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Reversal of Clopidogrel-Induced Bleeding with rFVIIa in Healthy Subjects: A Randomized, Placebo-Controlled, Double-Blind, Exploratory Study

Skolnick, Brett E. PhD*; Shenouda, Magdy MD; Khutoryansky, Naum M. PhD*; Pusateri, Anthony E. PhD; Gabriel, Don MD, PhD§; Carr, Marcus E. MD, PhD, FACP*

doi: 10.1213/ANE.0b013e318228c690
Cardiovascular Anesthesiology: Research Reports
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SDC

BACKGROUND: Clopidogrel (Plavix®) therapy, although effective for minimizing risk of thrombotic events, is also associated with potential bleeding risk. Recombinant activated FVII (rFVIIa, NovoSeven®) induces hemostasis in hemophilia patients with inhibitors (alloantibodies) and has been proposed as potential treatment for mitigating clopidogrel therapy–mediated bleeding.

METHODS: In this single-center, randomized, placebo-controlled, double-blind, dose-escalation, exploratory phase I trial, we assessed the safety and effects of rFVIIa in reversing clopidogrel-enhanced bleeding in an experimentally induced punch biopsy in healthy subjects. Efficacy assessments included the reversal of bleeding characteristics (bleed duration [BD], the primary end point and blood loss volume [BV] induced by punch biopsy, and thromboelastograph [TEG®] parameters) with rFVIIa or placebo after clopidogrel treatment.

RESULTS: A significant number of subjects (56%) had limited response to clopidogrel (defined as ≤30% platelet aggregation inhibition) and were discontinued from study. The remaining subjects continued and had 4 biopsies. Of 40 subjects randomized, 37 were evaluated for efficacy. Clopidogrel treatment increased BD and BV compared with the baseline biopsy. Recombinant FVIIa (10 and 20 μg/kg) significantly mitigated the clopidogrel-induced effects on BV (P = 0.007 and P = 0.001, respectively). Early trial termination limited the evaluation of effects of higher rFVIIa doses. Subgroup analyses of subjects biopsied by the same physician demonstrated significant reduction of clopidogrel-induced BD with 20 μg/kg rFVIIa (P = 0.048). Ex vivo analysis of rFVIIa demonstrated clotting dynamics presented by parameters time to clot onset (TEG®-R) and clot angle (TEG®-A) (P < 0.005).

CONCLUSIONS: In this clinical study, rFVIIa (10 and 20 μg/kg) reversed the effect of clopidogrel on blood loss.

Published ahead of print September 2, 2011 Supplemental Digital Content is available in the text.

From *Novo Nordisk Inc., Princeton, New Jersey; MDS Pharma Services, Neptune, New Jersey; Novo Nordisk Research Facility US, New Brunswick, New Jersey; and §Department of Medicine, Division of Hematology and Oncology, University of North Carolina, Chapel Hill, North Carolina.

Supported by the sponsor, Novo Nordisk Inc., Princeton, NJ. The sponsor was responsible for providing clinical study supplies, preparing the study protocol, data management, statistical analysis, and preparing the clinical study report.

Conflict of Interest: See Disclosures at the end of the article.

Authors' current affiliations are listed at the end of the article.

Reprints will not be available from the authors.

Address correspondence to Brett E. Skolnick, PhD, Novo Nordisk, 100 College Rd. West, Princeton, NJ 08540. Address e-mail to BRES@novonordisk.com.

