Marsha L. Fox

Circulating monocyte-platelet aggregates are an early marker of acute myocardial infarction

OBJECTIVES We investigated whether elevated levels of circulating monocyte-platelet aggregates (MPA) can be used to identify patients with acute myocardial infarction (AMI). BACKGROUND Commonly used blood markers of AMI reflect myocardial cell death, but do not reflect the earlier pathophysiologic processes of plaque rupture, platelet activation and resultant thrombus formation. Circulating MPA form after platelet activation. METHODS In a single center between October 1998 and November 1999, we measured circulating MPA in a blinded fashion by whole blood flow cytometry in 211 consecutive patients who presented to the emergency department (ED) with chest pain and were admitted to rule out AMI. Acute myocardial infarction was diagnosed by a CK-MB fraction greater than three times control. RESULTS Patients with AMI (n = 61), as compared with those without AMI (n = 150), had significantly higher numbers of circulating MPA (11.6 +/- 11.4 vs. 6.4 +/- 3.6, mean +/- SD, p < 0.0001). After controlling for age, the adjusted odds of developing AMI for patients in the 2nd, 3rd and 4th quartiles of MPA, in comparison with patients in the lowest quartile (odds ratio = 1.0), were 2.1 (95% confidence interval [CI]: 0.7, 6.8), 4.4 (95% CI: 1.5, 13.1) and 10.8 (95% CI: 3.6, 32.0), respectively. The number of circulating MPA in patients with AMI presenting within 4 h of symptom onset (14.4) was significantly greater than those presenting after 4 h (9.4) and after 8 h (7.0), (p < 0.001). Of the 61 patients with AMI, 35 (57%) had a normal creatine kinase isoenzyme ratio at the time of presentation to the ED, but had high levels of circulating MPA (13.3). CONCLUSIONS Circulating MPA are an early marker of AMI.

Residual Arachidonic Acid–Induced Platelet Activation via an Adenosine Diphosphate–Dependent but Cyclooxygenase-1– and Cyclooxygenase-2–Independent Pathway: A 700-Patient Study of Aspirin Resistance

Background— Thrombotic events still occur in aspirin-treated patients with coronary artery disease. Methods and Results— To better understand aspirin “resistance,” serum thromboxane B2 (TXB2) and flow cytometric measures of arachidonic acid–induced platelet activation (before and after the ex vivo addition of aspirin and indomethacin) were analyzed in 700 consecutive aspirin-treated patients undergoing cardiac catheterization. In 680 of 682 evaluable patients, serum TXB2 concentrations were reduced compared with nonaspirinated healthy donors. Twelve patients had serum TXB2 that was lower than nonaspirinated healthy donors but >10 ng/mL. Arachidonic acid stimulated greater platelet activation in patients with high serum TXB2 (>10 ng/mL) than in patients with low serum TXB2. Addition of ex vivo aspirin reduced arachidonic acid–induced platelet activation to similar levels regardless of serum TXB2 concentrations, which suggests that patients with high residual serum TXB2 concentrations were either noncompliant or underdosed with aspirin. Among the remaining 98% of patients, ex vivo administration of either aspirin or indomethacin failed to prevent platelet activation across all degrees of arachidonic acid–induced platelet activation and aspirin doses. Although the patients were not randomized with respect to clopidogrel treatment, multivariate analysis showed that arachidonic acid–induced platelet activation was less in patients receiving clopidogrel. Conclusions— There is a residual arachidonic acid–induced platelet activation in aspirin-treated patients that (1) is caused by underdosing and/or noncompliance in only ≈2% of patients and (2) in the remaining patients, occurs via a cyclooxygenase-1 and cyclooxygenase-2 independent pathway, in direct proportion to the degree of baseline platelet activation, and is mediated in part by adenosine diphosphate–induced platelet activation.

Association of cyclooxygenase-1-dependent and -independent platelet function assays with adverse clinical outcomes in aspirin-treated patients presenting for cardiac catheterization.

