Matthew D. Linden

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.

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

Summary.u2002 Background:u2002Aspirin ‘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:u2002(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:u2002To enable pre‐aspirin samples to be drawn, platelet function was measured in normal subjects (nu2003=u2003165) before and after aspirin 81u2003mg daily for sevenu2003days. Results:u2002The proportion of the post‐aspirin platelet function predicted by the pre‐aspirin platelet function was 28.3u2003±u20037.5% (meanu2003±u2003asymptotic standard error) for serum TXB2, 39.3u2003±u20036.8% for urinary 11‐dehydro TXB2, 4.4u2003±u20037.7% for arachidonic acid‐induced platelet aggregation, 40.4u2003±u20037.1% for adenosine diphosphate‐induced platelet aggregation, 26.3u2003±u20039.2% for the VerifyNow Aspirin Assay®, and 45.0u2003±u200310.9% 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:u2002(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.

Differences in platelet function in patients with acute myeloid leukemia and myelodysplasia compared to equally thrombocytopenic patients with immune thrombocytopenia

Summary.u2002 Background:u2002Severe thrombocytopenia is a major risk factor for hemorrhage, but platelet function and bleeding risk at low platelet counts are poorly understood, because of the limitations of platelet function testing at very low platelet counts. Objectives:u2002To examine and compare platelet function in severely thrombocytopenic patients with acute myeloid leukemia (AML) or myelodysplasia (MDS) with that in patients with immune thrombocytopenia (ITP). Methods:u2002Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms. Results:u2002Patients with AML/MDS had smaller platelets, lower IPF and substantially lower platelet surface expression of activated glycoprotein (GP)IIb–IIIa and GPIb, both with and without addition of ex vivo ADP or thrombin receptor‐activating peptide, than patients with ITP. In both ITP and AML/MDS patients, increased platelet surface GPIb on circulating platelets and expression of activated GPIIb–IIIa and GPIb on ex vivo activated platelets correlated with a higher IPF. Whereas platelet reactivity was higher for AML/MDS patients with bleeding than for those with no bleeding, platelet reactivity was lower for ITP patients with bleeding than for those with no bleeding. Conclusions:u2002AML/MDS patients have lower in vivo platelet activation and ex vivo platelet reactivity than patients with ITP. The proportion of newly produced platelets correlates with the expression of platelet surface markers of activation. These differences might contribute to differences in bleeding tendency between AML/MDS and ITP patients. This study is the first to define differences in platelet function between AML/MDS patients and ITP patients with equivalent degrees of thrombocytopenia.

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

Summary.u2002 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% (Pu2003<u20030.001) in the abciximab group and by 11% (Pu2003<u20030.02) in the eptifibatide group. Eighteen to 24 h after PCI, sCD40L was unchanged in the controls, reduced 30% (Pu2003<u20030.001) in the abciximab‐treated group and 9% (Pu2003<u20030.01) in the eptifibatide‐treated group. At the end of PCI, circulating monocyte‐platelet aggregates (MPA) were reduced by 12% (Pu2003=u2003NS) in the abciximab‐treated group, 13% in the eptifibatide‐treated group (Pu2003=u2003NS), but slightly increased in the controls (Pu2003=u2003NS). Eighteen to 24 h after PCI, MPA were reduced by 41% (Pu2003<u20030.001) compared to baseline in the abciximab‐treated group, by 23% (Pu2003=u2003NS) in the eptifibatide‐treated group, and 15% (Pu2003=u2003NS) 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 (Pu2003=u2003NS). 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.

In vivo effects of eltrombopag on platelet function in immune thrombocytopenia: no evidence of platelet activation.

The effects of eltrombopag, a thrombopoietin-receptor agonist, on platelet function in immune thrombocytopenia (ITP) are not fully characterized. This study used whole blood flow cytometry to examine platelet function in 20 patients receiving eltrombopag treatment at days 0, 7, and 28. Platelet surface expression of activated GPIIb/IIIa, P-selectin, and GPIb was measured with and without low and high adenosine diphosphate (ADP) and thrombin receptor activating peptide (TRAP) concentrations. Before eltrombopag treatment with no ex vivo agonist, platelet activation was higher in ITP patients than controls. Platelet GPIb and activated GPIIb/IIIa expression without added agonist was unchanged following eltrombopag treatment, whereas a slight increase in P-selectin was observed. Expression of P-selectin and activated GPIIb/IIIa in response to high-dose ADP was lower during eltrombopag treatment than at baseline. Eltrombopag led to a slight increase in platelet reactivity to TRAP only in responders to eltrombopag but not to levels above those in controls; whole blood experiments demonstrated that this increase was probably because of higher platelet counts rather than higher platelet reactivity. In conclusion, although thrombocytopenic ITP patients have higher baseline platelet activation than controls, eltrombopag did not cause platelet activation or hyper-reactivity, irrespective of whether the platelet count increased.