Accepted June 8, 2011

Published ahead of print September 2, 2011

Clopidogrel (Plavix®; Bristol-Myers Squibb Co., Princeton, NJ) is an antiplatelet drug indicated for acute coronary syndrome and peripheral artery disease. In recent years, clopidogrel has become widely used to minimize thrombosis risk after cardiovascular stenting. As such, many patients remain on long-term clopidogrel treatment. However, the use of clopidogrel carries the risk of bleeding as a potential side effect in patients with recent ischemic stroke, recent myocardial infarction, or peripheral arterial disease (CAPRIE Trial)1 and in patients symptomatic of coronary artery disease with evidence of ischemia (CREDO Trial).2

Recombinant activated FVII (rFVIIa) (NovoSeven®; Novo Nordisk A/S, Bagsværd, Denmark) is indicated for the treatment of bleeding episodes in hemophilia A or B patients with inhibitors (i.e., alloantibodies) to FVIII or FIX and in patients with acquired hemophilia, the prevention of bleeding in surgical interventions or invasive procedures in hemophilia A or B patients with inhibitors to FVIII or FIX and in patients with acquired hemophilia, and the treatment of bleeding episodes in patients with congenital factor VII deficiency and prevention of bleeding in surgical interventions or invasive procedures in patients with congenital FVII deficiency.3 A number of published ex vivo and retrospective studies have reported the use of rFVIIa to mitigate the bleeding associated with clopidogrel therapy.49 Several features make rFVIIa a candidate for the mitigation of antiplatelet-induced bleeding. These include rapid action localized to the site of vascular injury,10,11 low volume required for administration, allowing for rapid infusion, and demonstrations of effectiveness with good safety profiles.4,7,9

Data regarding bleed mitigation in clopidogrel-treated animals12,13 and in patients undergoing cardiac surgery14 are contradictory and provide little insight as to the use of rFVIIa in mitigating spontaneous or trauma-induced bleeds in patients receiving clopidogrel therapy. The current study was designed to assess the utility of rFVIIa, in vivo, to mitigate bleeding induced via a punch biopsy bleeding model in healthy volunteers treated with clopidogrel. The punch biopsy model has been shown to produce wounds of reproducible depths and widths to give measurable bleeding duration (BD) and blood volume (BV).15 Previous clinical trials using the punch biopsy technique to evaluate rFVIIa to mitigate bleeding provided BD sufficiently prolonged to allow for the detection of treatment differences.15 The results from the current study could provide additional information regarding the use of rFVIIa for clopidogrel-treated patients in instances where there is a need to mitigate spontaneous or trauma-induced bleeds.

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METHODS

This study was conducted from May 2008 to January 2009 in accordance with the Declaration of Helsinki.16 MDS Pharma Services IRB reviewed and approved the study conduct. Signed informed consent was obtained from each subject. This was a single-center, randomized, placebo-controlled, double-blind, dose-escalation, phase I study.

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Study Population

Healthy male subjects answering advertisements were recruited locally and paid for their participation in the study. All subjects were between 18 and 45 years of age, had normal platelet counts (150–352 × 109 cells/L), normal coagulation screening assays (prothrombin time of 9.4–12 seconds, and activated partial thromboplastin time of 25.4–38.4 seconds), and met the strict cardiovascular criteria [HDL-cholesterol ≥40 mg/dL, LDL-cholesterol <189 mg/dL, or apolipoprotein B 100 or lipoprotein(a) below the 90th percentile]. Subjects were withdrawn if there was excessive bleeding at the initial biopsy (>25 minutes, n = 5). Subjects taking investigational drugs, oral anticoagulant therapy, aspirin, or nonsteroidal antiinflammatory drugs were also excluded.

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Study Design

Novo Nordisk provided the randomization schedule. Subjects were randomized (6:2 or 6:6) to receive either the trial product (a single IV dose of rFVIIa) or placebo. The planned rFVIIa dose tiers were 5, 10, 20, 40, and 80 μg/kg. The punch biopsy was performed on the back of the thigh (after local anesthetic with 2% lidocaine without epinephrine) to a subcutaneous depth of 4 to 6 mm using a disposable punch biopsy instrument (Miltex Inc., York, PA) with a diameter of 5 mm. Subjects underwent 4 biopsies: before clopidogrel administration (biopsy Bx0), approximately 4 days after initiation of clopidogrel treatment (biopsy Bx1), 2 hours and 13 minutes after administration of the trial product (rFVIIa or placebo, biopsy Bx2), and then 5 hours after trial product (approximately 2 half-lives of rFVIIa) (biopsy Bx3).