Background— Poor clinical outcome in aspirin-treated patients has been termed aspirin resistance and may result from inadequate inhibition of platelet cyclooxygenase-1 (COX-1) by aspirin. The objectives of this study were to determine prospectively whether COX-1–dependent and other platelet function assays correlate with clinical outcomes in aspirin-treated patients. Methods and Results— Blood was collected before percutaneous coronary intervention from 700 consecutive aspirin-treated (81 or 325 mg for ≥3 days) patients. Platelet function was tested by (1) serum thromboxane B2; (2) arachidonic acid–stimulated platelet surface P-selectin and activated glycoprotein IIb/IIIa and leukocyte–platelet aggregates; and (3) platelet function analyzer (PFA)-100 collagen-epinephrine and collagen-ADP closure time (CT). Adverse clinical outcomes of all-cause death, cardiovascular death, and major adverse cardiovascular events (cardiovascular death, myocardial infarction, hospitalization for revascularization, or acute coronary syndrome) were assessed by telephone interview and/or medical record review. Clinical outcomes information was obtained at 24.8±0.3 months after platelet function testing. By univariate analysis, COX-1–dependent assays, including serum thromboxane B2 level, were not associated with adverse clinical outcomes, whereas the COX-1–independent assay, PFA-100 collagen-ADP CT <65 seconds, was associated with major adverse cardiovascular events (P=0.0149). After adjustment for covariables (including sex, aspirin dose, Thrombolysis in Myocardial Infarction risk score, clopidogrel use), both serum thromboxane B2 >3.1 ng/mL and PFA-100 collagen-ADP CT <65 seconds were associated with major adverse cardiovascular events. In contrast, indirect measures of platelet COX-1 (arachidonic acid–stimulated platelet markers, shortened PFA-100 collagen-epinephrine CT) were not significantly associated with adverse clinical outcomes even after adjustment for covariables. Conclusions— In this prospective study of 700 aspirin-treated patients presenting for angiographic evaluation of coronary artery disease, residual platelet COX-1 function measured by serum thromboxane B2 and COX-1–independent platelet function measured by PFA-100 collagen-ADP CT, but not indirect COX-1–dependent assays (arachidonic acid–stimulated platelet markers, shortened PFA-100 collagen-epinephrine CT), correlate with subsequent major adverse cardiovascular events. This study suggests that multiple mechanisms, including but not confined to inadequate inhibition of COX-1, are responsible for poor clinical outcomes in aspirin-treated patients, and therefore the term aspirin resistance is inappropriate.

Evidence that pre‐existent variability in platelet response to ADP accounts for ‘clopidogrel resistance’

Summary.  Background: Clopidogrel is a widely used antithrombotic agent that inhibits the platelet P2Y12 adenosine diphosphate (ADP) receptor. There is increasing interest in ‘clopidogrel resistance’. Objectives: To determine whether ‘clopidogrel resistance’ is accounted for by a pre‐existent variability in platelet response to ADP. Methods: Platelet response to 20 μm ADP was analyzed by four independent whole blood flow cytometric assays: platelet surface activated GPIIb‐IIIa, platelet surface P‐selectin, monocyte‐platelet aggregates and neutrophil‐platelet aggregates. In 25 consecutive, non‐aspirin‐treated healthy subjects, we studied platelet response before and after clopidogrel administration. In addition, we studied the platelet response in 613 consecutive aspirinated patients with or without coronary artery disease (CAD, as determined by angiography) who had or had not been treated with clopidogrel. In these patients, we tested for homogeneity of variance across all durations of clopidogrel exposure and severity of CAD by estimating the ‘goodness of fit’ of two independent models. Results: In the healthy subjects, pre‐clopidogrel response to ADP predicted post‐clopidogrel response to ADP. In the patients, clopidogrel, as expected, inhibited the platelet response to ADP. However, irrespective of the duration of clopidogrel administration, the severity of CAD, and the dose of aspirin, clopidogrel did not increase the variance in the platelet response to ADP in any of the four assays of platelet response. Conclusions: These studies provide evidence that ‘clopidogrel resistance’ is accounted for by a pre‐existent variability in platelet response to ADP and this variability is not increased by clopidogrel administration.

Indices of platelet activation and the stability of coronary artery disease

Aim:  To determine whether indices of platelet activation are associated with the stability of coronary artery disease (CAD).

Aspirin 'resistance': role of pre-existent platelet reactivity and correlation between tests.