Targeted inhibition of the serotonin 5HT2A receptor improves coronary patency in an in vivo model of recurrent thrombosis

Summary.u2002 Background: Release of serotonin and activation of serotonin 5HT2A receptors on platelet surfaces is a potent augmentative stimulus for platelet aggregation. However, earlier‐generation serotonin receptor antagonists were not successfully exploited as antiplatelet agents, possibly owing to their lack of specificity for the 5HT2A receptor subtype. Objective: To assess whether targeted inhibition of the serotonin 5HT2A receptor attenuates recurrent thrombosis and improves coronary patency in an in vivo canine model mimicking unstable angina. Methods: In protocol 1, anesthetized dogs were pretreated with a novel, selective inverse agonist of the 5HT2A receptor (APD791) or saline. Recurrent coronary thrombosis was then initiated by coronary artery injuryu2003+u2003stenosis, and coronary patency was monitored for 3u2003h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1u2003h after the onset of recurrent thrombosis; (ii) template bleeding time was measured; and (iii) blood samples were obtained for in vitro flow cytometric assessment of platelet responsiveness to serotonin. Results: APD791 attenuated recurrent thrombosis, irrespective of the time of treatment: in both protocols, flow–time area (index of coronary patency; normalized to baseline coronary flow) averaged 58–59% (Pu2003<u20030.01) following administration of APD791 vs. 21–28% in saline controls. Moreover, the in vivo antithrombotic effect of APD791 was not accompanied by increased bleeding, but was associated with significant and selective inhibition of serotonin‐mediated platelet activation. Conclusion: 5HT2A receptor inhibition with APD791, even when initiated after the onset of recurrent thrombosis, improves coronary patency in the in vivo canine model.

The Hemostatic Defect of Cardiopulmonary Bypass

Cardiac surgery involving cardiopulmonary bypass is a common yet complex procedure that results in considerable disruption of hemostasis during and following surgery. Despite the relatively common and widespread use of this procedure, there remains a significant peri-operative risk of both thrombosis and hemorrhage in some patients. This is known as the hemostatic defect of cardiopulmonary bypass.Strategies including the use of pharmacological agents, hemodilution, autologous blood transfusion, rapid in-theatre monitoring of hemostatic potential with fine-tuning of the degree of heparinization, minimally invasive surgery and the use of biologically coated cardiopulmonary bypass equipment have been employed to ameliorate the effects of cardiopulmonary bypass on hemostasis. However there exists a fine line between preventing hemorrhage and promoting thrombosis. Likewise attempts to prevent thrombosis may result in increased hemorrhage. Research into many strategies for minimizing the hemostatic defect of cardiopulmonary bypass is incomplete, with safety and efficacy the subjects of intensive investigation.

CHAPTER 30 – Flow Cytometry

Flow cytometry, a remarkably versatile tool for the study of platelet function, encompasses multiple assays for multiple purposes (Table 30-1). Flow cytometry can be used to: (a) measure the activation state of circulating platelets and their reactivity (by activation-dependent changes in platelet surface antigens, leukocyte-platelet aggregation, and procoagulant platelet-derived microparticles); (b) diagnose specific disorders (Bernard-Soulier syndrome, Glanzmann thrombasthenia, storage pool disease, and heparin-induced thrombocytopenia [HIT]); (c) monitor antiplatelet agents; (d) monitor thrombopoiesis (by the number of young, “reticulated” platelets); (e) perform assays relevant to blood banking (quality control of platelet concentrates, Identification of leukocyte contamination in platelet concentrates, immunophenotyping of platelet HPA-1a, detection of maternal and fetal anti-HPA-1a antibodies, and platelet cross-matching); (f) measure platelet-associated IgG; (g) measure the platelet count; and (h) perform other research assays (measurement of platelet survival and function in vivo, calcium flux, F-actin content, signal transduction, fluorescence resonance energy transfer, platelet recruitment, and bacteria-platelet interactions). All these applications of flow cytometry to the study of platelets are discussed in this chapter.

Monocyte-Platelet Aggregates in Patients With Ischemic Heart Disease

Platelets are a central cellular interface of the thrombotic and inflammatory processes of coronary atherosclerosis and modulate this interface by binding to leukocytes and altering their function. In addition, platelet activation; formation of leukocyte- platelet aggregates (monocyte-platelet aggregates and neutrophil-platelet aggregates); and platelet secretion of inflammatory modulators that affect leukocyte function, such as CD40 ligand, are associated with progression of disease across the entire spectrum of atherothrombosis. Monocyte-platelet aggregates are elevated in stable coronary artery disease and further elevate with plaque rupture, characteristic of percutaneous coronary intervention, acute coronary syndromes, and myocardial infarction. Through this interaction, activated platelets may contribute to the pathogensis of ischemic heart disease by localizing and activating monocytes to the site of the atherosclerotic lesion. These heterotypic aggregates have potential for a number of clinical applications, including measurement as an early marker for plaque rupture, evaluation of the efficacy of antiplatelet therapy, and as a potential target for therapeutic intervention. Additional investigations evaluating these applications as well as the relationship between monocyte-platelet aggregates and outcome are needed.

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:nnFrelinger 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 …