TR-05314; No of Pages 6 Thrombosis Research xxx (2013) xxx–xxx
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A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation☆ Joseph A. Jakubowski a,⁎, Chunmei Zhou a, Stipo Jurcevic b, Kenneth J. Winters a, D. Richard Lachno c, Andrew L. Frelinger III d, Neehar Gupta a, Jo Howard e, Christopher D. Payne c, Timothy G. Mant f a
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN USA King’s College, London UK Lilly Research Laboratories, Eli Lilly and Company, Windlesham, Surrey, UK d Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, MA USA e Guy’s and St. Thomas’ Hospital, London UK f Quintiles Drug Research Unit at Guy’s Hospital, London UK b c
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 20 November 2013 Accepted 3 December 2013 Available online xxxx Keywords: ADP Biomarkers P2Y12 receptor antagonist Platelets Prasugrel Sickle cell disease
a b s t r a c t Introduction: Prasugrel, a P2Y12 adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADPmediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD. Materials and Methods: Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B2 (TXB2), P-selectin, and TF. Results: Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB2 were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects. Conclusions: These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD. © 2013 Elsevier Ltd. All rights reserved.
Introduction Sickle cell disease (SCD) is a common hemoglobinopathy that affects millions of people worldwide and has substantial medical, social, and economic impact [1]. Much of the morbidity and mortality of SCD arises from complications of vaso-occlusive crisis (VOC), which can result in
Abbreviations: ADP, adenosine diphosphate; CD40L, CD40 ligand; EDTA, ethylenediaminetetraacetic acid; GPIIbIIIa, glycoprotein IIb-IIIa; PD, pharmacodynamics; PK, pharmacokinetics; RBCs, red blood cells; SCD, sickle cell disease; SD, standard deviation; TF, tissue factor; TXB2, thromboxane B2; VOC, vaso-occlusive crisis. ☆ This research was supported by Daiichi Sankyo Company, Ltd., and Eli Lilly and Company. ⁎ Corresponding author at: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA. Tel.: +1 317 276 9036; fax: +1 317 433 1996. E-mail address: [email protected] (J.A. Jakubowski).
ischemia and infarction of affected organs and which manifests clinically as episodic bouts of localized pain [2–4]. Microvascular vaso-occlusion and chronic inflammation are the hallmarks of VOC. Although the underlying pathophysiology remains unclear, it is thought to be the result of a complex and dynamic interplay between sickled red blood cells (RBCs), endothelial cells, leukocytes, platelets, and various plasma proteins. Evidence suggests that in patients with SCD, circulating platelets are chronically activated in the non-crisis steady state [5], and that this activation intensifies during VOC [6–8]. As sickled RBCs haemolyze, they release adenosine diphosphate (ADP) [6,9], which may drive platelet activation and aggregation [10]. In addition, activated platelets have increased expression of proteins such as CD40 ligand (CD40L) [5,8,11] and P-selectin [12], which may promote interaction with endothelial and inflammatory cells [11,12]. In addition to erythrocytes, platelet and leukocyte aggregates contribute to vessel occlusion [13]. Several studies have also found
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Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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coagulation markers including fibrin [7], tissue factor (TF), and thrombin [6,8] to be elevated in SCD in the non-crisis steady state and, to be further elevated during VOC in children and adult patients [7,12]. The potential role of platelets in VOC suggests that antiplatelet therapies may hold promise for prevention and treatment in SCD. Thus far, there have been two double-blind, placebo-controlled studies of the first-generation ADP-receptor antagonist ticlodipine. Though an ex vivo decrease in platelet aggregation was seen in a 4-week study of ticlodipine therapy, no difference in the frequency of VOC was observed [14]. However, in a larger study of ticlodipine lasting 6 months, a significant reduction in painful crises was seen in patients with SCD [15]. Prasugrel is a third generation platelet P2Y12 ADP-receptor antagonist, which has been shown to effectively inhibit platelet activation and aggregation via irreversible inhibition of the P2Y12 ADP receptor [10]. Given the potential role of ADP in the manifestation of VOC, prasugrel may reduce the frequency and severity of VOC and consistently decrease platelet activation to help reduce chronic low-level ischemia in these patients. The pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of prasugrel have been well-characterized in healthy control subjects and in patients with coronary artery disease [16,17], but were unknown in patients with SCD. However, these characteristics have now been evaluated before and after 12 days of prasugrel administration (5.0 mg/day or 7.5 mg/day) in a Phase 1b, open-label, single-center trial that included patients with SCD and healthy control subjects. In patients with SCD, prasugrel produced dose-dependent exposure to its active metabolite, which in turn reduced platelet reactivity to ADP, as measured using a variety of assays [18]. In the current analysis of data from the same study, we examine levels of cellular and soluble biomarkers of platelet activation and coagulation at baseline and following 12 days of prasugrel administration in patients with SCD and compare these values to those found in healthy subjects. Materials and Methods Study design This single-center, open-label trial was sponsored by Daiichi Sankyo Company, Ltd. and Eli Lilly and Company from July 2010 through February 2011 (NCT01178099). Patients with SCD and healthy controls were given a single 10-mg oral dose of prasugrel followed by administration for 12 ± 2 days of oral prasugrel, 5 mg/day for those weighing b60 kg and 7.5 mg/day for those weighing ≥60 kg. Participants Enrolled participants had to be between 18 and 60 years of age and weigh between 50 and 100 kg. Patients with SCD had to have the genotypes HbSS, HbSC, or HbSβ0 or HbSβ+ thalassemia, and were to have had no occurrence of VOC within 1 month of screening. Healthy control subjects were matched for gender, age (within 10 years), and weight (b 60 kg and ≥60 kg within 10 kg). Please refer to the primary publication for further detail [18].
assessing the binding of a platelet-specific antibody, CD61, to these cells. TF present on the surface of monocyte-platelet aggregates was measured using a CD142-specific antibody. The level of circulating platelet-erythrocyte aggregates was determined by gating on CD41a-positive platelets and measuring staining of the erythrocyte-specific glycophorin marker CD235a to determine the percent of platelet-erythrocyte aggregates [9]. The activation state of circulating platelets was determined by the detection of activation-dependent changes in the platelet surface markers by flow cytometry [22]. Platelets were identified by size, light scatter pattern, and the binding of a CD61-specific antibody. The expression of P-selectin (CD62P), activated glycoprotein IIb-IIIa (GPIIbIIIa [PAC-1]), CD40L (CD154), and TF (CD142) on the surface of platelets was measured by staining with antibodies specific for these markers (all antibodies were from BD Biosciences, Oxford, UK). Flow cytometric analysis was performed using a FACSCalibur cytometer and CellQuest Pro software (both BD Biosciences, San Jose, CA). Results were presented as percent of positive cells expressing a given marker above the background control level of staining. For platelet activation markers, this related to the proportion of platelets that may participate in aggregates or inflammatory processes. Soluble CD40L (Quantikine Human CD40 Ligand Immunoassay, R&D Systems, Minneapolis, MN) and P-selectin (Human P-Selectin ELISA, R&D Systems, Minneapolis, MN) were assessed in plasma prepared from blood samples collected into ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1000 g for 15 minutes within 30 minutes of collection. Samples for the CD40L assay were centrifuged for an additional 10 minutes at 10,000 g. Prothrombin fragment 1.2 (F1.2) was measured in plasma prepared from blood collected in 3.2% citrate using Dade-Behring Enzygnost F1 + 2 microkit (Marburg, Germany). Serum for thromboxane B2 (TXB2) was prepared from whole blood collected into glass tubes and allowed to clot for 1 hour at 37 °C [23] and assayed by immunoassay (Cayman Thromboxane B2 EIA kit, Cayman Chemical, Ann Arbor, MI). Blood samples for soluble coagulation Factor III/TF measurement were collected in 3.2% sodium citrate. After centrifugation at 1500 g for 15 minutes, plasma was taken off and frozen at −80 °C within 30 minutes of collection. Thawed samples were tested using an immunoassay method (R& D Systems, Abingdon, UK).