Summary.  Background: Aspirin ‘resistance’ is a widely used term for hyporesponsiveness to aspirin in a platelet function test. Serum thromboxane (TX) B2 is the most specific test of aspirin’s effect on platelets. Objectives: (i) To examine the role of pre‐existent platelet hyperreactivity in aspirin ‘resistance’. (ii) To determine the correlation between aspirin resistance defined by serum TXB2 and other assays of platelet function. Methods: To enable pre‐aspirin samples to be drawn, platelet function was measured in normal subjects (n = 165) before and after aspirin 81 mg daily for seven days. Results: The proportion of the post‐aspirin platelet function predicted by the pre‐aspirin platelet function was 28.3 ± 7.5% (mean ± asymptotic standard error) for serum TXB2, 39.3 ± 6.8% for urinary 11‐dehydro TXB2, 4.4 ± 7.7% for arachidonic acid‐induced platelet aggregation, 40.4 ± 7.1% for adenosine diphosphate‐induced platelet aggregation, 26.3 ± 9.2% for the VerifyNow Aspirin Assay®, and 45.0 ± 10.9% for the TEG® PlateletMapping™ System with arachidonic acid. There was poor agreement between aspirin‐resistant subjects identified by serum TXB2 vs. aspirin‐resistant subjects identified by the other five assays, irrespective of whether the analysis was based on categorical or continuous variables. Platelet count correlated with pre‐aspirin serum TXB2 and VerifyNow Aspirin Assay, but not with any post‐aspirin platelet function test. Conclusions: (i) Aspirin ‘resistance’ (i.e. hyporesponsiveness to aspirin in a laboratory test) is in part unrelated to aspirin but is the result of underlying platelet hyperreactivity prior to the institution of aspirin therapy. (ii) Aspirin resistance defined by serum TXB2 shows a poor correlation with aspirin resistance defined by other commonly used assays.

Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity

Summary.  Background: Monocytes and neutrophils form heterotypic aggregates with platelets initially via engagement of platelet surface P‐selectin with leukocyte surface P‐selectin glycoprotein ligand‐1 (PSGL‐1). The resultant intracellular signaling causes the leukocyte surface expression of tissue factor and activation of leukocyte surface Mac‐1 (integrin αMβ2, CD11b/CD18). The activation‐dependent conformational change in monocyte surface Mac‐1 results in the binding of coagulation factor Xa (FXa) and/or fibrinogen to Mac‐1. The aim of this study was to develop whole blood flow cytometry assays of these procoagulant activities and to investigate the effects of platelet binding to monocytes and neutrophils. Methods: Citrate or D‐Phe‐Pro‐Arg‐chloromethylketone (PPACK) anticoagulated whole blood was incubated with monoclonal antibodies against CD14 (PECy5), CD42a (PE), FITC‐conjugated test antibody and an agonist, and then fixed with FACS lyse. Appropriate isotype negative controls were prepared in parallel. A BD FACSCalibur was used to analyze monocytes and neutrophils, which were identified based on CD14 fluorescence, forward and 90° light scatter. These populations were further gated into CD42a‐positive (platelet‐bound) and CD42a‐negative (platelet‐free). Geometric mean fluorescence and per cent positive data were collected for each subpopulation to measure the binding of test antibodies directed at CD42a, tissue factor, coagulation FXa, bound fibrinogen, activated Mac‐1, and CD11b. Compensation controls were prepared on six normal donors prior to the study and these settings were used throughout the 10 donor study. Negative controls verified the lack of cross talk, particularly in the quantified FITC and PE parameters. Results: The physiologic agonists collagen and ADP increased monocyte‐platelet and neutrophil‐platelet aggregates and increased leukocyte surface Mac‐1/CD11b and surface‐bound tissue factor, FXa and fibrinogen. Whereas the increases in Mac‐1/CD11b were mainly independent of leukocyte‐platelet binding, the increases in surface‐bound tissue factor, FXa and fibrinogen were mainly dependent on leukocyte‐platelet binding. Conclusions: (i) We have developed novel whole blood flow cytometry assays to measure bound tissue factor, coagulation FXa, fibrinogen, activated Mac‐1 and CD11b on the surface of monocytes and neutrophils, allowing independent analysis of monocytes and neutrophils with and without surface‐adherent platelets. (ii) The monocyte and neutrophil surface binding of tissue factor, FXa and fibrinogen is mainly dependent on platelet adherence to monocytes and neutrophils, whereas the monocyte and neutrophil surface expression of CD11b and activated Mac‐1 is mainly independent of platelet adherence to monocytes and neutrophils.

