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(Circulation. 1997;95:63-68.)
© 1997 American Heart Association, Inc.

Articles

Prothrombotic Effects of Erythrocytes on Platelet Reactivity

Reduction by Aspirin

M. Teresa Santos, PhD; Juana Valles, PhD; Justo Aznar, MD; Aaron J. Marcus, MD; M. Johan Broekman, PhD; Lenore B. Safier, MS

the Research Center and Department of Clinical Pathology, University Hospital La Fe, Valencia, Spain (M.T.S., J.V., J.A.), and the Divisions of Hematology and Medical Oncology and Departments of Medicine, Veterans Affairs Medical Center and Cornell University Medical College, New York, NY (A.J.M., M.J.B., L.B.S.).

Correspondence to Aaron J. Marcus, MD, Thrombosis Research Laboratory, Room 13028W, Department of Veterans Affairs Medical Center, 423 E 23rd St, New York, NY 10010-5050. E-mail mjbroek@mail.med.cornell.edu.

	Abstract

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Background Aspirin effectively reduces the incidence of secondary vascular occlusive events in only 25% of patients. Low-dose aspirin as currently used blocks platelet production of prothrombotic thromboxane A₂ and allows endothelial synthesis of antithrombotic prostacyclin. This regimen minimizes gastrointestinal toxicity. We previously showed that intact erythrocytes markedly enhance platelet reactivity. Therefore we investigated whether supplementation of low-dose aspirin with a single high dose at 2-week intervals could more effectively block erythrocyte promotion of platelet reactivity.

Methods and Results Effects of different aspirin regimens on erythrocyte enhancement of platelet reactivity in normal volunteers were measured with the use of an assay that evaluates both platelet activation and recruitment. After 15 days of daily ingestion of 50 mg aspirin, reactivity of platelets alone was inhibited. However, erythrocyte promotion of platelet activation and recruitment was only inhibited by 50% and persisted in the total absence of thromboxane synthesis. In contrast, if 50 mg/d aspirin was preceded by a single loading dose of 500 mg aspirin, the erythrocyte prothrombotic effect was strongly inhibited (90%) for 2 to 3 weeks. However, over time, erythrocytes "escaped" from this inhibition, and once again became prothrombotic, even on a daily regimen of 50 mg aspirin.

Conclusions 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.

Key Words: aspirin • platelets • thromboxane • thrombosis • erythrocytes • cardiovascular diseases

	Introduction

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Clinical trials indicate that aspirin (ASA) is effective as an antithrombotic agent.^{1 2 3} This is especially true for secondary prevention of myocardial infarction.^{4 5} Nevertheless, ASA has reduced the incidence of vascular events by only 25% for a broad range of patients at risk for occlusive vascular diseases.^{1 2 5}

The accepted rationale for ASA treatment of cardiovascular disorders is that the compound inhibits synthesis of TXA₂, an eicosanoid with proaggregatory and vasoconstrictive properties.⁶ Such inhibition results in reduction of platelet reactivity as evidenced by prolongation of the bleeding time and reduced responsiveness to platelet agonists in vitro.⁷ When vascular occlusion occurs in patients treated with ASA, it takes place through mechanisms that overcome the aspirin-induced platelet functional defect in vivo. This may occur by exposure of platelets to high local concentrations of strong agonists such as collagen or thrombin or even high levels of released ADP, which do not require an active cyclooxygenase to promote platelet reactivity.^{8 9} These mechanisms may account for the failure of aspirin to protect a higher percentage of patients from cardiovascular events.

Cell-cell interactions between platelets and erythrocytes or platelets and neutrophils can significantly alter platelet reactivity.^{10 11 12 13 14 15 16} Our experiments demonstrated that this occurs through modulation of both platelet activation and recruitment.^{11 12 13} Metabolically intact erythrocytes markedly enhance the platelet release reaction, eicosanoid synthesis, and further platelet recruitment.^{12 13} These results extend the concept of thrombus formation as a multicellular event.^{15 16} In preliminary experiments, we found that erythrocyte enhancement of platelet reactivity was downregulated by aspirin.¹³ It was therefore of clinical relevance to establish a dose-response relationship between ASA administration and its effects on control of platelet responsiveness by erythrocytes ex vivo.

