Effect of eptifibatide on inflammation during acute pain episodes in sickle cell disease
Authors: Julia E. Brittain, Ciprian Anea, Payal Desai, Jack Delaney, Adam McDonald, Stephen W. Looney, Nigel S. Key, Leslie V. Parise, Kenneth I. Ataga
Journal: American Journal of Hematology (2018)

To the Editor: The contribution of platelets to sickle cell disease (SCD) pathophysiology remains incompletely defined. Platelet activation is increased during acute pain episodes, coincident with increased inflammation, endothelial activation, and platelet–leukocyte interaction. We conducted a single-center, phase II, double-blind, placebo-controlled study to evaluate the safety of eptifibatide, a glycoprotein IIb/IIIa inhibitor, in combination with "standard of care," for the treatment of patients during an acute pain episode. Details of the study design, randomization schema, and primary outcomes of the study have been previously reported.1 Here, we perform a secondary analysis of the effect of eptifibatide on ex vivo platelet function, platelet–leukocyte aggregates, coagulation activation, endothelial activation, and inflammatory markers in patients during the study. Details of the Methods are provided in the Supporting Information Data. Results are provided for 6 eptifibatide treated and 3 placebo patients for whom complete data are available. Baseline demographic and laboratory data are shown in Supporting Information Table 1. Eptifibatide significantly reduced plasma levels of platelet α-granule specific proteins, CD40L {ΔEptifibatide = −62.6% (interquartile range [IQR]: −76.3, −47.1) vs. ΔPlacebo = −12.7% (IQR: −25.5, −37.65); P = .041}, thrombospondin (TSP-1) (ΔEptifibatide = −44.1% [IQR: −73.9, 31.1] vs. ΔPlacebo = −23.7% [IQR: −34.3, 16.1]; P = .04), platelet factor 4 (PF4) (ΔEptifibatide = −50.85% [IQR −50.85, −48.2] vs. ΔPlacebo = 13.73% [IQR = −18.1, 9.8]; P < .001), and P-selectin (ΔEptifibatide = −81.45% [IQR: −88.09, −57.81], ΔPlacebo = 1.75% [IQR: −1.68, 2.60]; P = .023) compared to placebo (Supporting Information Table 2). Eptifibatide inhibited ADP-induced platelet aggregation in response to 20 mM and 5 mM (13.5% and 3.5% aggregation, respectively) (P < .001 for both concentrations of ADP) (Figure 1A). Patients in the eptifibatide arm expressed less platelet P-selectin, defined as (CD62P+CD41+)/(CD41+), (ΔΕ = 40.5% [30.0, 54.5] at 0 hours vs. 13.5% [10.3, 17.3] at 6-hours post-treatment; P < .001). Patients in the placebo group exhibited no significant change over the same period (0 hours = 45.0% [40.1, 56.1], 6 hours = 53.4% [38.2, 57.8]; P = .67) (Figure 1B). Effect of eptifibatide on plasma markers of platelet function and inflammation in SCD. A-C, Eptifibatide inhibits platelet function during pain crisis in SCD. A, Eptifibatide (blue bars) infusion inhibits ADP-induced platelet aggregation. Platelet aggregation was tested in platelet rich plasma using both low (5 µM) and high (20 µM) concentrations of ADP. The placebo group (red bars) showed no reduction in platelet aggregation after 6 hours of normal saline infusion. Data are shown as median percent difference ± interquartile range. P < .0001 for both concentrations of ADP. B, Platelet p-selectin expression is reduced following 6 hours of eptifibatide infusion (blue bars), but not placebo (red bars). Each line represents an individual subject. Measurement of platelet P-selectin expression was done in whole blood using two-color flow cytometry. Events that scored positive for both CD41 (platelet specific) and P-selectin (CD62p) are expressed over total labeled platelets (CD41+). C, Eptifibatide reduces platelet–leukocyte aggregates. Platelet–leukocyte aggregates were measured in whole blood using two-color flow cytometry. Events that scored positive for CD45 (pan-leukocyte) and CD41 were considered aggregates and expressed as percent of total leukocytes. The paired data from each subject are shown. Eptifibatide = blue bars, placebo = red bars. D-I, Eptifibatide-induced inhibition of platelets reduces inflammatory markers and MPO when compared with placebo. Eptifibatide = blue bars, Placebo = red bars. Data are shown as % change from time 0 to time 6 (ΔE vs. ΔP) and are shown as median with interquartile [IQR] range. Time 0 hour is prior to eptifibatide infusion, Time 6 hourr is immediately post eptifibatide infusion. Statistical significance indicated by *, and unless indicated otherwise P < .05. Abbreviations are defined in the text and Supporting Information Methods After 6-hours of eptifibatide infusion, the percent of leukocytes that were also platelet positive, defined as (CD45+ CD41+)/CD45 was significantly reduced to 23.04% [11.43, 36.5] vs. 70.1% [65.32, 74.15] in the placebo group (P = .002). Paired time points of these leukocyte–platelet aggregates in the eptifibatide arm (0 hours = 65.6% CD41+ [41.25, 70.2], 6 hours = 23.04% CD41+ [11.43, 36.5], P = .004) indicate that every treated patient experienced a reduction in platelet–leukocyte aggregates (Figure 1C). Furthermore, the percent decrease from 0 to 6 hours in leukocyte–platelet aggregates (ΔE leukocyte-platelet aggregate) was directly associated with the decreased percent expression of platelet P-selectin (ΔE platelet p-selectin) over the same time period (rspearman = 0.89, P = .03). Eptifibatide