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Treatment

All subjects received clopidogrel treatment orally with an initial 300-mg loading dose on day 1, followed by daily 75-mg doses of clopidogrel for 2 additional days. Two hours after Bx1, trial product (rFVIIa or placebo) was administered IV. There were no delays in performing the biopsies. Study subjects and all clinical staff involved in assessing outcomes were blinded. To maintain blinding, subjects were dosed based on their weights and given an equal weight-based volume of rFVIIa or placebo. The unblinded pharmacist prepared the trial product. Clopidogrel (clopidogrel bisulfate, Plavix®) was provided as 75-mg tablets for oral administration. Recombinant FVIIa and placebo were provided as identical freeze-dried powder in single-use vials (4.8 mg) to be reconstituted with sterile water for injection (USP) to be administered as a slow IV bolus injection. The reconstituted vials have a pH of approximately 6.0 and consist of sodium chloride (2.3 mg/mL), calcium chloride dehydrate (1.5 mg/mL), glycylglycine (1.3 mg/mL), polysorbate 80 (0.1 mg/mL), and mannitol (25 mg/mL). The rFVIIa vials also have 0.6 mg/mL rFVIIa.

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Assessment of Clopidogrel Effects on Platelet Inhibition

Platelet inhibition (PI) in subjects treated with clopidogrel was assessed using the VerifyNow® P2Y12 assay (Accumetrics®, San Diego, CA).17 Based on previous studies, the PI cutoff adopted in this study was a 20% PI at approximately 16 hours after the 300-mg initial loading dose of clopidogrel (day 1), and was set at 30% after a minimum of 2 additional days of 75 mg clopidogrel (day 3).17,18

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Assessment of Clopidogrel and rFVIIa Effects

BD and BV were evaluated after each biopsy as described previously.15 In addition to in vivo bleeding evaluations, thromboelastography (TEG®) provided ex vivo sequential coagulation profiles of whole blood coagulation.19,20 The primary end point was the punch biopsy–induced BD after trial product administration (biopsy Bx2). Secondary end points included BV and clot dynamics assessed by TEG®.

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Assessment of Safety

Safety was continuously monitored during the study, and blinded safety data (i.e., adverse events/serious adverse events, electrocardiograms, and safety laboratory values) were evaluated before dose escalation. Safety variables were assessed in the phase I unit that conducted the trial. As part of the discharge from the phase I unit, an electrocardiogram, troponin I measurement, and assessment of adverse events were performed. Subjects returned to the phase I unit after 8 to 15 days for suture removal and adverse event and vital sign assessment.

During the course of the study, there was a change in the physician performing the biopsies. Therefore, post hoc analyses included using the physician as a covariate in analyses for BD and BV, as well as an analysis of subjects biopsied only by the first physician. Analyses of TEG® parameters included TEG® values at Bx1 as a covariate. No adjustment for changes in physician was made because blood sampling is not influenced by biopsy technique. Duration of rFVIIa effects (as reflected by biopsy 3) was to be evaluated only if the preplanned analyses were significant.

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Statistical Analyses

The null hypothesis for the primary end point was that BD at Bx2 after rFVIIa treatment was more than or equal to placebo and the alternative hypothesis was that BD after rFVIIa treatment was less than that after placebo. All subjects who received clopidogrel and trial product or placebo were included in the safety analyses. All randomized subjects who underwent biopsies 1 and 2 were included in the intent-to-treat (ITT) analysis.

