Erythrocytes are known to influence hemostasis. Bleeding times are prolonged in anemia and corrected by normalizing the hematocrit. We now demonstrate that intact erythrocytes modulate biochemical and functional responsiveness of activated platelets. A two-stage procedure, permitting studies of cell-cell interactions and independently evaluating platelet activation and recruitment within 1 min of stimulation, was developed. Erythrocytes increased platelet serotonin release despite aspirin treatment, enzymatic adenosine diphosphate removal, protease inhibition, or combinations thereof. The data suggested that erythrocyte enhancement of platelet reactivity can reduce the therapeutic effectiveness of aspirin.Erythrocytes metabolically modified platelet arachidonate or eicosapentaenoate release and eicosanoid formation. They promoted significant increases in cyclooxygenase and lipoxygenase metabolites upon platelet stimulation with collagen or thrombin. However, with ionophore, erythrocytes strongly reduced platelet lipoxygenation. These erythrocyte modulatory effects were stimulus-specific. Activated platelet-erythrocyte mixtures, with or without aspirin, promoted 3-10-fold increases in extracellular free fatty acid, which would be available for transcellular metabolism. Erythrocyte-induced increases in free eicosapentaenoate may contribute to antithrombotic and anti-inflammatory effects of this fish oil derivative.

Neutrophil extracellular traps (NETs) are networks of DNA, histones, and proteolytic enzymes produced by activated neutrophils through different mechanisms. NET formation is promoted by activated platelets and can in turn activate platelets, thus favoring thrombotic processes. NETs have been detected in venous and arterial thrombosis, but data in stroke are scarce. The aim of this study was to evaluate NETs in the plasma of patients with acute ischemic stroke and their potential association with baseline clinical characteristics, stroke severity, and one-year clinical outcomes. The study included 243 patients with acute ischemic stroke. Clinical and demographic data and scores of stroke severity (NIHSS and mRs) at onset and discharge were recorded. Markers of NETs (cell-free DNA, nucleosomes, and citrullinated histone 3 (citH3)), were determined in plasma. Patients were followed-up for 12 months after the ischemic event. NETs were significantly elevated in the plasma of patients with acute ischemic stroke when compared to healthy subjects. NETs were increased in patients who were over 65 years of age and in those with a history of atrial fibrillation (AF), cardioembolic stroke, high glucose levels, and severe stroke scores at admission and discharge. In multivariate analysis, elevated levels of citH3, the most specific marker of NETs, at onset were independently associated with AF and all-cause mortality at one-year follow-up. NETs play a role in the pathophysiology of stroke and are associated with severity and mortality. In conclusion, citH3 may constitute a useful prognostic marker and therapeutic target in patients with acute stroke.

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.