Results of this study indicate that erythrocyte promotion of platelet responsiveness is maximally inhibited by a single dose of 500 mg ASA, followed by daily ingestion of a 50-mg dose. However, erythrocytes "escape" from this inhibition and again become prothrombotic after 2 to 3 weeks. On the basis of these data, we suggest that maximal ASA therapeusis can be achieved with a single dose in the range of 500 mg ASA every 2 to 3 weeks, supplemented with a daily dose of 50 mg. This regimen drastically reduces the enhancing effect of erythrocytes on platelet reactivity and may thereby improve the effectiveness of ASA as an antithrombotic modality.

	Methods

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Blood Cell Collection and Processing
Citrate-anticoagulated venous blood (129 mmol/L; 9:1 [vol:vol]) was collected from male and female volunteers (20 to 50 years of age) after an overnight fast into 6 siliconized glass tubes (Vacutainer, Becton Dickinson), according to a protocol approved by the Institutional Review Board. Donor blood chemistries and hematological parameters were within the normal range (Hematolog-D and SMAC, Technicon). Volunteers had not taken any medication for at least 15 days before blood donation. For ex vivo aspirin studies, the drug was ingested 2 hours before venipuncture with the use of standard European dosage forms.

PRP and PPP were prepared by differential centrifugation (200g and 2500g, respectively, 15 minutes, 22°C). After removal of PRP, PPP, and buffy coat, 1 mL of erythrocytes was removed from the central area of the erythrocyte zone from each tube. The absence of platelets in erythrocyte preparations was verified by phase contrast microscopy. Moreover, TX was undetectable when these erythrocyte suspensions were exposed to thrombin. 5HT release was the parameter for measuring platelet activation. Platelets were radiolabeled with serotonin (¹⁴C-5HT, 56 mCi/mmol, Amersham) as previously described.^{12 17} Supernatants of PRP or PRP plus erythrocytes to which agonist buffer alone was added served as controls, and their 5HT content was subtracted from sample values.

Measurement of Platelet Activation and Recruitment
We developed a two-stage in vitro system for evaluation of platelet activation (5HT release) and recruitment (capacity of a cell-free releasate to act as agonist for platelet aggregation in PRP).^{12 13 14} PRP (1.8x10⁸ platelets/mL), PRP plus intact erythrocytes (hematocrit 40%), or whole blood was preincubated (37°C, 10 minutes). Collagen (1 µg/mL) was added, and the tube contents were mixed by inversion (10 seconds) and centrifuged (13 000g, 50 seconds) to obtain a cell- and collagen-free releasate. An aliquot of releasate was immediately transferred to a PRP assay system for testing as agonist for platelet aggregation (recruitment) by aggregometry.^{12 13 14} Recruitment was expressed as maximal height (mm) of the aggregation response. For each group of experiments, PRP for the assay system was obtained from a single donor to avoid intersubject variations. Recruitment was also evaluated using PRP autologous to that used in the generating system from the aspirin-treated donors. In addition, aliquots of the releasate from the generating system were used to evaluate platelet activation, monitored by ¹⁴C-5HT release and TXB₂ formation (radioimmunoassay).¹³ We have previously demonstrated that the enhancing effect of erythrocytes on platelet reactivity only occurs with metabolically intact erythrocytes.^{12 13}

Statistical Analyses
All data are presented as mean±SEM. The paired Student's t test was used to compare effects of platelets versus platelets plus erythrocytes. Simultaneous comparisons of more than two means were performed with ANOVA followed by Duncan's multiple rank test.

