assay [16,37,38], and the TEG PlateletMapping System . However, the present study demonstrates that in aspirin-treated subjects none of these five assays is significantly correlated with the most definitive marker of aspirin resistance: serum TXB2 (Table 4B, first row). However, three of these assays – arachidonic acid-induced platelet aggregation, ADP-induced platelet aggregation, and the VerifyNow Aspirin Assay – correlated with each other (Table 4B). These findings suggest that the residual post-aspirin activity measured by these three assays may be partly COX-1-independent, consistent with other recent data from our group [26,39] and others [35,40]. Furthermore, given that arachidonic acid-induced platelet aggregation, ADP-induced platelet aggregation, and the VerifyNow Aspirin Assay results have been shown to correlate with clinical outcomes [15,16,37,38], but serum TXB2 has not , our present data demonstrate that hyporesponsiveness to aspirin as defined by platelet aggregation and/or the VerifyNow Aspirin Assay cannot be assumed to reflect aspirin resistance as defined by aspirin’s inability to suppress TXB2.