The planned sample size allowed up to 108 subjects based on an adaptive study design that accommodated incremental progression of rFVIIa dose based on the results of the interim analyses and safety review. The design involved systematic evaluation at each rFVIIa dose tier and accommodated small initial sample sizes while allowing rapid determination of the optimally effective rFVIIa dose, compared with a more conventional study design. The study started with 8 subjects (6 rFVIIa and 2 placebo) in the 5 μg/kg dose tier and was planned to progress in ascending dose tiers to 80 μg/kg. It was planned that statistical comparisons should start at the first dose where the normalization of the clopidogrel-induced BD was assessed or at the highest dose (80 μg/kg) if there was no normalization at lower doses. Normalization (ratio of BD at Bx2 to BD at initial biopsy ≤1.1) was not achieved at any completed dose tier in this study. The statistical inferential comparisons of the 3 rFVIIa dose groups (5, 10, and 20 μg/kg) with placebo for the BD and blood loss were performed using a generalized linear model with the logarithmic link and the γ distribution for errors. This approach is preferable for data with constant coefficient of variation (CV).21 The assumption of constant CV is approximately satisfied for BD and blood loss data in the different dose groups of the trial. The model for BD and blood loss at Bx2 included adjustments for logarithmically transformed baseline values (at Bx1) of the dependent variable as a covariate and investigator as a fixed factor. The statistical analysis for repeated TEG® data was based on a mixed-effect model with subject as a random effect. This model allows for the accounting of correlations between data for different time points.

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RESULTS

This study was terminated to align with the sponsor's (Novo Nordisk A/S) decision to only pursue the development of rFVIIa within the licensed indications. At the time the study was stopped, only 3 subjects were enrolled in the 40 μg/kg dose tier, and no subjects in the 80 μg/kg dose tier. The results of the 40 μg/kg dose tier were not included in the analyses because of the small sample size, although no apparent differences were noted in safety variables. Efficacy end points for each rFVIIa dose tier (5, 10, and 20 μg/kg rFVIIa) were tested separately with no estimation of the α level inflation because of multiplicity of testing, which is often the case for exploratory analyses. Therefore, these results should be interpreted with caution.

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Subject Disposition

Subject disposition is presented in Figure 1. One hundred seventeen subjects fulfilled the screening criteria and were enrolled in the study. Of the 104 subjects who received clopidogrel, 56% (58 of 103) did not meet the initial PI cutoff level of 30% on day 3, and were therefore removed from the study. The safety analysis set comprised 40 subjects who received trial product (rFVIIa or placebo).

Figure 1

Figure 1

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Demographics

Demographics and baseline characteristics of the clopidogrel nonresponders and clopidogrel responders are presented in Table 1. The mean age of the clopidogrel nonresponders (32.5 years) was older than the mean age of the clopidogrel responders (28.1 years) (P < 0.05). The mean body mass index of the clopidogrel nonresponders (26.8 kg/m2) was higher than the mean body mass index of the clopidogrel responders (25.1 kg/m2) (P < 0.05). All other demographic and baseline characteristics are presented in Table 1.

Table 1

Table 1

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Effect of Clopidogrel on PI

Of the 103 subjects who received the 300-mg clopidogrel initial loading dose on day 1, 55 did not reach the initial PI cutoff level of 20% and 2 subjects were withdrawn from the study because of excessive bleeding at Bx0. In the total population of subjects (n = 46), mean ± SD PI levels increased from 44.0% ± 18.1% after the 300-mg loading dose on day 1 to 58.9% ± 20.3% on day 3 after a minimum of 2 additional maintenance doses of 75 mg clopidogrel (Table 2). Three subjects from the total subject population did not meet the PI cutoff level of 30% on day 3 and 3 subjects were withdrawn from the study because of noncompliance with the investigator's instructions or excessive bleeding (i.e., BD [Bx1] >60 minutes) The mean ± SD PI level for the randomized population on day 3 (n = 40) was 60.1% ± 18.1%. Total clopidogrel exposure ranged from 450 to 525 mg in the randomized subjects, dependent on whether subjects received a third 75-mg dose.

