Thrombosis Research 135 (2015) 485–491

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Regular Article

An international, multicenter, prospective study of a prothrombin complex concentrate, Prothromplex Total®, in anticoagulant reversal Áron Altorjay a, Éva Szabó b, Zoltán Boda c, Ludwig Kramer d, Leock Y. Ngo e, Werner Engl f, Clair L. Firth f, Erik R. Ahlstrom e,1, David M. Gelmont e, Ingrid Pabinger g,⁎ a

Department of Surgery, Saint George University Teaching Hospital, Székesfehérvár, Hungary Pediatric Department, Veszprém County Hospital, Veszprém, Hungary Department of Internal Medicine, Thrombosis and Hemostasis Center, University of Debrecen, Debrecen, Hungary d 1st Department of Medicine with Gastroenterology, Wien-Hietzing Hospital, Vienna, Austria e Baxter Healthcare Corporation, Westlake Village, CA, USA f Baxter Innovations GmbH, Vienna, Austria g Department of Internal Medicine I, Division of Hematology and Hemostaseology, Medical University of Vienna, Vienna, Austria b c

a r t i c l e

i n f o

Article history: Received 15 September 2014 Received in revised form 15 December 2014 Accepted 23 December 2014 Available online 2 January 2015 Keywords: Prothrombin complex concentrate Prothromplex total Anticoagulant reversal Oral anticoagulant Vitamin K antagonists

a b s t r a c t Introduction: Prothrombin complex concentrates (PCCs) are a common treatment option for the reversal of oral anticoagulation with vitamin K antagonists (VKAs). This study assessed efficacy and safety of Prothromplex Total®. Materials and Methods: Patients (≥18 years) with acquired prothrombin complex coagulation factor deficiency (international normalized ratio [INR] ≥2 at screening) due to oral VKAs, requiring reversal of anticoagulation, were treated with 25, 35, or 50 IU/kg BW PCC. After infusion, efficacy was assessed for 72 ± 4 hours. Adverse events (AEs) were captured for 15 days. Results: Sixty-one subjects, 48 requiring interventional procedures and 13 with acute bleeds, received a single infusion of PCC. Of 59 subjects analyzed, all achieved normalization of INR (≤1.3) within 30 ± 5 minutes of infusion, demonstrating effective anticoagulant reversal. IVRs of factors II, VII, IX, and X ranged from 1.12-2.03 IU/dL: IU/kg. Median INRs remained between 1.00 and 1.18 for up to 6 hours. Overall efficacy of treatment was rated “excellent” for 60 subjects. Three AEs were deemed possibly related to treatment: 1 serious AE (SAE) of acute myocardial infarction (rated severe), 1 SAE of deep vein thrombosis (rated mild), and 1 AE of pyrexia (rated mild). Thrombotic adverse events (2/61, 3.3%) reported here are comparable to rates observed in other PCC studies. Conclusions: While there is a risk of thromboembolic events following treatment with PCC products, the number of events reported here was low and could have occurred without PCC treatment. The individualized, INR-based dosing of PCC used here for VKA anticoagulant reversal produces rapid normalization of INR to ≤ 1.3 within 30 minutes. © 2015 Elsevier Ltd. All rights reserved.

Introduction Long-term oral anticoagulation therapy with vitamin K antagonists (VKAs) is widely prescribed in patients with an increased risk of thromboembolic events (risk factors include atrial fibrillation, heart valve replacement, and history of thromboembolic disease) [1,2]. However, with a 13.4% case-fatality rate of major bleeding and a risk for intracranial hemorrhage of 1.1 in 100 patient-years, bleeding is a serious concern in these patients [3]. VKAs exert their anticoagulation effect by ⁎ Corresponding author at: Medical University of Vienna, Währinger Gürtel 18–20, Ebene 6 I, 1090 Vienna, Austria. Tel.: +43 1 40400 44480. E-mail address: [email protected] (I. Pabinger). 1 Present address: 1885 Las Gallinas Ave, San Rafael, CA 94903, USA.

http://dx.doi.org/10.1016/j.thromres.2014.12.026 0049-3848/© 2015 Elsevier Ltd. All rights reserved.

inhibiting the functional maturation of the prothrombinase complex, which consists of the vitamin K-dependent factor Xa, factor Va and calcium on a phospholipid membrane [4]. Rapid reversal of oral anticoagulation can be achieved by administering a prothrombin complex concentrate, containing coagulation factors II, VII, IX and X, protein C and protein S. Clinical guidelines recommend the use of agents such as fresh frozen plasma (FFP) or prothrombin complex concentrates (PCCs) to manage acute bleeding and as prophylaxis prior to invasive procedures in orally anticoagulated patients [1,5,6]. The recommended dose of 15 ml FFP per kg body weight translates to infusion volumes of 1050 ml for a 70 kg patient, however, and does not completely reverse the anticoagulant effect [7]. PCCs have been shown to be more effective at achieving rapid and reliable elevation of prothrombin complex factor levels with smaller infusion volume than FFP, thereby reducing the risk