Statistical analysis Unless otherwise noted, values are presented as mean and standard deviation (SD) for continuous variables and as percentages for categorical variables. The two dose groups (5 mg/day and 7.5 mg/day) were combined for all analyses since there were only 4 subjects in each group receiving 5 mg/day and the doses were similar when considered on a mg/kg basis (0.107 ± 0.015 mg/kg for SCD patients and 0.104 ± 0.012 mg/kg for healthy subjects, p = 0.66). Platelet biomarker levels Table 1 Baseline Demographic Characteristics for Healthy Subjects and Patients with Sickle Cell Disease.
Cellular and soluble platelet and coagulation biomarkers
Characteristic/measurement
Healthy Subjects (N = 13)
Patients with SCD (N = 13)a
Within groups, we assessed the response to 12 ± 2 days of prasugrel administration, and between groups, we compared biomarker levels before and after prasugrel administration. Venous blood samples were collected at baseline (1 hour before the first dose of prasugrel) and on Day 12, 24 hours after the previous 5-mg or 7.5-mg dose of prasugrel. Whole blood samples were collected into 3.2% citrate and, within 2 hours of collection, were assessed for biomarker expression by flow cytometry. As previously described, monocyte and neutrophil cell populations were distinguished within whole blood on the basis of their differential expression of CD14 and light scatter properties [19–21]. The presence of heterotypic platelet aggregates was then determined by
Age (years), mean (SD) Range (years) Male gender, n (%) Ethnicity, n (%) African descent Asian White Weight (kg) Mean (SD) Range (minimum – maximum)
26.7 (5.7) 19-42 7 (53.8)
29.1 (9.3) 19-50 8 (61.5)
3 (23.1) 1 (7.7) 9 (69.2)
13 (100.0) 0 (0.0) 0 (0.0)
64.5 (9.6) 50.5 – 82.1
63.3 (9.5) 52.2 – 87.2
Abbreviations: N = total number of subjects/patients; n = number of subjects/patients in that category; SCD = sickle cell disease; SD = standard deviation. a N = 13 at baseline and 12 at study end.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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at baseline and Day 12 were compared between patients with SCD and healthy subjects using a two sample t-test. Platelet reactivity changes from baseline to Day 12 within each population were analyzed using a one sample t-test. For all statistical comparisons, a p-value b0.05 was considered statistically significant.
The majority of healthy subjects were white, while all patients with SCD were of African descent. All patients with SCD were of the HbSS genotype. Due to poor vein quality, one patient with SCD discontinued the study following the baseline venipuncture, immediately after the first dose of prasugrel. All healthy subjects completed the study.
Results
Cellular platelet biomarkers (Fig. 1 and Table 2)
Demographics (Table 1)
All data are presented as values (mean [SD]) for patients with SCD vs. values for healthy subjects. At baseline, patients with SCD had a greater percentage of monocyte-platelet aggregates compared with healthy controls (78% [26%] vs. 51% [18%], p = 0.004). Patients with SCD also had a greater percentage of neutrophil-platelet aggregates
Twenty-six subjects were enrolled; 13 healthy subjects and 13 patients with SCD. The mean age was 27 years for healthy subjects (range 19-42 years) and 29 years for patients with SCD (range 19–50 years).
Fig. 1. Whole Blood Platelet Biomarkers at Baseline and after 12 Days of Prasugrel Administration in Healthy Subjects and Patients with Sickle Cell Disease. Boxes represent the 25th through 75th percentiles and whiskers represent the 10th and 90th percentiles. The solid line is the median and the dashed line is the mean. Results are expressed as percent of cells positive for the respective biomarker. Abbreviations: CD40L = CD40 ligand.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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Table 2 Whole Blood Biomarkers of Platelet Activation and Coagulation at Baseline and Day 12 of Prasugrel Administration in Healthy Subjects and Patients with Sickle Cell Disease. Biomarker
Monocyte-platelet aggregates (%) Neutrophil-platelet aggregates (%) CD40L (% cell CD40L)
P-Selectin (% CD62P expression) Activated GPIIbIIIa (% PAC-1) Tissue factor expression on circulating platelets (%) Monocyte-platelet tissue factor expression (%) Platelet-erythrocyte aggregates (%)
Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea
Healthy Subjects (N = 13) mean (SD)
Patients with SCD (N = 13)c mean (SD)
p-valueb
50.7 (17.9) 40.4 (23.5) 0.254 17.7 (4.0) 16.1 (8.2) 0.528 13.0 (4.6) 17.8 (4.8) 0.011 10.3 (6.4) 5.4 (2.1) 0.012 5.8 (4.7) 4.9 (7.6) 0.803 4.2 (3.3) 5.5 (3.6) 0.379 7.3 (3.3) 9.9 (6.7) 0.105 8.5 (4.6) 8.2 (2.2) 0.819
78.0 (25.6) 62.2 (24.3) 0.045 27.5 (15.2) 25.9 (17.8) 0.837 17.3 (4.0) 16.4 (3.8) 0.504 15.8 (7.5) 12.0 (12.7) 0.324 9.3 (9.8) 7.8 (12.5) 0.300 5.0 (3.4) 4.8 (3.3) 0.506 5.1 (3.1) 5.8 (1.8) 0.593 5.9 (1.3) 5.6 (2.7) 0.683
0.004 0.032 0.035 0.086 0.017 0.441 0.058 0.079 0.277 0.479 0.522 0.602 0.082 0.051 0.060 0.017
Abbreviations: CD40L = CD40 ligand; GPIIbIIIa = glycoprotein IIb-IIIa; N = number of subjects/patients; SCD = sickle cell disease; SD = standard deviation. a p-values derived from one sample t-test. b p-values derived from two sample t-test. c N = 13 at baseline and 12 at study end.
(27% [15%] vs. 18% [4%], p = 0.035) and expression of platelet CD40L (17% [4%] vs. 13% [5%], p = 0.017). Following 12 ± 2 days of prasugrel administration, patients with SCD demonstrated a significant decrease in the number of monocyte-platelet aggregates (from 78% [26%] to 62% [24%], p = 0.045) that was not seen in healthy subjects. In addition, at the end of the treatment period, patients with SCD maintained a higher percentage of monocyte-platelet aggregates (62% [24%] vs. 40% [23%], p = 0.032) and a lower percentage of platelet-erythrocyte aggregates (6% [3%] vs. 8% [2%], p = 0.017) than healthy subjects. Following prasugrel administration, platelet surface P-selectin had decreased in healthy subjects (from 10% [6%] to 5% [2%], p = 0.012) and CD40L had increased (from 13% [5%] to18% [5%], p = 0.011), changes that were not observed in the SCD group. Other biomarkers did not differ significantly between groups at baseline or following prasugrel treatment, or within groups from baseline to Day 12.
