Nuisance and alarming bleeding do not correlate with on-treatment platelet reactivity

Abstract

Aims

We hypothesized that patients with a history of either alarming or nuisance bleeding events, compared to those with no history of bleeding, would have lower levels of on-treatment platelet reactivity (aspirin and a thienopyridine).

Methods and Results

In total, 42 patients with no bleeding, 34 with nuisance bleeding, and 14 with alarming bleeding underwent platelet reactivity testing 1 month to 1 year after PCI with light transmission aggregometry (LTA 5 and 20 μM adenosine disphosphate [ADP]), vasodilator stimulated phosphoprotein phosphorylation (VASP) and VerifyNow P2Y12. Clinical and demographic characteristics of the 3 groups were generally similar, except that patients with alarming bleeding were less likely to be Caucasian; only 6 patients (6.7%) were taking prasugrel. There was considerable overlap between no bleeding, nuisance bleeding and alarming bleeding groups with respect to on-treatment platelet reactivity. Furthermore, there was no difference in the median platelet reactivity values for all assays. Prevalence of high on-treatment platelet reactivity did not differ among the 3 groups; 32.6% of patients had high on-treatment platelet reactivity as measured by LTA with 5 μM ADP ( P = .91); 40.0% as measured by VASP ( P = .35); and 35.6% as measured by VerifyNow P2Y12 ( P = .61).

Conclusion

The use of platelet reactivity assays to identify patients on thienopyridine therapy at higher risk of bleeding is an unfounded strategy.

1 Introduction

An extensive literature demonstrates that high on-treatment platelet reactivity in patients taking clopidogrel is associated with major adverse cardiac events and periprocedural myonecrosis following percutaneous coronary intervention (PCI) . The importance of effective platelet inhibition is further supported by recent trials of novel antiplatelet agents such as prasugrel and ticagrelor, more potent P2Y12 inhibitors that reduce thrombotic events in acute coronary syndrome patients when compared to clopidogrel . Bleeding risk and complications, however, rise in concert with more effective platelet inhibition. The importance of mitigating bleeding risk is obvious, as numerous studies have demonstrated increased mortality in patients with major bleeding events . In addition, even so-called “insignificant” or nuisance bleeding may increase the risk of major adverse cardiac events and clopidogrel noncompliance .

The concept of a “therapeutic window” for antiplatelet agents, within which both thrombotic and bleeding events are minimized, is thus appealing. Such a strategy would hold the most promise for clopidogrel, which demonstrates significant inter-individual variability in its antiplatelet effect but remains the most widely utilized P2Y12 inhibitor following PCI . The utility of platelet reactivity testing to assess bleeding risk in patients on thienopyridine therapy, however, is relatively unknown. We therefore sought to examine the correlation between on-treatment platelet reactivity as measured by light transmission aggregometry (LTA), vasodilator stimulated phosphoprotein phosphorylation (VASP), and VerifyNow P2Y12, and the occurrence of both nuisance and alarming bleeding events in patients on dual antiplatelet therapy with aspirin and clopidogrel or prasugrel following PCI. We hypothesized that patients with any bleeding events, compared to those without bleeding events, would have lower levels of on-treatment platelet reactivity.

2 Methods

Patients ≥ 18 years of age and on a maintenance dose of clopidogrel 75 mg or prasugrel 10 mg (for a minimum of 5 days) were consecutively enrolled in this study from April 2010 through May 2011. All patients were enrolled within 1 month to 1 year after elective or urgent PCI at a single center and were also taking a maintenance dose of aspirin 325 mg. There were 3 predefined study arms based upon bleeding events while on clopidogrel: patients with a history of nuisance bleeding; alarming bleeding; or no significant bleeding. After assignment to the appropriate study arm based upon bleeding history, patients then underwent platelet reactivity testing with LTA, VASP, and VerifyNow P2Y12.