Table 2

Table 2

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Effect of rFVIIa on Bleeding Characteristics

After clopidogrel treatment, BD increased from 10.7 ± 4.8 minutes to 32.3 ± 15.8 minutes (the mean difference was statistically significant; P < 0.001) and BV increased from 5.0 ± 4.8 mL to 17.7 ± 18.8 mL (P < 0.001). Treatment with rFVIIa had no significant effect on BD compared with placebo (Table 3, Fig. 2). However, BV was significantly reduced with the 10 and 20 μg/kg rFVIIa doses compared with placebo (Table 3, Fig. 2).

Table 3

Table 3

Figure 2

Figure 2

The mean values and variation in the measurements for BD and BV were higher for the second physician performing the biopsies. Subjects in the 5 and 10 μg/kg rFVIIa treatment groups were biopsied only by the first physician. The treatment groups had smaller standard deviations for BD (3.6 and 6.6 minutes) and BV (8.7 and 5.6 mL). The subjects in the placebo and 20 μg/kg rFVIIa groups were biopsied either by the first or second physician and had larger standard deviations for BD (16.8 and 19.7 minutes) and BV (12.0 and 30.4 mL).

In an attempt to account for the influence of the variability between physicians, analyses of rFVIIa effects on BD and BV incorporated adjustment for physician. In addition, the statistical analysis model used for BD and BV performs satisfactorily even for large changes in standard deviation if the CV is approximately constant across treatment groups. In these analyses, monotonicity was observed for the effect of rFVIIa dose on BD and BV, although this was only significantly different from placebo for blood loss in the 10 μg/kg (P = 0.007) and 20 μg/kg (P = 0.001) dose groups (Table 3). However, a separate analysis for physician 1, who was completely responsible for the 5 and 10 μg/kg dose groups, can be useful. The effects of rFVIIa on BV in subjects biopsied by physician 1 were significant for the 10 and 20 μg/kg dose groups for physician 1 (P = 0.006 and P = 0.013, respectively) (Table 4). The effect of rFVIIa on BD was significant only in the 20 μg/kg dose group for physician 1 (P = 0.048) (Table 4).

Table 4

Table 4

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Effect of rFVIIa on Clot Dynamics

Clot dynamics were assessed using TEG®. A longitudinal approach was performed using a mixed-effect model. Treatment with rFVIIa significantly decreased time to clot onset (R) and increased the clot angle (A), compared with placebo (P < 0.005 for all dose groups) (Fig. 3). The effects of rFVIIa on any parameter of clot dynamics were not observed 3 hours after Bx2 or 1 hour after Bx3. No significant differences were observed in other TEG® parameters across treatment groups.

Figure 3

Figure 3

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Safety

The adverse events reported in this study were burning sensation (placebo, n = 3), suture-related complications (placebo, n = 2), eczema (40 μg/kg rFVIIa, n = 2), catheter site hemorrhage (20 μg/kg rFVIIa, n = 1), vessel puncture at site of hematoma (20 μg/kg rFVIIa, n = 1), postprocedural complication (10 μg/kg rFVIIa, n = 1), dizziness (20 μg/kg rFVIIa, n = 1), headache (placebo, n = 1), agitation (20 μg/kg rFVIIa, n = 1), ecchymosis (placebo, n = 1), erythema (40 μg/kg rFVIIa, n = 1), and rash (placebo, n = 1). All adverse events were graded as mild or moderate in severity and were related to the biopsy procedure. No thromboembolic complications were reported.