Soluble biomarkers (Fig. 2 and Table 3) Patients with SCD had higher plasma concentrations of F1.2 at baseline than healthy controls (1777 nmol/L [2487 nmol/L] vs. 206 nmol/L [292 nmol/L], p = 0.033). Baseline levels of serum TXB2 were also higher in patients with SCD (221 ng/ml [114 ng/ml] vs. 129 ng/ml [82 ng/ml], p = 0.027). While prasugrel administration lowered the mean level of F1.2 in patients with SCD, the change from baseline did not meet statistical significance (from 1777 nmol/L [2487 nmol/L] to 403 nmol/L [266 nmol/L], p = 0.120). Compared to healthy subjects, patients with SCD had higher levels of F1.2 (403 nmol/L [266 nmol/L] vs. 136 nmol/L [58 nmol/L], p = 0.002) and lower levels of TF (26 pg/ml [12 pg/ml] vs. 37 pg/ml [9 pg/ml], p = 0.017) following prasugrel administration.
Fig. 2. Soluble Biomarkers at Baseline and Day 12 in Healthy Subjects and Patients with Sickle Cell Disease See text of Fig. 1 for key. Abbreviations: TXB2 = thromboxane B2.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
J.A. Jakubowski et al. / Thrombosis Research xxx (2013) xxx–xxx Table 3 Soluble Biomarkers of Platelet Activation and Coagulation at Baseline and Day 12 of Prasugrel Administration in Healthy Subjects and Patients with Sickle Cell Disease. Biomarker
Time
Healthy Subjects (N = 13) mean (SD)
Patients with SCD (N = 13)c mean (SD)
p-valueb
Soluble CD40L (pg/ml)
Baseline Day 12 p-value a Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea
266.6 (184.2) 155.6 (83.8) 0.046 205.8 (291.9) 135.6 (57.6) 0.405 129.4 (82.0) 151.0 (74.5) 0.426 29.5 (11.1) 26.7 (8.0) 0.082 31.9 (11.4) 36.8 (8.8) 0.066
610.2 (644.2) 419.4 (640.2) 0.477 1777.0 (2487.4) 403.0 (265.5) 0.120 221.5 (114.0) 185.6 (65.9) 0.167 37.6 (9.2) 32.8 (12.3) 0.215 25.2 (9.5) 25.7(12.0) 0.700
0.120 0.385
Soluble F1.2 (nmol/L)
Serum thromboxane B2 (ng/mL) Soluble P-Selectin (ng/ml) Soluble tissue factor (pg/ml)
0.033 0.002 0.027 0.233 0.054 0.157 0.115 0.017
Abbreviations: CD40L = CD40 ligand; F1.2 = prothrombin fragment 1.2; N = number of subjects/patients; SCD = sickle cell disease; SD = standard deviation. a p-values derived from one sample t-test. b p-values derived from two sample t-test. c N = 13 at baseline and 12 at study end.
Discussion In a previous study, we used multiple measures of platelet aggregation to demonstrate that compared to healthy subjects, patients with SCD generally exhibited greater platelet aggregation in response to ADP, but that aggregation was lowered by a similar extent in both groups following prasugrel administration [18]. In the present report, we provide results of an analysis of cellular and soluble biomarkers of platelet activation and coagulation that were a component to that study. At baseline, that is, prior to prasugrel administration, patients with SCD showed evidence of activation of both platelets and coagulation compared to healthy subjects. This was reflected by an increase in the percentage of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. In addition, levels of the soluble biomarkers F1.2 and serum TXB2 were elevated at baseline in patients with SCD. These elevations in biomarkers of hemostasis in the noncrisis steady state are consistent with the findings of others [2,6,9,12] and suggest platelet/coagulation involvement in the pathophysiology of SCD. After 12 days of prasugrel, a specific platelet P2Y12 ADP receptor antagonist, percentages of monocyte-platelet aggregates were significantly lowered in patients with SCD, suggesting that platelet activation was dependent, at least in part, on ADP signaling through P2Y12. Although the source of ADP is unknown, it may originate from hemolysis of sickled erythrocytes, a hallmark of SCD. Alternatively, ADP may be released by platelets themselves as a consequence of thrombin stimulation, and thus not be a direct result of erythrocyte-derived ADP. Regardless of its source, ADP stimulates platelet activation and contributes to surface exposure of negatively charged phospholipids (phosphatidylserine) that are required for assembly of Factors Xa and Va into a prothrombinase complex, thereby enhancing the rate of thrombin generation by a factor of 105 compared to the activity of Factor Xa alone [24]. Indeed, plasma F1.2, which is released when prothrombin is cleaved to generate thrombin, was elevated at baseline in patients with SCD compared to healthy subjects. Thus, the large numerical decrease in F1.2 in patients with SCD who are treated with prasugrel suggests that activated platelets contribute to thrombin generation in SCD, and that reducing platelet activation with prasugrel may help to limit thrombotic events. After 12 days of prasugrel administration, patients with SCD had a significant reduction in platelet-monocyte aggregates. In addition to their potential inflammatory role [25] these aggregates may also contribute to coagulation through expression of TF factor on their surface.
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The active metabolite of prasugrel, R-138727, has been shown to reduce TF expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates [26]. Taken together, these results suggest that patients with SCD are in a hypercoaguable state and also exhibit heightened platelet reactivity/activation, and that prasugrel may, to a certain extent, dampen SCD-related activation of both platelets and the coagulation system. Prior studies of patients with SCD in the non-crisis steady state that included biomarkers of platelet activation and coagulation found similar results [2,6,9,12]. Similar to Wun et al. [9,27], we found elevated levels of monocyte-platelet aggregates in patients with SCD compared to healthy controls, and also noted elevated levels of neutrophil-platelet aggregates. Elevations in activation-dependent platelet expression of GPIIbIIIa, P-selectin, and CD40L were previously identified in flow cytometric studies of patients with SCD [9,12]. This analysis confirmed significant elevations of CD40L at baseline, and an elevation in platelet surface expressed P-selectin that approached statistical significance (p = 0.058). We also observed elevations in levels of soluble F1.2 and TXB2 in patients with SCD. We did not, however, see the elevation in soluble CD40L and TF that had been previously described by Lee et al. [5]. Some of the discrepancies noted above may be a reflection of the relatively small number of patients in the current study, which we acknowledge as a limitation. For example, the difference in soluble CD40L for healthy subjects and patients with SCD appeared large, but was not statistically significant. Few studies of patients with SCD have looked at response to treatment with anticoagulants or antiplatelet agents and included examination of the biomarkers highlighted in this analysis, and/or have considered the impact of prasugrel, a third generation thienopyridyl inhibitor of ADPmediated platelet activation [28]. Two prior studies have demonstrated a significant reduction in F1.2 in patients with SCD treated with the vitamin K antagonist, acenocoumarol [29,30]. We did not observe this effect with prasugrel; however, acenocoumarol specifically and potently targets components of the coagulation cascade, while prasugrel is primarily an antiplatelet agent. We did, however, find that in patients with SCD, prasugrel produced reductions in monocyte-platelet aggregates, presumably reflecting a reduction in circulating levels of monocyteplatelet aggregates in vivo. Limitations of our study include the possibility that the impact of prasugrel treatment on biomarker levels in patients with SCD may have been underrepresented due to a small sample size. Also, although a 12-day study allowed prasugrel to reach steady state, more prolonged administration may be needed to see the true effect of prasugrel treatment in this population. Though not presented here, it should also be noted that in our patients with SCD, platelet and leukocyte counts were significantly higher than counts in healthy subjects, and this, or other differences in baseline hematologic state, might account for differences in activation markers such as those noted for platelet leukocyte aggregates. Also, although healthy subjects were matched by weight and age to patients in the SCD group, they were not matched for ethnicity. In conclusion, baseline elevations of biomarkers of platelet activation and coagulation found in this study confirm the findings of previous studies of patients with SCD. Attenuation of several of these markers following prasugrel administration suggests a role for ADP as a driver of in vivo activation in SCD and supports further investigation of prasugrel as a potential therapy for ischaemic complications of this disease. Conflict of interest statement Joseph A. Jakubowski, Chunmei Zhou, Kenneth J. Winters, D. Richard Lachno, Neehar Gupta, Christopher D. Payne, and Kenneth J. Winters had employment with and held stock in Eli Lilly and Company in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work; Andrew L. Frelinger III had consultancy fees and grants from Eli Lilly and Company and Daiichi
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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Sankyo Company, Ltd. in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work. Timothy G. Mant had employment with and held stock in Quintiles, Inc., who were contracted with Eli Lilly for assistance on this investigation. Stipo Jurcevic received funding from Eli Lilly for consumables and work hours of laboratory staff. Jo Howard declared no conflicts of interest.