Patients on warfarin or non-steroidal anti-inflammatory drugs, in addition to patients known to be pregnant, with a history of bleeding diathesis, active bleeding, platelet count < 100 x 10 ⁹/L, hematocrit < 25%, or those who had received a blood transfusion in the preceding 10 days were excluded. In addition, patients with an alarming bleeding event (definition below) during hospitalization for the index PCI were excluded in order to exclude bleeding more likely to be related to periprocedural medications. Written, informed consent was obtained prior to platelet reactivity testing. The Institutional Review Board at MedStar Washington Hospital Center and MedStar Health Research Institute (Washington, DC) approved this study.

Patients were identified from a database in which bleeding history had been previously ascertained by phone interview. More specifically, patients were queried as to whether they had suffered any bleeding events since hospital discharge from the index PCI. Alarming bleeding was defined as a bleeding event self-reported by a patient with any of the following characteristics: intracranial, life-threatening (i.e. requiring hospitalization), requiring transfusion, hematoma, epistaxis, from the mouth or vagina, eye bleeding, hematuria, hematemesis, or melena. Nuisance bleeding was defined as a bleeding event self-reported by a patient with any of the following: easy bruising, bleeding from small cuts, petechia, and ecchymosis, in the absence of a history of alarming bleeding. Patients without bleeding had neither alarming nor nuisance bleeding events. High on-treatment platelet reactivity was defined according to the consensus white paper definitions: > 46% maximal platelet aggregation for LTA with 5 μM adenosine disphosphate (ADP), > 235 P2Y12 reactivity units for VerifyNow P2Y12, and > 50% platelet reactivity index for VASP . Although there is no consensus definition for high on-treatment platelet reactivity as measured by LTA with 20 μmol ADP, we selected MPA > 60% based upon previous studies .

All patients underwent platelet reactivity testing with 3 assays simultaneously: LTA (ChronoLog, Havertown, PA, USA), VASP phosphorylation analysis (FACSCalibur flow cytometer, BD Biosciences, San Jose, CA, USA), and the VerifyNow P2Y12 assay (Accumetrics, San Diego, CA, USA). The objective was to correlate on-treatment platelet reactivity with both nuisance and alarming bleeding events. Whole blood samples were drawn through an 18 gauge (or larger) needle into 3.2% sodium citrate tubes, including one 1.8 mL Greiner tube for the VerifyNow P2Y12 assay. Two highly trained research scientists performed the platelet reactivity testing within 3 hours of blood sampling.

LTA was performed at 37° Celsius using platelet-rich plasma obtained by centrifugation of citrated whole blood for 10 minutes at 1000 revolutions per minute. LTA results were not adjusted for baseline platelet count, as this time-consuming process has been shown to be unnecessary . Aggregation parameters were measured with both 5 and 20 μM of ADP as agonists; these on-treatment platelet reactivity values are reported as percentages of maximal platelet aggregation.

VASP phosphorylation analysis was performed using the PLT VASP/P2Y12 assay (BioCytex, Marseille, France). First, whole blood was incubated with ADP with or without prostglandin E1. A monoclonal antibody to label the VASP protein in its phosphorylated state was then added. This antibody was then stained with a fluorescein reagent, which can be detected as mean fluorescence intensity (MFI) by flow cytometry. The ratio of (MFI ^PGE1– MFI ^ADP+PGE1)/MFI ^PGE1was then calculated, in order to estimate the ratio of activated versus nonactivated platelets. This value is reported as the platelet reactivity index.

The VerifyNow P2Y12 assay was performed by addition of whole blood to dedicated cartridges containing fibrinogen-coated beads, 20 μmol of ADP (as the agonist), and 22 nmol of prostaglandin E1 (to reduce the nonspecific contribution of P2Y1 receptors). This point-of-care system utilizes turbidemetric optical detection to measure changes in light transmittance that result from clumping of the fibrinogen-coated beads, with these changes reported as P2Y12 reaction units.

Continuous variables are presented as either mean ± standard deviation or median ± interquartile range; categorical variables are presented as counts and percentages. Normality was assessed with the Kolmogorov-Smirnov test. Categorical variables were compared using the χ ²test or Fisher’s exact test. Continuous variables, including platelet reactivity values, were compared among the 3 groups using the Kruskal-Wallis test. A p value < 0.05 was considered statistically significant. Statistical analyses were performed using SAS version 9.1 (SAS Institute, Cary, NC, USA).