Justo Aznar

BACKGROUND: Aspirin (acetylsalicylic acid, ASA) is widely used for secondary prevention of ischemic vascular events, although its protection only occurs in 25% of patients. We previously demonstrated that platelet reactivity is enhanced by a prothrombotic effect of erythrocytes in a thromboxane-independent manner. This diminishes the antithrombotic therapeutic potential of ASA. Recent data from our laboratory indicate that the prothrombotic effect of erythrocytes also contains an ASA-sensitive component. In accordance with this observation, intermittent treatment with high-dose ASA reduced the prothrombotic effects of erythrocytes ex vivo in healthy volunteers. In the present study, the effects of platelet-erythrocyte interactions were evaluated ex vivo in 82 patients with vascular disease: 62 patients with ischemic heart disease treated with 200 mg ASA/d and 20 patients with ischemic stroke treated with 300 mg ASA/d. METHODS AND RESULTS: Platelet activation (release reaction) and platelet recruitment (fluid-phase proaggregatory activity of cell-free releasates from activated platelets) were assessed after collagen stimulation (1 microg/mL) of platelets, platelet-erythrocyte mixtures, or whole blood. Platelet thromboxane A2 synthesis was inhibited by >94% by ASA administration in all patients. Importantly, platelet recruitment followed one of three distinct patterns. In group A (n=32; 39%), platelet recruitment was blocked by ASA both in the presence and absence of erythrocytes. In group B (n=37; 45%), recruitment was abolished when platelets were evaluated alone but continued in the presence of erythrocytes, indicating a suboptimal effect of ASA on erythrocytes of this patient group. In group C (n= 13; 16%), detectable recruitment in stimulated platelets alone persisted and was markedly enhanced by the presence of erythrocytes. CONCLUSIONS: In two thirds of a group of patients with vascular disease, 200 to 300 mg ASA was insufficient to block platelet reactivity in the presence of erythrocytes despite abolishing thromboxane A2 synthesis. Platelet activation in the presence of erythrocytes can induce the release reaction and generate biologically active products that recruit additional platelets into a developing thrombus. Insufficient blockade of this proaggregatory property of erythrocytes can lead to development of additional ischemic complications.

Activated platelets release biologically active compounds, which then recruit additional platelets into an evolving thrombus. We studied activation of alpha(IIb)beta(3) and exposure of P-selectin on platelets recruited by releasates obtained from collagen-treated platelets and evaluated modifications in prothrombotic effects of releasates induced by platelet-erythrocyte interactions and aspirin treatment. Releasates from collagen-stimulated platelets induced alpha(IIb)beta(3) activation and P-selectin exposure (monitored by flow cytometry using fluorescein isothiocyanate-PAC-1 and phycoerythrin-CD62 antibodies). These responses were markedly amplified by releasates from combined platelet-erythrocyte suspensions. This finding demonstrates a novel mechanism(s) by which erythrocytes intensify platelet aggregability and mediate increased platelet recruitment. Because P-selectin and alpha(IIb)beta(3) are potential sites for platelet-leukocyte interactions, erythrocytes may also modulate leukocyte recruitment. Following aspirin ingestion both the recruiting capacity of platelet releasates and erythrocyte-induced amplification of platelet recruitment were down-regulated. These events represent an additional antithrombotic property of aspirin. We also examined the possibility that arachidonic acid, or eicosanoids derived therefrom, can induce a prothrombotic activity of erythrocytes. The TXA(2)-analog U46 619 and free arachidonate, but not PGI(2) or 12-HETE, induced increases in cytosolic Ca(++) and promoted phosphatidylserine (PS) exposure on a subpopulation of erythrocytes. PS exposure and increases in erythrocyte [Ca(++)](i) are associated with enhanced procoagulant activity, increased endothelial adhesion, and reduced erythrocyte deformability. Our findings, therefore, suggest that TXA(2) and arachidonic acid, derived from activated platelets, induce a prothrombotic phenotype on erythrocytes in proximity. We conclude that by these mechanisms, erythrocytes can actively contribute to platelet-driven thrombogenesis and microvascular occlusion.