Acknowledgements This research was supported by Daiichi Sankyo Company, Ltd., and Eli Lilly and Company. Sponsors were involved in the study design, in the collection, analysis and interpretation of data; in the writing of the manuscript; and in the decision to submit the manuscript for publication. Appreciation is expressed to Tamara Ball, MD, (inVentiv Health Clinical) for writing and editorial contributions. Also acknowledged is Julie Sherman, AAS, of Eli Lilly for assistance with graphics. Dr. Mant is supported by the National Institute for Health Research Biomedical Research Centre at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London. References [1] World Health Organization. Genes and Human Disease: Sickle Cell Anemia. c2012 [cited 2013 Apr 24]. Available from: http://www.who.int/genomics/public/ geneticdiseases/en/index2.html#SCA. [2] Charneski L, Congdon HB. Effects of antiplatelet and anticoagulant medications on the vasoocclusive and thrombotic complications of sickle cell disease: A review of the literature. Am J Health Syst Pharm 2010;67:895–900. [3] Serjeant GR. Sickle-cell disease. Lancet 1997;350:725–30. [4] Steinberg MH. Management of sickle cell disease. N Engl J Med 1999;340:1021–30. [5] Lee SP, Ataga KI, Orringer EP, Phillips DR, Parise LV. Biologically active CD40 ligand is elevated in sickle cell anemia: potential role for platelet-mediated inflammation. Arterioscler Thromb Vasc Biol 2006;26:1626–31. [6] Ataga KI, Cappellini MD, Rachmilewitz EA. Beta-thalassaemia and sickle cell anaemia as paradigms of hypercoagulability. Br J Haematol 2007;139:3–13. [7] Francis Jr RB. Platelets, coagulation, and fibrinolysis in sickle cell disease: their possible role in vascular occlusion. Blood Coagul Fibrinolysis 1991;2:341–53. [8] Ataga KI, Orringer EP. Hypercoagulability in sickle cell disease: a curious paradox. Am J Med 2003;115:721–8. [9] Wun T, Paglieroni T, Tablin F, Welborn J, Nelson K, Cheung A. Platelet activation and platelet-erythrocyte aggregates in patients with sickle cell anemia. J Lab Clin Med 1997;129:507–16. [10] Jakubowski JA, Winters KJ, Naganuma H, Wallentin L. Prasugrel: A novel thienopyridine antiplatelet agent. A review of preclinical and clinical studies and the mechanistic basis for its distinct antiplatelet profile. Cardiovasc Drug Rev 2007;25:358–74. [11] Inwald DP, Kirkham FJ, Peters MJ, Lane R, Wade A, Evans JP, Klein NJ. Platelet and leucocyte activation in childhood sickle cell disease: association with nocturneal hypoxaemia. Br J Haematol 2000;111:474–81. [12] Tomer A, Harker LA, Kasey S, Eckman JR. Thrombogenesis in sickle cell disease. J Lab Clin Med 2001;137:398–407.
[13] Beurling-Harbury C, Schade SG. Platelet activation during pain crisis in sickle cell anemia patients. Am J Hematol 1989;31:237–41. [14] Semple MJ, Al-Hasani SF, Savidge GF. A double-blind trial of ticlopidine in sickle cell disease. Thromb Haemost 1984;51:303–6. [15] Cabannes R, Lonsdorfer J, Castaigne JP, Ondo A, Plassard A, Zohoun I. Clinical and biological double-blind-study of ticlopidine in preventive treatment of sickle-cell disease crises. Agents Actions Suppl 1984;15:199–212. [16] Small DS, Li YG, Ernest CS, April JH, Farid NA, Payne CD, Winters KJ, Rohatagi S, Ni L. Integrated analysis of pharmacokinetic data across multiple clinical pharmacology studies of prasugrel, a new thienopyridine antiplatelet agent. J Clin Pharmacol 2011;51:321–32. [17] Jakubowski JA, Payne CD, Li YG, Brandt JT, Small DS, Farid NA, Salazar DE, Winters KJ. The use of the VerifyNow P2Y12 point-of-care device to monitor platelet function across a range of P2Y12 inhibition levels following prasugrel and clopidogrel administration. Thromb Haemost 2008;99:409–15. [18] Jakubowski JA, Zhou C, Small DS, Winters KJ, Lachno DR, Frelinger III AL, Howard J, Mant TG, Jurcevic S, Payne CD. A Phase 1 Study of Prasugrel in Patients with Sickle Cell Disease: Pharmacokinetics and Effects on ex Vivo Platelet Reactivity. Br J Clin Pharmacol 2013;75:1433–44. [19] Barnard MR, Linden MD, Frelinger III AL, Li Y, Fox ML, Furman MI, Michelson AD. Effects of platelet binding on whole blood flow cytometry assays of monocyte and neutrophil procoagulant activity. J Thromb Haemost 2005;3:2563–70. [20] Frelinger III AL, Jakubowski JA, Li Y, Barnard MR, Linden MD, Tarnow I, Fox ML, Sugidachi A, Winters KJ, Furman MI, Michelson AD. The active metabolite of prasugrel inhibits ADP-stimulated thrombo-inflammatory markers of platelet activation: Influence of other blood cells, calcium, and aspirin. Thromb Haemost 2007;98:192–200. [21] Furman MI, Kereiakes DJ, Krueger LA, Mueller MN, Pieper K, Broderick TM, Schneider JF, Howard WL, Fox ML, Barnard MR, Frelinger AL, Michelson AD, et al. Leukocyteplatelet aggregation, platelet surface P-selectin, and platelet surface glycoprotein IIIa after percutaneous coronary intervention: Effects of dalteparin or unfractionated heparin in combination with abciximab. Am Heart J 2001;142:790–8. [22] Berny-Lang M, Frelinger ALI, Barnard MR, Michelson AD. Flow Cytometry. In: Michelson AD, editor. Platelets. 