administration produced significant percent reductions in plasma levels of IL-8, IL-6, monocyte chemotactic protein-1 (MCP-1), IL-1β, myeloperoxidase (MPO), and TNF-α compared to placebo (Figure 1D-I). However, eptifibatide had no effect on levels of CRP (P = .65). Reductions in the plasma levels of fibrinogen were not statistically different (P = .058) (Supporting Information Table 2). In the eptifibatide group, the percent reduction of TNF-α over 6 hours was significantly correlated with percent reduction of platelet P-selectin expression (rspearman = 0.81, P = .01). The percent reduction of platelet–leukocyte aggregates and percent reduction of TNF-α were also significantly correlated (rspearman = 0.86, P = .023). The decrease in MPO correlated with decreases in both platelet P-selectin and platelet–leukocyte aggregates (rspearman of 0.79, P = .02 and 0.90, P = .03, respectively) (Supporting Information Table 3). However, neither the difference in vWF antigen between the paired eptifibatide subjects at 0- and 6-hours, nor the difference between eptifibatide versus placebo groups at 6-hours was statistically significant (P = .13 and .75, respectively). Similarly, interrogation of other soluble markers of endothelial activation, sVCAM, sICAM, and sE-selectin, revealed no significant effect of eptifibatide administration at 6-hours (Supporting Information Table 2). The complete list of evaluated biomarkers is shown in Supporting Information Table 4. Patients with SCD exhibit increased platelet activation and inflammation at steady state, with further increases during pain crises.2 In this study, we found that eptifibatide significantly inhibited platelet aggregation, platelet release, and platelet P-selectin expression compared to placebo following 6 hours of therapy. We also noted significant reductions in platelet–leukocyte aggregates, multiple inflammatory cytokines, and MPO. The decline in inflammatory cytokines correlated with a decrease in platelet P-selectin expression as well as a decline in platelet–leukocyte aggregates. P-selectin-mediated interaction between platelets and leukocytes may contribute to the inflammatory state during pain crises. However, few studies have examined the relationship between platelet–leukocyte aggregates and inflammatory factors in SCD. Platelets release multiple pro-inflammatory mediators upon activation. In addition, P-selectin mediated-platelet adhesion to leukocytes may induce innate immune cells to release inflammatory factors such as TNF-α, IL-1β, MCP-1, and IL-6. Consistent with these observations, we found that decreasing P-selectin expression and platelet–leukocyte aggregates are associated with the reduction of several inflammatory factors. Reduction in the frequency of pain crises with the P-selectin inhibitor, Crizanlizumab,3 as well as the reduction in the duration of acute pain episodes with the pan-selectin inhibitor, Rivipansel,4 confirms the important role of P-selectin in SCD pathophysiology. Patients with SCD have elevated plasma levels of MPO even at "steady state," likely due to granulocyte activation. MPO is implicated in neutrophil net formation5 and can permanently incorporate 3-chlorotyrosine into proteins, thus exerting long-term effects on vascular and cellular function.6 Treatments that reduce MPO may provide clinical benefits to patients with SCD. Despite its multiple effects, eptifibatide did not reduce the plasma levels of markers of endothelial cell activation, possibly due to the short infusion period and few measured time points. Our study is limited by the small number of subjects evaluated. However, this weakness is offset by the paired nature of the analyses, allowing for direct comparisons in the same patient. The paired analysis (pre- and post-eptifibatide) eliminates the profound patient-to-patient variability often observed during pain crisis. Although the study was designed to collect blood samples at multiple time points, we were unable to collect samples at all the designated times due to issues of poor venous access. Based on its demonstrated safety and anti-inflammatory effects, the conduct of adequately powered studies of eptifibatide and similar antiplatelet drugs during pain crises are warranted. The authors would like to acknowledge SK Jones, for her assistance, the Clinical and Translational Research Center at UNC, Chapel Hill and the UNC Comprehensive Sickle Cell Program. At the time of study conduct the authors claim no conflict of interest. Dr. Ataga serves on Advisory Boards for Novartis Pharmaceuticals, Global Blood Therapeutics, Modus Therapeutics and Bioverativ J.E.B: Wrote manuscript, interpreted data, conducted experiments, analyzed data, and collected data. C.B.A: Analyzed data, wrote manuscript, interpreted data. P.D: Collected data, recruited patients, performed study data collection. J.D & A.M: Performed experiments, analyzed data. S.W.L: Performed statistical analysis. N.S.K & L.V.P: Wrote manuscript. K.I.A: Designed study, wrote manuscript, interpreted data, conducted experiments, analyzed data, and collected data. Additional Supporting Information may be found online in the supporting information tab for this article. Supporting Information Supporting Information Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.