GPIIb–IIIa antagonists reduce thromboinflammatory processes in patients with acute coronary syndromes undergoing percutaneous coronary intervention

Summary.  Objective: To investigate the effects of abciximab, eptifibatide and no GPIIb–IIIa antagonist (control) on soluble CD40 ligand (sCD40L) and the formation of leukocyte‐platelet aggregates (LPA) in 98 ACS patients undergoing percutaneous coronary intervention (PCI). Background: sCD40L and LPA are increased in patients with ACS. Methods: sCD40L was measured by enzyme‐linked immunosorbent assay (ELISA) and LPA by whole blood flow cytometry. Results: There were no baseline differences between the three groups in sCD40L and LPA. At the end of PCI, sCD40L was unchanged in the controls, decreased by 30% (P < 0.001) in the abciximab group and by 11% (P < 0.02) in the eptifibatide group. Eighteen to 24 h after PCI, sCD40L was unchanged in the controls, reduced 30% (P < 0.001) in the abciximab‐treated group and 9% (P < 0.01) in the eptifibatide‐treated group. At the end of PCI, circulating monocyte‐platelet aggregates (MPA) were reduced by 12% (P = NS) in the abciximab‐treated group, 13% in the eptifibatide‐treated group (P = NS), but slightly increased in the controls (P = NS). Eighteen to 24 h after PCI, MPA were reduced by 41% (P < 0.001) compared to baseline in the abciximab‐treated group, by 23% (P = NS) in the eptifibatide‐treated group, and 15% (P = NS) in the controls. In contrast to control patients presenting while on clopidogrel, control patients presenting not on clopidogrel demonstrated a reduction in sCD40L and LPA 18–24 h post‐PCI (P = NS). At low receptor occupancy, GPIIb–IIIa antagonists did not augment the release of sCD40L or the number of circulating LPA. Conclusions: GPIIb–IIIa antagonists reduce circulating sCD40L and LPA formation in patients with ACS undergoing PCI. At low receptor occupancy, GPIIb–IIIa antagonists do not activate platelets.

Response to Letter Regarding Article, “Residual Arachidonic Acid–Induced Platelet Activation via an Adenosine Diphosphate–Dependent but Cyclooxygenase-1– and Cyclooxygenase-2–Independent Pathway: A 700-Patient Study of Aspirin Resistance”

To the Editor: Frelinger et al1 conclude that nonadherence is associated with aspirin resistance in a small minority (≈2%) of aspirin-treated patients and that a cyclooxygenase-independent pathway may mediate aspirin resistance in the remainder of patients. Although we agree that there may be a cyclooxygenase-independent pathway that allows platelets to remain active in some aspirin-treated patients, we believe that the authors underestimate the role nonadherence plays in aspirin resistance, because of their restricted sample of acute coronary …

The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin

The novel thienopyridine prodrug prasugrel, a platelet P2Y(12) ADP receptor antagonist, requires in vivo metabolism for activity. Although pharmacological data have been collected on the effects of prasugrel on platelet aggregation, there are few data on the direct effects of the prasugrels active metabolite, R-138727, on other aspects of platelet function. Here we examined the effects of R-138727 on thrombo-inflammatory markers of platelet activation, and the possible modulatory effects of other blood cells, calcium, and aspirin. Blood (PPACK or citrate anticoagulated) from healthy donors pre- and post-aspirin was incubated with R-138727 and the response to ADP assessed in whole blood or platelet-rich plasma (PRP) by aggregometry and flow cytometric analysis of leukocyte-platelet aggregates, platelet surface P-selectin, and GPIIb-IIIa activation. Low-micromolar concentrations of R-138727 resulted in a rapid and consistent inhibition of these ADP-stimulated thrombo-inflammatory markers. These rapid kinetics required physiological calcium levels, but were largely unaffected by aspirin. Lower IC(50) values in whole blood relative to PRP suggested that other blood cells affect ADP-induced platelet activation and hence the net inhibition by R-138727. R-138727 did not inhibit P2Y(12)-mediated ADP-induced shape change, even at concentrations that completely inhibited platelet aggregation, confirming the specificity of R-138727 for P2Y(12). In conclusion, R-138727, the active metabolite of prasugrel, results in rapid, potent, consistent, and selective inhibition of P2Y(12)-mediated up-regulation of thrombo-inflammatory markers of platelet activation. This inhibition is enhanced in the presence other blood cells and calcium, but not aspirin.