3rd ed. San Diego: Academic Press; 2012. p. 581–602. [23] Panara MR, Renda G, Sciulli MG, Santini G, Di Giamberardino M, Rotondo MT, Tacconelli S, Seta F, Patrono C, Patrignani P. Dose-dependent inhibition of platelet cyclooxygenase-1 and monocyte cyclooxygenase-2 by meloxicam in healthy subjects. J Pharmacol Exp Ther 1999;290:276–80. [24] Nesheim ME, Taswell JB, Mann KG. The contribution of bovine Factor V and Factor Va to the activity of prothrombinase. J Biol Chem 1979;254:10952–62. [25] Ziegler-Heitbrock L. The CD14+ CD16+ blood monocytes: their role in infection and inflammation. J Leukoc Biol 2007;81:584–92. [26] Frelinger III AL, Jakubowski JA, Li Y, Barnard MR, Linden MD, Tarnow I, Fox ML, Sugidachi A, Winters KJ, Furman MI, Michelson AD. The active metabolite of prasugrel inhibits adenosine diphosphate- and collagen-stimulated platelet procoagulant activities. J Thromb Haemost 2008;6:359–65. [27] Wun T, Soulieres D, Frelinger AL, Krishnamurti L, Novelli EM, Kutlar A, Ataga KI, Knupp CL, McMahon L, Strouse JJ, Zhou C, Heath LE, Nwachuku CE, Jakubowski JA, Riesmeyer JS, Winters KJ. A Double-Blind, Randomized, Multicenter Phase 2 Study of Prasugrel versus Placebo in Adult Patients with Sickle Cell Disease. J Hematol Oncol 2013;6:17. [28] Wiviott SD, Braunwald E, McCabe CH, Montalescot G, Ruzyllo W, Gottlieb S, Neumann FJ, Ardissino D, De Servi S, Murphy SA, Riesmeyer J, Weerakkody G, Gibson CM. Antman EM; TRITON-TIMI 38 Investigators. Prasugrel versus clopidogrel in patients with acute coronary syndromes. N Engl J Med 2007;357:2001–15. [29] Schnog JB, Kater AP, Mac Gillavry MR, Duits AJ, Lard LR, van Der Dijs FP, Brandjes DP, ten Cate H, van Eps LW, Rojer RA. Low adjusted-dose acenocoumarol therapy in sickle cell disease: a pilot study. Am J Hematol 2001;68:179–83. [30] Wolters HJ, ten Cate H, Thomas LL, Brandjes DP, van der Ende A, van der Heiden Y. Statius van Eps LW. Low-intensity oral anticoagulation in sickle-cell disease reverses the prethrombotic state: promises for treatment? Br J Haematol 1995;90:715–7.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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Regular Article
A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation☆ Joseph A. Jakubowski a,⁎, Chunmei Zhou a, Stipo Jurcevic b, Kenneth J. Winters a, D. Richard Lachno c, Andrew L. Frelinger III d, Neehar Gupta a, Jo Howard e, Christopher D. Payne c, Timothy G. Mant f a
Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, IN USA King’s College, London UK Lilly Research Laboratories, Eli Lilly and Company, Windlesham, Surrey, UK d Center for Platelet Research Studies, Division of Hematology/Oncology, Boston Children’s Hospital, Dana-Farber Cancer Institute, Harvard Medical School, MA USA e Guy’s and St. Thomas’ Hospital, London UK f Quintiles Drug Research Unit at Guy’s Hospital, London UK b c
a r t i c l e
i n f o
Article history: Received 20 August 2013 Received in revised form 20 November 2013 Accepted 3 December 2013 Available online xxxx Keywords: ADP Biomarkers P2Y12 receptor antagonist Platelets Prasugrel Sickle cell disease
a b s t r a c t Introduction: Prasugrel, a P2Y12 adenosine diphosphate (ADP) receptor antagonist effectively inhibits ADPmediated platelet activation and aggregation, and may be useful in reducing vaso-occlusive crises in sickle cell disease (SCD). In this study, we assess the effect of prasugrel on biomarkers of platelet activation and coagulation in patients with SCD. Materials and Methods: Twelve adult patients with SCD and 13 healthy subjects were examined before and after 12 ± 2 days of 5.0 or 7.5 mg/day oral prasugrel. Assessed cellular biomarkers included monocyte- and neutrophil-platelet aggregates, activated glycoprotein IIb-IIIa (GPIIbIIIa), P-selectin, CD40 ligand (CD40L), tissue factor (TF) expression on circulating platelets and on monocyte-platelet aggregates, and platelet-erythrocyte aggregates. Soluble biomarkers included CD40L, prothrombin fragment 1.2 (F1.2), thromboxane B2 (TXB2), P-selectin, and TF. Results: Patients with SCD had increased platelet baseline activation compared to healthy subjects, as measured by percentages of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. Likewise, baseline levels of soluble F1.2 and TXB2 were elevated in patients with SCD compared to healthy subjects. After 12 days of prasugrel, patients with SCD had a significant reduction in platelet-monocyte aggregates that was not observed in healthy subjects. Following prasugrel administration, those with SCD maintained higher levels of monocyte-platelet aggregates and soluble F1.2, but had lower levels of platelet-erythrocyte aggregates and soluble TF compared to healthy subjects. Conclusions: These results provide evidence for chronic platelet activation in the SCD steady state, activation that was in part attenuated by prasugrel, thereby suggesting that ADP may mediate platelet activation in SCD. © 2013 Elsevier Ltd. All rights reserved.
Introduction Sickle cell disease (SCD) is a common hemoglobinopathy that affects millions of people worldwide and has substantial medical, social, and economic impact [1]. Much of the morbidity and mortality of SCD arises from complications of vaso-occlusive crisis (VOC), which can result in
Abbreviations: ADP, adenosine diphosphate; CD40L, CD40 ligand; EDTA, ethylenediaminetetraacetic acid; GPIIbIIIa, glycoprotein IIb-IIIa; PD, pharmacodynamics; PK, pharmacokinetics; RBCs, red blood cells; SCD, sickle cell disease; SD, standard deviation; TF, tissue factor; TXB2, thromboxane B2; VOC, vaso-occlusive crisis. ☆ This research was supported by Daiichi Sankyo Company, Ltd., and Eli Lilly and Company. ⁎ Corresponding author at: Lilly Research Laboratories, Lilly Corporate Center, Indianapolis, IN 46285, USA. Tel.: +1 317 276 9036; fax: +1 317 433 1996. E-mail address: [email protected] (J.A. Jakubowski).