	Results

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Platelet-Erythrocyte Interactions After Daily Ingestion of 50 mg Aspirin
The effects of low-dose ASA (50 mg/d, 15 days) on platelet-erythrocyte interactions were studied in 5 normal volunteers. Before ASA ingestion, cell-free releasates from collagen-stimulated platelet-erythrocyte mixtures exhibited far more activation (5-HT release, TXB₂ formation) and recruitment (aggregation induced by adding the releasate to a separate PRP sample) than did releasates from platelets alone (Fig 1, A through C, "Prior to ASA").^{12 13} These results are illustrative of the ability of erythrocytes to enhance platelet reactivity.

View larger version (19K): [in this window] [in a new window]   Figure 1. Erythrocyte enhancement of platelet reactivity persists after low-dose aspirin (50 mg/d). Platelet reactivity was measured in normal donors before ASA, 2 hours after, and 4, 8, and 15 days later. Samples were tested 2 hours after the last dose, as described in "Methods." In these experiments, PRP from the same donor was used at all time points to evaluate recruitment. Within 4 days of ASA treatment, TXB2 formation was blocked in platelets and in platelet-erythrocyte mixtures (A). In contrast, erythrocytes continued to promote appreciable platelet activation (5HT release, B) and recruitment (C), even after 15 days. This could reduce the therapeutic effectiveness of this dose of ASA. Data are mean±SEM of triplicate determinations in 5 normal donors. P<.001 (PRP vs PRP + RBC) by paired t test in all instances.

Two hours after ingestion of 50 mg ASA, TXB₂ formation was reduced both in platelets alone (38%) and in platelet-erythrocyte mixtures (52%; Fig 1A). However, at this time point, platelet 5HT release and recruitment were unaffected in the presence as well as absence of erythrocytes (Fig 1, B and C). Thus, at this time point, a single low-dose ASA tablet reduced TX formation but did not affect platelet reactivity.

Within 4 days of aspirin ingestion, TX was undetectable in releasates obtained from either platelets or platelet-erythrocyte mixtures (Fig 1A). Whereas activation was reduced time-dependently in platelets alone, it remained markedly enhanced in the presence of erythrocytes at all time points (Fig 1B). Over a 15-day period, ASA ingestion gradually decreased platelet recruitment in collagen-stimulated platelets alone until it was totally abolished. However, even after 15 days, the presence of erythrocytes resulted in appreciable recruitment (Fig 1C). This indicated that even in the total absence of TX formation, serotonin release and platelet recruitment occurred when erythrocytes were present in the system.

Effects of Different Doses of Aspirin on Erythrocyte Promotion of Platelet Reactivity
Since low-dose ASA did not abolish the enhancing effects of erythrocytes on platelet recruitment, we studied the effects of different single doses of ASA on platelet recruitment ex vivo, 2 hours after ASA. Recruitment by releasates from platelets alone was reduced to a greater extent after a single dose of 100 mg compared with 50 mg of aspirin and abolished at ASA doses of 300 to 500 mg (Table). Marked promotion of recruiting activity by erythrocytes was observed after 50 to 100 mg ASA. However, this promoting effect was strongly reduced after ingestion of 300 mg ASA (88% inhibition) and abolished (97% inhibition) after 500 mg. These results indicated that quantities of ASA >100 mg were required to effectively prevent erythrocyte promotion of platelet recruitment ex vivo.

View this table: [in this window] [in a new window]   Table 1. Effect of Single Doses of Aspirin on Platelet Proaggregatory Activity (Recruitment)

In addition to recruitment, we also measured platelet activation in the presence or absence of erythrocytes 2 hours after ingestion of 500 mg ASA in another group of donors. TXB₂ formation was completely blocked and 5HT release greatly reduced by 500 mg ASA both in the presence (84% inhibition) and absence (97% inhibition) of erythrocytes (n=10).