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DISCUSSION

Clopidogrel Response

Because platelet response to clopidogrel (i.e., inhibition of platelet aggregation by clopidogrel) is highly variable,2224 with poor response rates occurring in as many as 40% to 60% of patients,2527 it was critical to include reliable PI screening to exclude subjects nonresponsive to clopidogrel. In the current study, 56% of subjects failed to demonstrate an antiplatelet effect measured as PI. Previous studies report that up to 60% of clopidogrel-treated subjects exhibit a poor clopidogrel response, a poor response being arbitrarily defined as less than 30% to 40% inhibition of platelet aggregation.2527 Furthermore, the variability (20.3% SD) in PI in those subjects who met the cutoff levels was similar to the variability reported from secondary post hoc analyses of clopidogrel-treated subjects by Serebruany et al.28 (20.8% SD). Factors such as age (older than 55 years),27 increased body weight,26,29 and conditions that increase platelet reactivity, including diabetes,27,30 acute coronary syndrome, and acute stroke31 may affect response to clopidogrel. It is more likely that other factors affected subject response to clopidogrel because most subjects in this study did not have any of these characteristics that affect response to clopidogrel. Factors that influence the absorption and biotransformation of clopidogrel and/or variability in the P2Y12 adenosine diphosphate receptor affecting platelet activation and aggregation32 may explain the clopidogrel response pattern. Furthermore, there are several other mechanisms and pathways for platelet aggregation (e.g., activation of platelet aggregation by thromboxane A2).33,34 Therefore, targeting activation of the P2Y12 adenosine diphosphate–receptor pathway alone may not sufficiently attenuate the entire aggregation process.

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Bleeding Characteristics

The punch biopsy model has advantages over the original Ivy and Simplate bleeding tests. The Ivy method has limited use in the clinic because of its poor correlation with actual patient bleeding.35 When healthy volunteers were treated with acetylsalicylic acid, bleeding times did not significantly increase using the Simplate bleeding test (P > 0.05).36 The punch biopsy model produced significantly longer BD and BV in healthy volunteers after anticoagulant therapy.15 For these reasons, the punch biopsy model was deemed a suitable model to investigate the ability of rFVIIa to mitigate the effects of clopidogrel on BD and BV. The reasons for the limited ability of the punch biopsy model to demonstrate an effect of rFVIIa on mitigating drug-induced (warfarin15 or clopidogrel) coagulopathies measured by BD remain unclear. Other than the inter-investigator variability that was specific to this study, the nature of the injury (capillary effects), the interaction of the local anesthetic with rFVIIa at the area of injury, or simply the limited effects of rFVIIa in such a small vessel bleeding model are possible explanations.

The effects of rFVIIa on BV, unlike BD, were consistent with the ex vivo time to clot onset (TEG®-R) and clot angle (TEG®-A) results (Fig. 3). These results are similar to the previously reported findings in which rFVIIa corrected the effect of warfarin on all ex vivo TEG® parameters,15 and suggest that time to clot formation affects BV but not BD.

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Limitations of the Study

Dual therapy with aspirin and clopidogrel is the common practice for inhibition of platelet aggregation for preventing cardiovascular events.34 This study might have benefited from the use of dual antiplatelet drugs and/or increased loading/maintenance dose26,37,38 to allow a larger proportion of subjects to achieve the PI cutoff levels and possibly a more uniform clopidogrel response. However, the percentage of subjects who did not respond to clopidogrel treatment (56%) was similar to that reported in a previous study that used dual therapy with aspirin.18

A signal on BV for the 10 and 20 μg/kg rFVIIa dose groups and for BD in the 20 μg/kg rFVIIa dose group, for the initial physician, clearly underscores the impact of technical skill in this bleeding model. The premature termination of the study restricted enrollment of subjects in the 40 μg/kg dose group and did not allow for the investigation of the 80 μg/kg dose group, a dose that is within the trial product label. Even though a signal showed an rFVIIa effect for BV in the 10 and 20 μg/kg rFVIIa dose groups as compared with placebo, the results for BD at these doses were not convincing.

Despite these noted limitations of the punch biopsy model, rFVIIa mitigated the effects of clopidogrel-induced bleeding on BV. It is possible that the inhibition of platelet aggregation was compensated by rFVIIa-mediated enhancement of thrombin generation and thrombin-mediated activation of platelet aggregation,33,39 which was reflected more in the BV measurements than in BD. As such, the results of this study cannot be extrapolated to clinical hemorrhages because the effectiveness of rFVIIa to mitigate clopidogrel-associated bleeding has not been tested.

