More on: aspirin resistance: position paper of the Working Group on Aspirin Resistance. Proposal for a Laboratory Test Guiding Algorithm


Fabio M. Pulcinelli, Dipartimento di Medicina Sperimentale e Patologia, Università degli Studi ‘La Sapienza’, Viale Regina Elena 324, 00161 Rome, Italy.
Tel.: +39 6 49973002; fax: +39 6 4454820; e-mail:

The position paper of the Working Group on Aspirin Resistance has recently been published, indicating that there is evidence that major adverse clinical events (MACE) can be predicted by some tests of aspirin resistance [1]. However, the low number of MACE and the variability in the laboratory tests so far employed in the different trials [1–3] have not allowed a unequivocal interpretation of the results obtained, nor universal cut-off values of all the in vitro assays performed to assess the platelet function.

For this reason, ad hoc clinical trials are necessary not only to understand the clinical relevance of the phenomenon of aspirin resistance, but also to identify in vitro measures of platelet activation that, either alone or in combination, might hint at predicting vascular events and even at altering an aspirin-based therapy in ‘resistant’ patients. Therefore, in our opinion, the first step that has to be taken is the identification of a standardized laboratory procedure that future research trials should follow in order to address the problem.

By analyzing the different mechanisms of platelet activation, and taking into account the classification of Weber et al. [4], we can assume that platelet resistance to aspirin can be ascribed to two main causes:

  • 1the capability of platelets still to produce thromboxane A2 (TxA2), even at very low concentrations, despite aspirin treatment, because of pharmacokinetic or pharmacodynamic problems;
  • 2platelet activation independently of TxA2 formation, possibly linked to the presence of polymorphisms of platelet receptors or of pro-aggregating molecules.

Moreover, the involvement of other circulating cells in providing precursors (PGH2) or biosynthesized TxA2, bypassing COX-1, should be considered [1].

The impetus of gaining important information that might reliably predict treatment failure and clarify the role of ‘aspirin resistance’ in MACE [1,5] makes combined platelet functional and biochemical laboratory tests desirable.

In the present letter, we propose a procedure that, with a single blood sample, allows to investigation of most of the platelet metabolic pathways involved in the phenomenon.

Patient's non-compliance to the drug has to be also regarded as a confounding problem and has been taken into account.

Platelet aggregation (Born's method) should be evaluated on platelet-rich plasma (PRP) in response to ADP, collagen and arachidonic acid (AA). The supernatant of these samples can be collected in order to measure the amount of TxA2 produced by the platelets in response to such agonists. To further investigate the source of an eventual persistence of TxA2, platelet aggregation and TxA2 formation should also be evaluated in samples treated in vitro with aspirin as a COX-1 inhibitor, with a COX-2 inhibitor, or with both inhibitors.

Samples of plasma, buffy coats, and platelet lysates can also be stored for later additional studies. The complete procedure is depicted in Fig. 1.

Figure 1.

Flow chart depicting the procedure described in the text. PA, platelet aggregation; TxA2, thromboxane A2; >, unmodified; <, reduced. The suggested laboratory tests are indicated in bold, while the possible mechanisms involved are indicated in capital letters.

The inhibition of both agonist-induced platelet aggregation and TxA2 production should identify those patients sensitive to the inhibitory action of aspirin. A consistent platelet aggregation occurring despite the inhibition of platelet TxA2 indicates a TxA2-independent platelet activation and the need for more investigation concerning the presence of glycoprotein polymorphisms (buffy coat) and of eventual pro-aggregating molecules (plasma). The lack of inhibition of collagen- and AA-induced platelet aggregation and of TxA2 production points to dose salicylates on plasma samples, in order to assess patient's compliance to aspirin. While the absence of salicylates can be ascribed to non-compliance or to inadequate dosage, detectable levels indicate an ineffective inhibitory action of aspirin on TxA2 production. A further in vitro treatment with aspirin or with a COX-2 inhibitor might be useful to identify the source of the persistent TxA2 generation. However, in order to ascribe TxA2 production to COX-2 activation, the evaluation of in vitro treatment with only the COX-2 inhibitor cannot prove sufficient, and therefore aspirin should be added concomitantly; the results should be compared with those obtained by adding aspirin alone. The lack of inhibition of platelet aggregation and TxA2 production despite the further in vitro addition of aspirin suggests investigation of the potential presence of COX-1 polymorphism (buffy coat). If the inhibition of platelet aggregation and TxA2 production is achieved by concomitantly inhibiting COX-1 and COX-2, the presence of COX-2 in platelet lysates should be evaluated.

Other biological fluids, such as serum and urine samples, allows the study of TxA2 biosynthesis that involves both platelet and extraplatelet COX activation. The method presented here appears more suitable for the study of platelet-derived TxA2, as it allows study of the effects of in vitro treatment with several inhibitors. However, serum and urine samples should, nonetheless, be collected and stored for comparative studies. In the case of a significant discrepancy between serum and agonist-stimulated platelet TxA2, COX-2 expression in inflammatory cells should be analyzed.

Another advantage that makes the above-described procedure a good first-level test is that it can be easily reproduced by any laboratory simply competent for platelet aggregation, as all additional analysis can be performed by more specialized centers on stored material.

In conclusion, we believe that if future clinical trials take into consideration, even in part, the presented procedure, it will be possible to obtain much information concerning ‘aspirin resistance’.


This paper was partially supported by a Grant from the Italian Ministry of Education, University and Scientific Research (MIUR) ‘ex 60% Ateneo 2002, to F.M.P’.