To separate the effect of ASA on erythrocytes from that on platelets, we isolated "ASA-free" erythrocytes and erythrocytes obtained from donors who had ingested single doses of ASA (50 to 500 mg). These erythrocytes were tested for their ability to promote reactivity of fully ASA-treated platelets (500 mg). Recruitment was markedly enhanced by ASA-free erythrocytes (Fig 2). Single low doses of ASA (50 to 100 mg) reduced but did not abolish the erythrocyte enhancing effect on platelet recruitment. To completely block the prothrombotic effect of erythrocytes, 500 mg ASA was required (Fig 2). Thus, aspirin dose-dependently inhibited erythrocyte promotion of platelet recruitment as measured ex vivo. These data also indicated that an appropriate dose of ASA could prevent the prothrombotic effect of erythrocytes on platelet reactivity.

View larger version (15K): [in this window] [in a new window]   Figure 2. Effect of ingestion of different doses of aspirin on erythrocyte-platelet interactions. Platelet recruitment was markedly enhanced by ASA-free erythrocytes when combined with PRP obtained from a donor 2 hours after ingestion of 500 mg ASA. In contrast, single doses of 50 or 100 mg ASA reduced the enhancing effect of the erythrocytes on platelet recruitment. However, the 500-mg dose totally abolished erythrocyte enhancement. Thus, ASA dose-dependently inhibits promotion of platelet recruitment by erythrocytes. Aspirin-free erythrocytes (0 mg) n=33; RBC (50 mg) n=9; RBC (100 mg) n=16; RBC (500 mg) n=8. Data are mean±SEM. *P<.05 vs RBC (0 mg).

Time Course of Aspirin Inhibition of Erythrocyte Promotion of Platelet Reactivity
Erythrocytes were isolated 2 hours and 1 and 2 weeks after ingestion of a single dose of 500 mg ASA (3 donors). Combined suspensions of these erythrocytes and fully ASA-treated platelets (obtained from a separate donor, 2 hours after ingestion of 500 mg ASA) were stimulated with collagen and studied in triplicate. The results indicated that 1 and 2 weeks after ingestion of a single 500-mg aspirin tablet, erythrocytes "escaped" from this initial aspirin inhibition and were again capable of promoting activation and recruitment of fully ASA-treated platelets (data not shown).

Does 50 mg ASA per Day Exert a Cumulative Inhibitory Effect on Erythrocyte Promotion of Platelet Reactivity?
Since low-dose ASA exerts a cumulative inhibitory effect on platelet TX formation,^{18 19 20} we tested whether ASA would comparably inhibit the ability of erythrocytes to increase platelet reactivity. ASA-treated PRP (500 mg) from one donor was combined with erythrocytes obtained from other donors who had ingested 50 mg of aspirin daily. After 15 days of ASA treatment, prevention of the erythrocyte prothrombotic effect by ASA was no better than at 4 or 8 days of ASA treatment: Recruitment was inhibited by only 30% in comparison to preaspirin values (Fig 3A), and activation was reduced by 59% at 4 to 15 days (Fig 3B). Therefore, low-dose aspirin as currently used reduces the enhancing effect of erythrocytes on platelet activation and recruitment but does not abolish it.

View larger version (21K): [in this window] [in a new window]   Figure 3. Cumulative effect of 50 mg/d aspirin on erythrocyte promotion of platelet reactivity. Erythrocytes were obtained before (-ASA), and 2 hours after ingestion of 50 mg aspirin on day 1 (2 h). Additional erythrocyte samples were similarly obtained after 4, 8, and 15 days of 50 mg/d aspirin. These erythrocytes were combined with PRP obtained from a donor 2 hours after ingestion of a single dose of 500 mg aspirin. Platelet reactivity (recruitment [A] and activation, 5HT release [B]) was measured as described in "Methods." These data indicate that low-dose aspirin does not block the prothrombotic effects of erythrocytes on platelet activation and recruitment. *P<.01 vs RBC (-ASA). Data are mean±SEM of triplicate determinations in 5 normal donors.

