YouFu Li

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.

Pharmacodynamic assessment of platelet inhibition by prasugrel vs. clopidogrel in the TRITON-TIMI 38 trial

AIMS To examine the extent of platelet inhibition by prasugrel vs. clopidogrel in a TRITON-TIMI 38 substudy. METHODS AND RESULTS TRITON-TIMI 38 randomized acute coronary syndrome (ACS) patients undergoing percutaneous coronary intervention (PCI) to prasugrel or standard dose clopidogrel. Selected sites prospectively enrolled TRITON-TIMI 38 patients to evaluate adenosine diphosphate (ADP)-attenuated phosphorylation of platelet vasodilator-stimulated phosphoprotein (VASP) (n = 125 patients) and, in a subset (n = 31 patients), ADP-stimulated platelet aggregation. VASP platelet reactivity index (PRI) was lower in prasugrel-treated patients than in clopidogrel-treated patients at 1-2 h post-PCI (>or=1 h after loading dose) (P < 0.001) and at 30 days (P < 0.001). Maximal platelet aggregation to 20 microM ADP was lower in prasugrel-treated patients than in clopidogrel-treated patients at 1-2 h (P = 0.004) and 30 days (P = 0.03). Results were similar with 5 microM ADP. Thienopyridine hyporesponsiveness, prespecified as VASP PRI >50%, was more frequent in clopidogrel-treated patients than in prasugrel-treated patients at 1-2 h (P < 0.001) and 30 days (P = 0.03). CONCLUSIONS The TRITON-TIMI 38 platelet substudy shows that prasugrel results in greater inhibition of ADP-mediated platelet function in ACS patients than clopidogrel, supporting the hypothesis that greater platelet inhibition leads to a lower incidence of ischaemic events and more bleeding both early and late following PCI.

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.

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

Summary.  Background: Severe 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: To 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: Whole blood flow cytometric measurement of platelet activation and platelet reactivity to agonists was correlated with the immature platelet fraction (IPF) and bleeding symptoms. Results: Patients 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: AML/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.

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.  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 injury + stenosis, and coronary patency was monitored for 3 h. Protocol 2 was similar, except that: (i) treatment with APD791 or saline was begun 1 h 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% (P < 0.01) 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.