The present data indicate that administration of 200/300 mg ASA OD is insufficient for optimal inhibition of platelet recruitment in the presence of RBC or WB in a high proportion of patients with vascular disease (51 and 41% respectively - ). This is in agreement with our previous research 11
. The extent of recruitment is strongly modulated by platelet-erythrocyte interactions3,8
, in a manner which is partially inhibitable by ASA10,11
. Therefore, we studied ASA regimens that would inhibit platelet COX-1-dependent responses as well as the ASA-inhibitable RBC effect in patients with vascular diseases. This would enhance down-regulation of platelet reactivity in the physiological milieu of WB. This had previously been achieved in normal subjects with an intermittent loading dose (500 mg) at 2-week intervals supplemented with 50 mg ASA OD10
. In sharp contrast to the reduction in platelet responsiveness observed in normal subjects10
, the L+50−OD or L+100−OD regimens resulted in a significant increase in the number of patients with insufficient inhibition of platelet recruitment. The latter was evaluated in PRP alone, in PRP plus RBC, and in whole blood, and compared to 200/300 mg OD (). We observed that 87% (L+50−OD) and 58% (L+100−OD) of patients had insufficient inhibition of platelet recruitment in WB (). This paralleled results with combined PRP plus RBC () and increased 14
C-5HT release (). These less than optimal ex vivo
results may be clinically relevant because 81–100 mg ASA OD is widely used for secondary prevention of occlusive vascular diseases. These patients may have had a greater proportion of activated platelets in their circulation, and may represent a group of patients at increased risk for a vascular event.
The difference in optimal dosage with normal subjects10
in all probability reflects greater platelet reactivity in patients. This could be a consequence of underlying vascular disease or increased platelet turnover12
, which results in a greater number of ASA-free platelets into the circulation. The latter is certainly a contributory factor as indicated by the strong reduction in percentage of patients with insufficient inhibition with L+50−BID compared to L+100−OD, especially in PRP alone (14.1% vs.
48.5%), but also in the presence of erythrocytes: PRP+RBC (47.4 vs.
72%) or WB (39 vs.
57.6%) (). This confirms our previous experimental data in which 10% of ASA-free platelets (the estimated daily platelet turnover) mixed with autologous ASA-treated platelets and RBC induced a marked increase in platelet recruitment8
. A similar phenomenon was demonstrated during induction of experimental thrombosis in animals in vivo13
. Furthermore, newly formed platelets with an active cyclooxygenase are detectable as early as 4 hours after ASA ingestion14
. Therefore, ensuring that circulating platelets are fully aspirin-treated is critical for optimal control of platelet reactivity.
Increasing the daily dose from L+50−BID to L+100−BID did not modify the percentage of patients with insufficient inhibition of recruitment evaluated in PRP alone (13–14%, ) or 14
C-5HT release (). Since TXA2
synthesis was blocked (), these platelet responses are COX-independent. As previously shown, such responses can be increased by either ASA-free or ASA-treated RBC8
. Importantly, the L+100-BID regimen drastically reduced by 50% the percentage of patients with insufficient inhibition when recruitment was evaluated in PRP plus RBC or WB as compared to other regimens (). This demonstrates increased blockade of the ASA-sensitive prothrombotic effect of RBC in the patients by this ASA regimen, which is also shown by a reduction in 14
C-5HT release. The enhancing effect of erythrocytes on platelet reactivity may be a contributory factor to the phenomenon known as “aspirin resistance”, defined as a higher degree of platelet reactivity than expected in ASA-treated patients. As demonstrated herein, this could be markedly reduced by altering the ASA regimen. With the L+100−BID treatment, 80% of patients had optimal inhibition in WB. Interestingly, this is achieved with a change in timing of ASA administration, rather than in the total amount of ASA ingested. If we calculate the loading dose (500 mg) plus the daily dose (100 mg BID for 14 days), the ASA ingested is equivalent to 221 mg/day in each cycle, within the range (75–325 mg) of ASA recommended for treatment15–17
. Remarkably, with the L+50−BID regimen (equivalent to an average of 129 mg/day in the 14-day cycle), the proportion of patients with optimal inhibition was similar to the 200/300 mg OD regimen, with only about half the amount of ASA administered.
We did not further increase the aspirin dose in the remaining 20% of patients who did not achieve optimal inhibition with the L+100−BID regimen in WB (). Of those patients, optimal control was not achieved in 13% due to responsiveness in platelets alone (). In the remaining 7%, platelets were inhibited, but blockade of the ASA-sensitive prothrombotic effect of RBC was not fully achieved. This resulted in an increased response in WB (). Since TXA2
generation was blocked (), the platelet responses were elicited by mechanisms independent of COX-1 and/or COX-2. These would include formation of aspirin-insensitive eicosanoids such as 8-iso-PGF2α18
, protein tyrosine phosphorylation4
, other signal transduction mechanisms or genetic variations in platelet proteins19
Platelet activation as evaluated by 14
C-5HT secretion from dense granules was increased by RBC and reduced by ASA (), as previously reported3,10,11
. The reduction was greatest in patients treated with the L+100−BID regimen and as a result, 5HT release was lowest in the presence of RBC or in WB in this group of patients. Reduction of released 5HT from activated platelets (a vasoconstrictor and platelet agonist20
) reflects a reduction in the detrimental effects of continuously elevated platelet reactivity.
We observed an individual variability of aspirin effects in patients depending on the specific dose-regime and the prothrombotic effect of RBC. However, in our patient population, all ASA regimes significantly reduce platelet activation (5HT, TXA2
) and recruitment. The latter is in accordance with the beneficial effect of aspirin at any dose found in a recent meta-analysis of ASA clinical trials1
, resulting from the fact that ASA always reduces platelet reactivity to some extent. However, composite data from such studies may tend to mask individual variability in responsiveness to the drug23
A correlation between the effect of aspirin on platelet reactivity ex vivo
and clinical outcomes was reported by others5,21,22
. Thus, the parallel blockade of platelet reactivity and the aspirin-sensitive component of the prothrombotic effect of erythrocytes in a large proportion of patients with vascular disease may enhance the clinical benefit of aspirin treatment.
In conclusion, the administration of an intermittent biweekly dose of 500 mg ASA along with 100 mg ASA every 12 hours provides optimal inhibition of platelet recruitment in WB in 80% of patients with previous cardiovascular or cerebrovascular events. This reduced by 50% the proportion of patients who did not achieve optimal inhibition in WB from 41% on a regimen of 200 or 300 mg ASA OD to 20% on our L+100−BID regimen. Patients not achieving optimal inhibition may require additional platelet inhibitory therapy, alone or in combination with ASA. Nevertheless, further studies would be necessary concerning the association between ex vivo platelet reactivity and clinical events.