Effect of a Loading Dose of 500 mg Followed by 50 mg ASA Daily on Platelet- Erythrocyte Promotion of Platelet Reactivity
As demonstrated above, an initial single dose of 500 mg ASA blocked erythrocyte enhancement of platelet reactivity. Since this dose is not suitable for long-term therapeusis, we evaluated whether 50 mg/d ASA for up to 5 weeks, after a 500-mg loading dose, would sustain ASA inhibition of erythrocyte prothrombotic activity.

Erythrocyte enhancement of platelet recruitment was drastically reduced (95%) 2 hours after a loading dose of 500 mg ASA (Fig 4A). This reduction persisted during maintenance treatment with 50 mg/d ASA for only 2 to 3 weeks. Erythrocyte enhancement of platelet activation (5HT release) followed a similar pattern: 5HT release was inhibited by 77% 2 hours after the 500-mg loading dose and persisted during 2 to 3 weeks of the 50-mg/d maintenance period. However, at 5 weeks, both recruiting activity and platelet activation approached pre-ASA levels (Fig 4, A and B), indicating (undesirable) erythrocyte "escape" from aspirin inhibition.

View larger version (22K): [in this window] [in a new window]   Figure 4. Effect of a loading dose of 500 mg aspirin plus 50 mg/d aspirin on erythrocyte promotion of platelet reactivity. Erythrocytes were obtained from different donors 2 hours after a single loading dose of 500 mg aspirin and after 1, 2, 3, and 5 weeks of supplementation with 50 mg/d aspirin. Each sample was combined with PRP obtained from a separate donor 2 hours after the ingestion of 500 mg aspirin. Platelet reactivity (recruitment [A] and activation, 5HT release [B]) was measured as described in "Methods." Inhibition of erythrocyte enhancement of platelet reactivity was initially effective. However, after 5 weeks of 50 mg/d ASA, the prothrombotic activity of erythrocytes was less inhibited and approached pre-ASA levels. Data are mean±SEM of triplicate determinations on 7 donors. *P<.01 vs RBC (-ASA).

Effect of a Loading Dose of 500 mg Followed by 50 mg ASA Daily on Platelet Reactivity in Whole Blood Ex Vivo
With the use of the same loading and maintenance doses of ASA (500 mg followed by 50 mg/d), similar results were obtained in a whole blood system with erythrocytes and platelets from the same donor. The loading dose of 500 mg aspirin abolished TX formation in whole blood, and this inhibition was maintained by 50 mg/d aspirin over a 5-week period (Fig 5A). Although inhibition of recruitment in PRP induced by the loading dose was maintained for 2 weeks, the inhibition gradually lost effectiveness thereafter (Fig 5B). In these experiments, recruitment was measured in ASA-free PRP as well as autologous ASA-treated PRP. Results with the ASA-treated system (not shown) closely paralleled those in Fig 5B. Platelet activation followed a pattern similar to that observed for recruitment (Fig 5C). These data, through the use of the more physiological setting of a whole blood system, confirm erythrocyte "escape" from ASA inhibition and a return to their prothrombotic activity.

View larger version (17K): [in this window] [in a new window]   Figure 5. Effect of a loading dose of 500 mg aspirin plus 50 mg daily on platelet reactivity as tested in whole blood. Whole blood was obtained from donors before aspirin, 2 hours after a loading dose of 500 mg, and after 1, 2, 3, and 5 weeks of supplementation with 50 mg/d. TXB2 synthesis (A), recruiting activity (B), and activation (5HT release, C) were measured as described in "Methods." Results with this system indicate that effective aspirin therapeusis involves an initial loading dose in the range of 500 mg, followed by daily administration of 50 mg. As can be seen, platelet reactivity is effectively suppressed for up to 3 weeks. Since erythrocytes regain their ability to promote platelet recruitment (B) and activation (C) after 3 weeks of 50 mg/d, repetition of the 500-mg loading dose at 2- to 3-week intervals is indicated. In these experiments, the phenomenon was demonstrable in a whole blood system, indicating physiological significance. Data are mean±SEM of triplicate determinations on 7 donors. P<.01 in all instances vs before ASA; *P<.01 vs 2 hours after 500 mg ASA.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
Thrombotic events usually occur at sites of pathological vascular damage (fissured or ruptured atherosclerotic plaques). High shear forces created by stenotic lesions in the vessel wall also promote platelet accumulation at these sites. These phenomena play a major role in the pathogenesis of occlusive vascular diseases.^{21 22 23} Early inhibition of platelet reactivity (before thrombin formation) is essential for prophylaxis of thrombotic events.

