Erythrocytes promoted platelet reactivity in a plasma medium, as demonstrated in an in vitro system that independently evaluated the biochemistry of platelet activation and recruitment. The prothrombotic erythrocyte effects were metabolically regulated, as evidenced by lack of activity of ATP-depleted or glutaraldehyde-fixed erythrocytes. They occurred in the absence of cell lysis as verified by lactate dehydrogenase assays, and had an absolute requirement for platelet activation. The presence of erythrocytes induced a twofold increase in platelet thromboxane B2 (TXB2) synthesis upon collagen stimulation, indicating that erythrocytes modulated platelet eicosanoid formation. Cell-free releasates from stimulated platelet-erythrocyte suspensions, which exhibited increased recruiting capacity, contained 6.9-fold more ADP and 4.9-fold more ATP than releasates from stimulated platelets alone. Following aspirin ingestion, TXB2 formation was blocked, but erythrocyte promotion of platelet reactivity persisted at those doses of collagen that reinduced platelet activation. Moreover, when platelet mixtures consisted of as little as 10% obtained before aspirin plus 90% obtained post-aspirin ingestion, significant erythrocyte enhancement of platelet reactivity occurred, even at low agonist concentrations. These erythrocyte effects would decrease the therapeutic potential of inhibition of platelet cyclooxygenase by aspirin. The erythrocyte- induced modulation of platelet biochemistry and function emphasizes the importance of cell-cell interactions in stimulus-response coupling.

Activated platelets release biologically active compounds, which then recruit additional platelets into an evolving thrombus. We studied activation of ␣ IIb ␤ 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 ␣ IIb ␤ 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 Pselectin and ␣ IIb ␤ 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 downregulated. 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. IntroductionWhen platelets interact with the subendothelial matrix of an injured vessel, they become activated, release components of their intracellular granules, and generate metabolic products. These products include adenine nucleotides, eicosanoids, and serotonin. 1 In turn, the platelet releasate functions as agonist for recruitment of additional platelets into the evolving thrombus. [1][2][3][4][5] By using an experimental approach to evaluate platelet activation and recruitment independently, we previously demonstrated that cell-cell interactions between activated platelets and intact erythrocytes (RBCs) amplify both platelet activation and the proaggregatory capacity of cell-free releasates. [2][3][4] In contrast, we found that platelet-neutrophil interactions down-regulate platelet reactivity in our system. 5 One of the earliest events in platelet reactivity is activation of platelet ␣ IIb ␤ 3 integrin receptors. Such activation is a prerequisite for fibrinogen binding to platelets, which culminates ...

For clinical purposes, we recommend a loading dose of aspirin (500 mg), followed by daily administration of 50 mg. The loading dose should be repeated at 2-week intervals. This regimen blocks recovery of the erythrocyte capacity to promote platelet reactivity and may amplify the therapeutic potential of aspirin in cardiovascular disease.