ischemia and infarction of affected organs and which manifests clinically as episodic bouts of localized pain [2–4]. Microvascular vaso-occlusion and chronic inflammation are the hallmarks of VOC. Although the underlying pathophysiology remains unclear, it is thought to be the result of a complex and dynamic interplay between sickled red blood cells (RBCs), endothelial cells, leukocytes, platelets, and various plasma proteins. Evidence suggests that in patients with SCD, circulating platelets are chronically activated in the non-crisis steady state [5], and that this activation intensifies during VOC [6–8]. As sickled RBCs haemolyze, they release adenosine diphosphate (ADP) [6,9], which may drive platelet activation and aggregation [10]. In addition, activated platelets have increased expression of proteins such as CD40 ligand (CD40L) [5,8,11] and P-selectin [12], which may promote interaction with endothelial and inflammatory cells [11,12]. In addition to erythrocytes, platelet and leukocyte aggregates contribute to vessel occlusion [13]. Several studies have also found
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Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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coagulation markers including fibrin [7], tissue factor (TF), and thrombin [6,8] to be elevated in SCD in the non-crisis steady state and, to be further elevated during VOC in children and adult patients [7,12]. The potential role of platelets in VOC suggests that antiplatelet therapies may hold promise for prevention and treatment in SCD. Thus far, there have been two double-blind, placebo-controlled studies of the first-generation ADP-receptor antagonist ticlodipine. Though an ex vivo decrease in platelet aggregation was seen in a 4-week study of ticlodipine therapy, no difference in the frequency of VOC was observed [14]. However, in a larger study of ticlodipine lasting 6 months, a significant reduction in painful crises was seen in patients with SCD [15]. Prasugrel is a third generation platelet P2Y12 ADP-receptor antagonist, which has been shown to effectively inhibit platelet activation and aggregation via irreversible inhibition of the P2Y12 ADP receptor [10]. Given the potential role of ADP in the manifestation of VOC, prasugrel may reduce the frequency and severity of VOC and consistently decrease platelet activation to help reduce chronic low-level ischemia in these patients. The pharmacokinetic (PK) and pharmacodynamic (PD) characteristics of prasugrel have been well-characterized in healthy control subjects and in patients with coronary artery disease [16,17], but were unknown in patients with SCD. However, these characteristics have now been evaluated before and after 12 days of prasugrel administration (5.0 mg/day or 7.5 mg/day) in a Phase 1b, open-label, single-center trial that included patients with SCD and healthy control subjects. In patients with SCD, prasugrel produced dose-dependent exposure to its active metabolite, which in turn reduced platelet reactivity to ADP, as measured using a variety of assays [18]. In the current analysis of data from the same study, we examine levels of cellular and soluble biomarkers of platelet activation and coagulation at baseline and following 12 days of prasugrel administration in patients with SCD and compare these values to those found in healthy subjects. Materials and Methods Study design This single-center, open-label trial was sponsored by Daiichi Sankyo Company, Ltd. and Eli Lilly and Company from July 2010 through February 2011 (NCT01178099). Patients with SCD and healthy controls were given a single 10-mg oral dose of prasugrel followed by administration for 12 ± 2 days of oral prasugrel, 5 mg/day for those weighing b60 kg and 7.5 mg/day for those weighing ≥60 kg. Participants Enrolled participants had to be between 18 and 60 years of age and weigh between 50 and 100 kg. Patients with SCD had to have the genotypes HbSS, HbSC, or HbSβ0 or HbSβ+ thalassemia, and were to have had no occurrence of VOC within 1 month of screening. Healthy control subjects were matched for gender, age (within 10 years), and weight (b 60 kg and ≥60 kg within 10 kg). Please refer to the primary publication for further detail [18].
assessing the binding of a platelet-specific antibody, CD61, to these cells. TF present on the surface of monocyte-platelet aggregates was measured using a CD142-specific antibody. The level of circulating platelet-erythrocyte aggregates was determined by gating on CD41a-positive platelets and measuring staining of the erythrocyte-specific glycophorin marker CD235a to determine the percent of platelet-erythrocyte aggregates [9]. The activation state of circulating platelets was determined by the detection of activation-dependent changes in the platelet surface markers by flow cytometry [22]. Platelets were identified by size, light scatter pattern, and the binding of a CD61-specific antibody. The expression of P-selectin (CD62P), activated glycoprotein IIb-IIIa (GPIIbIIIa [PAC-1]), CD40L (CD154), and TF (CD142) on the surface of platelets was measured by staining with antibodies specific for these markers (all antibodies were from BD Biosciences, Oxford, UK). Flow cytometric analysis was performed using a FACSCalibur cytometer and CellQuest Pro software (both BD Biosciences, San Jose, CA). Results were presented as percent of positive cells expressing a given marker above the background control level of staining. For platelet activation markers, this related to the proportion of platelets that may participate in aggregates or inflammatory processes. Soluble CD40L (Quantikine Human CD40 Ligand Immunoassay, R&D Systems, Minneapolis, MN) and P-selectin (Human P-Selectin ELISA, R&D Systems, Minneapolis, MN) were assessed in plasma prepared from blood samples collected into ethylenediaminetetraacetic acid (EDTA) and centrifuged at 1000 g for 15 minutes within 30 minutes of collection. Samples for the CD40L assay were centrifuged for an additional 10 minutes at 10,000 g. Prothrombin fragment 1.2 (F1.2) was measured in plasma prepared from blood collected in 3.2% citrate using Dade-Behring Enzygnost F1 + 2 microkit (Marburg, Germany). Serum for thromboxane B2 (TXB2) was prepared from whole blood collected into glass tubes and allowed to clot for 1 hour at 37 °C [23] and assayed by immunoassay (Cayman Thromboxane B2 EIA kit, Cayman Chemical, Ann Arbor, MI). Blood samples for soluble coagulation Factor III/TF measurement were collected in 3.2% sodium citrate. After centrifugation at 1500 g for 15 minutes, plasma was taken off and frozen at −80 °C within 30 minutes of collection. Thawed samples were tested using an immunoassay method (R& D Systems, Abingdon, UK).
Statistical analysis Unless otherwise noted, values are presented as mean and standard deviation (SD) for continuous variables and as percentages for categorical variables. The two dose groups (5 mg/day and 7.5 mg/day) were combined for all analyses since there were only 4 subjects in each group receiving 5 mg/day and the doses were similar when considered on a mg/kg basis (0.107 ± 0.015 mg/kg for SCD patients and 0.104 ± 0.012 mg/kg for healthy subjects, p = 0.66). Platelet biomarker levels Table 1 Baseline Demographic Characteristics for Healthy Subjects and Patients with Sickle Cell Disease.
Cellular and soluble platelet and coagulation biomarkers
Characteristic/measurement
Healthy Subjects (N = 13)
Patients with SCD (N = 13)a
Within groups, we assessed the response to 12 ± 2 days of prasugrel administration, and between groups, we compared biomarker levels before and after prasugrel administration. Venous blood samples were collected at baseline (1 hour before the first dose of prasugrel) and on Day 12, 24 hours after the previous 5-mg or 7.5-mg dose of prasugrel. Whole blood samples were collected into 3.2% citrate and, within 2 hours of collection, were assessed for biomarker expression by flow cytometry. As previously described, monocyte and neutrophil cell populations were distinguished within whole blood on the basis of their differential expression of CD14 and light scatter properties [19–21]. The presence of heterotypic platelet aggregates was then determined by
Age (years), mean (SD) Range (years) Male gender, n (%) Ethnicity, n (%) African descent Asian White Weight (kg) Mean (SD) Range (minimum – maximum)
26.7 (5.7) 19-42 7 (53.8)
29.1 (9.3) 19-50 8 (61.5)
3 (23.1) 1 (7.7) 9 (69.2)
13 (100.0) 0 (0.0) 0 (0.0)
64.5 (9.6) 50.5 – 82.1
63.3 (9.5) 52.2 – 87.2
Abbreviations: N = total number of subjects/patients; n = number of subjects/patients in that category; SCD = sickle cell disease; SD = standard deviation. a N = 13 at baseline and 12 at study end.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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at baseline and Day 12 were compared between patients with SCD and healthy subjects using a two sample t-test. Platelet reactivity changes from baseline to Day 12 within each population were analyzed using a one sample t-test. For all statistical comparisons, a p-value b0.05 was considered statistically significant.