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CONCLUSIONS

This exploratory study was designed to investigate the effect of escalating doses of rFVIIa in clopidogrel-mediated bleeding in a punch biopsy model. Despite study limitations, rFVIIa (10 and 20 μg/kg) significantly mitigated clopidogrel-induced effects on BV. Furthermore, in subgroup analyses of subjects, 20 μg/kg rFVIIa showed a significant reduction of clopidogrel-induced BD as well as on BV. The clinical enhancement of coagulation by rFVIIa was also reflected in ex vivo clotting parameters (TEG®-R and TEG®-A).

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CURRENT AFFILIATIONS

Brett E. Skolnick, PhD, is currently affiliated with Novo Nordisk Inc, Princeton, NJ; Magdy Shenouda, MD, is currently affiliated with Iberia Clinical Research, Eatontown, NJ; Anthony E. Pusateri, PhD, is currently affiliated with US Army Medical Research and Materiel Command, Fort Detrick, MD; and Marcus E. Carr, MD, PhD, FACP, is currently affiliated with Pfizer Inc., Collegeville, PA.

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DISCLOSURES

Name: Brett E. Skolnick, PhD.

Contribution: This author helped design the study, conduct the study, analyze the data, and write the manuscript.

Attestation: Brett E. Skolnick designed the study, has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.

Conflicts of Interest: Brett E. Skolnick is an employee of Novo Nordisk.

Name: Magdy Shenouda, MD.

Contribution: This author helped conduct the study and write the manuscript.

Attestation: Magdy Shenouda has seen the original study data and approved the final manuscript.

Conflicts: Magdy Shenouda received financial compensation for his role as investigator of the study.

Name: Naum M. Khutoryansky, PhD.

Contribution: This author helped design the study, analyze the data, perform statistical analysis, and write the manuscript.

Attestation: Naum M. Khutoryansky has seen the original study data, supervised the analysis and review of the data, and approved the final manuscript.

Conflicts of Interest: Naum M. Khutoryansky is an employee of Novo Nordisk.

Name: Anthony E. Pusateri, PhD.

Contribution: This author helped conduct the study, write the manuscript, and contributed to acquisition of laboratory data.

Attestation: Anthony E. Pusateri has seen the original study data and approved the final manuscript.

Conflicts of Interest: Anthony E. Pusateri was an employee of Novo Nordisk at the time of study.

Name: Don Gabriel, MD, PhD.

Contribution: This author helped conduct the study, write the manuscript, and served as an external independent Safety Officer.

Attestation: Don Gabriel has seen the original study data and approved the final manuscript.

Conflicts of Interest: Don Gabriel received financial compensation for his role as Safety Officer of the study.

Name: Marcus E. Carr, MD, PhD, FACP.

Contribution: This author helped write the manuscript.

Attestation: Marcus E. Carr has seen the original study data and approved the final manuscript.

Conflicts of Interest: Marcus E. Carr was an employee of Novo Nordisk at the time of study.

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ACKNOWLEDGMENTS

We thank all the study subjects for their participation. We also thank Dr. Sandra Connolly and other study staff at MDS Pharma Services, Neptune, NJ, and the facility staff at Novo Nordisk Research Facility US (NNRUS), New Brunswick, NJ, including Jun D. Guzman, and Petula Fraser-Davies at Novo Nordisk, Princeton, NJ, for acquisition of data, study supervision, and administrative, technical, and/or material support. Dr. Alvin Estilo, Novo Nordisk's Safety Officer, assembled a safety group (including internal Novo Nordisk members not involved in the study conduct and an external independent Safety Officer, Dr. Don Gabriel) to review safety findings at the conclusion of each dose tier. We thank Abha Chandra, PhD, Neal Okarter, PhD, and Charlotte Yap, MSc, of Novo Nordisk, for providing writing and editorial assistance with the manuscript. We also thank Hongli Wang, MSc, and Thomas Henschel, employees of Novo Nordisk, for performing the statistical analyses.

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