In a recent overview of randomized trials of antiplatelet therapy, "medium dose" ASA (75 to 325 mg/d) was found to reduce vascular death by 20% and nonfatal vascular events by 30%.² Since these doses of ASA obliterated formation of the platelet agonist TX, one could have anticipated better protection from occlusive events.¹⁶ Among other possible explanations for the lower-than-expected results are (1) erythrocytes continuously promote platelet activation during this dosage regimen; and (2) the prothrombotic activity of the pathological vessel surface (including plaque rupture and fibrin formation) could not be overcome by the inhibitory action of aspirin.

In an evolving thrombus, multicellular events modulate platelet reactivity through cell-cell interactions. Endothelial cells and neutrophils downregulate the prothrombotic activity(ies) of platelets, whereas erythrocytes act in a prothrombotic manner.^{14 15 16 22} We previously observed that ASA treatment reduces the prothrombotic effect of erythrocytes on platelet responsiveness.¹³ Since erythrocyte promotion of platelet reactivity would contribute to ischemic vascular events, we evaluated different doses of ASA for their ability to inhibit prothrombotic erythrocyte-platelet interactions.

We found that administration of 50 mg/d aspirin to normal recipients abolished platelet TX synthesis within 4 days (Fig 1A). This agrees with previous reports on the action of low-dose ASA (20 to 40 mg/d).^{18 19 20} In our experiments, low-dose aspirin reduced platelet activation and recruitment time-dependently in platelets alone. However, platelet reactivity was still enhanced by the presence of erythrocytes, even after 15 days of low-dose aspirin administration (Fig 1,B and C). Thus, in the presence of erythrocytes, as might occur in vivo, low-dose aspirin is less effective in preventing platelet responsiveness. Erythrocyte enhancement of platelet reactivity, even in the context of ASA treatment, may be of clinical relevance. Such enhancement of the platelet release reaction would still serve to promote cell proliferation in the vascular wall and acceleration of the atherosclerotic process.^{23 26}

Conclusions
We suggest that low-dose antithrombotic therapy with aspirin can be rendered more efficient and the prothrombotic effect of erythrocytes neutralized by a regimen consisting of an initial loading dose of 500 mg ASA followed by 50 mg daily. To prevent undesirable recovery of the aspirin-sensitive prothrombotic activity of erythrocytes, the loading dose should be repeated at 2-week intervals. Further clinical studies testing this therapeutic regimen of aspirin for the prophylaxis of thrombosis would be of interest.

	Selected Abbreviations and Acronyms

 

ASA (or aspirin) = acetylsalicylic acid 5HT = serotonin PPP = platelet-poor plasma PRP = platelet-rich plasma RBC = red blood cell(s) TX = thromboxane (also TXA2, TXB2)

	Acknowledgments

 
This study was supported in part by grants from the Spanish Fondo de Investigaciones Sanitarias de la Seguridad Social 95/9603 (M.T.S., J.V., J.A.), the Department of Veterans Affairs, and the National Institutes of Health (HL-18828 SCOR, HL-47073, and HL-46403) (A.J.M, M.J.B.). The technical assistance of M.C. Insa, M.D. Lopez, and M.J. Solano is gratefully acknowledged.

Received January 1, 1996; revision received April 22, 1996; accepted May 6, 1996.

	References

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