The majority of healthy subjects were white, while all patients with SCD were of African descent. All patients with SCD were of the HbSS genotype. Due to poor vein quality, one patient with SCD discontinued the study following the baseline venipuncture, immediately after the first dose of prasugrel. All healthy subjects completed the study.
Results
Cellular platelet biomarkers (Fig. 1 and Table 2)
Demographics (Table 1)
All data are presented as values (mean [SD]) for patients with SCD vs. values for healthy subjects. At baseline, patients with SCD had a greater percentage of monocyte-platelet aggregates compared with healthy controls (78% [26%] vs. 51% [18%], p = 0.004). Patients with SCD also had a greater percentage of neutrophil-platelet aggregates
Twenty-six subjects were enrolled; 13 healthy subjects and 13 patients with SCD. The mean age was 27 years for healthy subjects (range 19-42 years) and 29 years for patients with SCD (range 19–50 years).
Fig. 1. Whole Blood Platelet Biomarkers at Baseline and after 12 Days of Prasugrel Administration in Healthy Subjects and Patients with Sickle Cell Disease. Boxes represent the 25th through 75th percentiles and whiskers represent the 10th and 90th percentiles. The solid line is the median and the dashed line is the mean. Results are expressed as percent of cells positive for the respective biomarker. Abbreviations: CD40L = CD40 ligand.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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Table 2 Whole Blood Biomarkers of Platelet Activation and Coagulation at Baseline and Day 12 of Prasugrel Administration in Healthy Subjects and Patients with Sickle Cell Disease. Biomarker
Monocyte-platelet aggregates (%) Neutrophil-platelet aggregates (%) CD40L (% cell CD40L)
P-Selectin (% CD62P expression) Activated GPIIbIIIa (% PAC-1) Tissue factor expression on circulating platelets (%) Monocyte-platelet tissue factor expression (%) Platelet-erythrocyte aggregates (%)
Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea
Healthy Subjects (N = 13) mean (SD)
Patients with SCD (N = 13)c mean (SD)
p-valueb
50.7 (17.9) 40.4 (23.5) 0.254 17.7 (4.0) 16.1 (8.2) 0.528 13.0 (4.6) 17.8 (4.8) 0.011 10.3 (6.4) 5.4 (2.1) 0.012 5.8 (4.7) 4.9 (7.6) 0.803 4.2 (3.3) 5.5 (3.6) 0.379 7.3 (3.3) 9.9 (6.7) 0.105 8.5 (4.6) 8.2 (2.2) 0.819
78.0 (25.6) 62.2 (24.3) 0.045 27.5 (15.2) 25.9 (17.8) 0.837 17.3 (4.0) 16.4 (3.8) 0.504 15.8 (7.5) 12.0 (12.7) 0.324 9.3 (9.8) 7.8 (12.5) 0.300 5.0 (3.4) 4.8 (3.3) 0.506 5.1 (3.1) 5.8 (1.8) 0.593 5.9 (1.3) 5.6 (2.7) 0.683
0.004 0.032 0.035 0.086 0.017 0.441 0.058 0.079 0.277 0.479 0.522 0.602 0.082 0.051 0.060 0.017
Abbreviations: CD40L = CD40 ligand; GPIIbIIIa = glycoprotein IIb-IIIa; N = number of subjects/patients; SCD = sickle cell disease; SD = standard deviation. a p-values derived from one sample t-test. b p-values derived from two sample t-test. c N = 13 at baseline and 12 at study end.
(27% [15%] vs. 18% [4%], p = 0.035) and expression of platelet CD40L (17% [4%] vs. 13% [5%], p = 0.017). Following 12 ± 2 days of prasugrel administration, patients with SCD demonstrated a significant decrease in the number of monocyte-platelet aggregates (from 78% [26%] to 62% [24%], p = 0.045) that was not seen in healthy subjects. In addition, at the end of the treatment period, patients with SCD maintained a higher percentage of monocyte-platelet aggregates (62% [24%] vs. 40% [23%], p = 0.032) and a lower percentage of platelet-erythrocyte aggregates (6% [3%] vs. 8% [2%], p = 0.017) than healthy subjects. Following prasugrel administration, platelet surface P-selectin had decreased in healthy subjects (from 10% [6%] to 5% [2%], p = 0.012) and CD40L had increased (from 13% [5%] to18% [5%], p = 0.011), changes that were not observed in the SCD group. Other biomarkers did not differ significantly between groups at baseline or following prasugrel treatment, or within groups from baseline to Day 12.
Soluble biomarkers (Fig. 2 and Table 3) Patients with SCD had higher plasma concentrations of F1.2 at baseline than healthy controls (1777 nmol/L [2487 nmol/L] vs. 206 nmol/L [292 nmol/L], p = 0.033). Baseline levels of serum TXB2 were also higher in patients with SCD (221 ng/ml [114 ng/ml] vs. 129 ng/ml [82 ng/ml], p = 0.027). While prasugrel administration lowered the mean level of F1.2 in patients with SCD, the change from baseline did not meet statistical significance (from 1777 nmol/L [2487 nmol/L] to 403 nmol/L [266 nmol/L], p = 0.120). Compared to healthy subjects, patients with SCD had higher levels of F1.2 (403 nmol/L [266 nmol/L] vs. 136 nmol/L [58 nmol/L], p = 0.002) and lower levels of TF (26 pg/ml [12 pg/ml] vs. 37 pg/ml [9 pg/ml], p = 0.017) following prasugrel administration.
Fig. 2. Soluble Biomarkers at Baseline and Day 12 in Healthy Subjects and Patients with Sickle Cell Disease See text of Fig. 1 for key. Abbreviations: TXB2 = thromboxane B2.
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
J.A. Jakubowski et al. / Thrombosis Research xxx (2013) xxx–xxx Table 3 Soluble Biomarkers of Platelet Activation and Coagulation at Baseline and Day 12 of Prasugrel Administration in Healthy Subjects and Patients with Sickle Cell Disease. Biomarker
Time
Healthy Subjects (N = 13) mean (SD)
Patients with SCD (N = 13)c mean (SD)
p-valueb
Soluble CD40L (pg/ml)
Baseline Day 12 p-value a Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea Baseline Day 12 p-valuea
266.6 (184.2) 155.6 (83.8) 0.046 205.8 (291.9) 135.6 (57.6) 0.405 129.4 (82.0) 151.0 (74.5) 0.426 29.5 (11.1) 26.7 (8.0) 0.082 31.9 (11.4) 36.8 (8.8) 0.066
610.2 (644.2) 419.4 (640.2) 0.477 1777.0 (2487.4) 403.0 (265.5) 0.120 221.5 (114.0) 185.6 (65.9) 0.167 37.6 (9.2) 32.8 (12.3) 0.215 25.2 (9.5) 25.7(12.0) 0.700
0.120 0.385
Soluble F1.2 (nmol/L)
Serum thromboxane B2 (ng/mL) Soluble P-Selectin (ng/ml) Soluble tissue factor (pg/ml)
0.033 0.002 0.027 0.233 0.054 0.157 0.115 0.017
Abbreviations: CD40L = CD40 ligand; F1.2 = prothrombin fragment 1.2; N = number of subjects/patients; SCD = sickle cell disease; SD = standard deviation. a p-values derived from one sample t-test. b p-values derived from two sample t-test. c N = 13 at baseline and 12 at study end.
Discussion In a previous study, we used multiple measures of platelet aggregation to demonstrate that compared to healthy subjects, patients with SCD generally exhibited greater platelet aggregation in response to ADP, but that aggregation was lowered by a similar extent in both groups following prasugrel administration [18]. In the present report, we provide results of an analysis of cellular and soluble biomarkers of platelet activation and coagulation that were a component to that study. At baseline, that is, prior to prasugrel administration, patients with SCD showed evidence of activation of both platelets and coagulation compared to healthy subjects. This was reflected by an increase in the percentage of monocyte-platelet aggregates, neutrophil-platelet aggregates, and platelets expressing CD40L. In addition, levels of the soluble biomarkers F1.2 and serum TXB2 were elevated at baseline in patients with SCD. These elevations in biomarkers of hemostasis in the noncrisis steady state are consistent with the findings of others [2,6,9,12] and suggest platelet/coagulation involvement in the pathophysiology of SCD. After 12 days of prasugrel, a specific platelet P2Y12 ADP receptor antagonist, percentages of monocyte-platelet aggregates were significantly lowered in patients with SCD, suggesting that platelet activation was dependent, at least in part, on ADP signaling through P2Y12. Although the source of ADP is unknown, it may originate from hemolysis of sickled erythrocytes, a hallmark of SCD. Alternatively, ADP may be released by platelets themselves as a consequence of thrombin stimulation, and thus not be a direct result of erythrocyte-derived ADP. Regardless of its source, ADP stimulates platelet activation and contributes to surface exposure of negatively charged phospholipids (phosphatidylserine) that are required for assembly of Factors Xa and Va into a prothrombinase complex, thereby enhancing the rate of thrombin generation by a factor of 105 compared to the activity of Factor Xa alone [24]. Indeed, plasma F1.2, which is released when prothrombin is cleaved to generate thrombin, was elevated at baseline in patients with SCD compared to healthy subjects. Thus, the large numerical decrease in F1.2 in patients with SCD who are treated with prasugrel suggests that activated platelets contribute to thrombin generation in SCD, and that reducing platelet activation with prasugrel may help to limit thrombotic events. After 12 days of prasugrel administration, patients with SCD had a significant reduction in platelet-monocyte aggregates. In addition to their potential inflammatory role [25] these aggregates may also contribute to coagulation through expression of TF factor on their surface.
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The active metabolite of prasugrel, R-138727, has been shown to reduce TF expression on ADP-, collagen-, and ADP plus collagen-stimulated monocyte-platelet aggregates [26]. Taken together, these results suggest that patients with SCD are in a hypercoaguable state and also exhibit heightened platelet reactivity/activation, and that prasugrel may, to a certain extent, dampen SCD-related activation of both platelets and the coagulation system. Prior studies of patients with SCD in the non-crisis steady state that included biomarkers of platelet activation and coagulation found similar results [2,6,9,12]. Similar to Wun et al. [9,27], we found elevated levels of monocyte-platelet aggregates in patients with SCD compared to healthy controls, and also noted elevated levels of neutrophil-platelet aggregates. Elevations in activation-dependent platelet expression of GPIIbIIIa, P-selectin, and CD40L were previously identified in flow cytometric studies of patients with SCD [9,12]. This analysis confirmed significant elevations of CD40L at baseline, and an elevation in platelet surface expressed P-selectin that approached statistical significance (p = 0.058). We also observed elevations in levels of soluble F1.2 and TXB2 in patients with SCD. We did not, however, see the elevation in soluble CD40L and TF that had been previously described by Lee et al. [5]. Some of the discrepancies noted above may be a reflection of the relatively small number of patients in the current study, which we acknowledge as a limitation. For example, the difference in soluble CD40L for healthy subjects and patients with SCD appeared large, but was not statistically significant. Few studies of patients with SCD have looked at response to treatment with anticoagulants or antiplatelet agents and included examination of the biomarkers highlighted in this analysis, and/or have considered the impact of prasugrel, a third generation thienopyridyl inhibitor of ADPmediated platelet activation [28]. Two prior studies have demonstrated a significant reduction in F1.2 in patients with SCD treated with the vitamin K antagonist, acenocoumarol [29,30]. We did not observe this effect with prasugrel; however, acenocoumarol specifically and potently targets components of the coagulation cascade, while prasugrel is primarily an antiplatelet agent. We did, however, find that in patients with SCD, prasugrel produced reductions in monocyte-platelet aggregates, presumably reflecting a reduction in circulating levels of monocyteplatelet aggregates in vivo. Limitations of our study include the possibility that the impact of prasugrel treatment on biomarker levels in patients with SCD may have been underrepresented due to a small sample size. Also, although a 12-day study allowed prasugrel to reach steady state, more prolonged administration may be needed to see the true effect of prasugrel treatment in this population. Though not presented here, it should also be noted that in our patients with SCD, platelet and leukocyte counts were significantly higher than counts in healthy subjects, and this, or other differences in baseline hematologic state, might account for differences in activation markers such as those noted for platelet leukocyte aggregates. Also, although healthy subjects were matched by weight and age to patients in the SCD group, they were not matched for ethnicity. In conclusion, baseline elevations of biomarkers of platelet activation and coagulation found in this study confirm the findings of previous studies of patients with SCD. Attenuation of several of these markers following prasugrel administration suggests a role for ADP as a driver of in vivo activation in SCD and supports further investigation of prasugrel as a potential therapy for ischaemic complications of this disease. Conflict of interest statement Joseph A. Jakubowski, Chunmei Zhou, Kenneth J. Winters, D. Richard Lachno, Neehar Gupta, Christopher D. Payne, and Kenneth J. Winters had employment with and held stock in Eli Lilly and Company in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work; Andrew L. Frelinger III had consultancy fees and grants from Eli Lilly and Company and Daiichi
Please cite this article as: Jakubowski JA, et al, A phase 1 study of prasugrel in patients with sickle cell disease: Effects on biomarkers of platelet activation and coagulation, Thromb Res (2013), http://dx.doi.org/10.1016/j.thromres.2013.12.008
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J.A. Jakubowski et al. / Thrombosis Research xxx (2013) xxx–xxx
Sankyo Company, Ltd. in the previous 3 years and no other relationships or activities that could appear to have influenced the submitted work. Timothy G. Mant had employment with and held stock in Quintiles, Inc., who were contracted with Eli Lilly for assistance on this investigation. Stipo Jurcevic received funding from Eli Lilly for consumables and work hours of laboratory staff. Jo Howard declared no conflicts of interest.
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