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of volume overload [8–10]. A recent study has demonstrated the statistical superiority of PCC administration compared to FFP (noninferiority p b 0.0001), with respect to normalization of INR, and also confirmed the comparable safety profile of a PCC product to that of FFP [9]. Prothromplex Total® (Baxter Healthcare Corporation, Deerfield, IL, USA) is a human plasma-derived PCC containing coagulation factors II (480–900 IU/vial), VII (500 IU/vial), IX (600 IU/vial), and X (600 IU/ vial), as well as the endogenous anticoagulant protein C (400 IU/vial). Excipients include heparin (max 0.5 IU/IU FIX), antithrombin III (15 to 30 IU/vial), sodium chloride (80 mg/vial), and sodium citrate (unspecified amount). The presence of adventitious viruses in the PCC preparation is prevented by plasma donor selection, steam treatment, and nanofiltration. Reported here are the results of an international, multicenter, prospective, open-label, non-randomized study to collect new and additional efficacy and safety information on the use of Prothromplex Total for oral VKA anticoagulant reversal in patients with acute bleeding or requiring urgent surgery. Materials and Methods This Phase 4 study was conducted at 3 centers in Austria and 3 centers in Hungary between July 2010 and April 2012. The study was conducted in accordance with the principles set forth in the European Clinical Trials Directive, the Declaration of Helsinki, and the International Committee on Harmonisation Guidelines for Good Clinical Practice. This study was registered with the U.S. National Institutes of Health website (ClinicalTrials.gov) as NCT01159210. The study protocol and its amendments were approved by the local ethics committee at each participating center and by the regulatory authorities of the corresponding countries. Written informed consent was obtained from each patient and/or their legally authorized representative prior to entering into the study. Patient Selection Patients were enrolled concurrently across study sites, according to predefined inclusion and exclusion criteria. Patient enrollment was entirely at the independent clinicians’ discretion. Patients ≥18 years old receiving oral anticoagulation with VKA, with an INR ≥2 at screening, were eligible to participate in this study if they required reversal of oral anticoagulation for urgent surgery, a planned or urgent invasive procedure, or acute bleeding episode. To ensure comparability between patients, it was decided to only enroll those who were receiving a stable dose of VKAs, or those whose INR had been stable for ≥72 hours. Patients who had not yet been assigned a stable and constant VKA dose were considered to be unlikely to provide robust data with respect to the reversibility of anticoagulant therapy through administration of PCC. Major exclusion criteria included: laboratory tests and/or clinical symptoms clearly indicative of disseminated intravascular coagulation (DIC); treatment with whole blood, FFP, or platelets within 6 hours prior to study enrollment (NB: transfusion of packed red blood cells was not excluded); blood loss of ≥5 units of blood; known congenital protein C, protein S, or antithrombin deficiency, or hereditary bleeding disorder; oral anticoagulant treatment for a period of b4 weeks for the treatment of a thrombotic event such as deep vein thrombosis or pulmonary embolism; acute ischemic cardiovascular disorder; additionally, subjects with acute or chronic liver failure (hepatic cirrhosis ChildPUGH score C) or renal failure and undergoing dialysis were excluded from the study.

administration of PCC. The use of INR to measure the efficacy of PCCs is standard practice worldwide [11–15]. The secondary outcome measures were the number of doses required to achieve normalization of INR, the shortening of the prothrombin time (PT; expressed as percent of normal coagulation activity), and the in-vivo recovery of coagulation factors II, VII, IX, and X at 30 ± 5 minutes after administration of PCC. Safety outcome measures included the incidence of treatment-related adverse events (AEs) and levels of coagulation markers including fibrinogen, D-dimer, protein C activity, and antithrombin activity. Hemostatic Efficacy In addition, the treating physicians could rate the hemostatic efficacy of Prothromplex Total® treatment in subjects undergoing interventional procedures (including urgent surgery and invasive procedures) and in subjects treated for acute bleeding episodes using the four predefined assessment categories defined below. 1. Excellent – meets or exceeds efficacy expectations based on experience with previous hemostatic therapy, i.e., bleeding episodes typically respond to the same or a lesser number of infusions and the same or a lower dose of PCC as compared to previous therapy, if applicable 2. Good – efficacy is somewhat less than expected, but adequate compared to previous hemostatic therapy, i.e., most bleeding episodes respond to the same number of infusions and doses of PCC but some require more infusions or a higher dose than expected based on previous experience, if applicable 3. Fair – efficacy is significantly less than expected as compared to previous hemostatic therapy, i.e., the majority of bleeding episodes require more infusions and/or higher doses of PCC than expected based on previous experience, if applicable 4. Poor – routine failure to control bleeding, and/or requirement of additional agents or different therapeutics for hemostatic management Efficacy assessments in bleed treatment were based upon professional opinion and experience with the subject, current health status (co-morbidities), concomitant medication (especially other anticoagulants and/or anti-platelets with long half-life), and considered the anatomical site of bleeds (arterial, venous, capillary, diffuse oozing), or the response to therapy in relation to experience with prior therapies, if applicable. If there was more than one bleeding site, each site was evaluated separately for clinical assessment of hemostasis. The time taken for resolution of bleeding was also recorded. Efficacy in bleed treatment and during surgeries or interventions was assessed at the study completion visit. Treatment The PCC product was administered as a single intravenous infusion, with dosing based on INR measurements performed at local laboratories prior to initiating treatment, according to the dosage described by Makris et al. [8] Baseline INR determined which of the 3 possible doses the subject should receive according to the following table:

INR

Dose (IU/kg Factor IX)

2.0-3.9 4.0-6.0 N6.0

25 35 50

Outcome Measures The primary outcome measure was the proportion of subjects who achieved normalization of INR to ≤ 1.3 within 30 ± 5 minutes after

Additional doses of PCC could be administered at any time at the discretion of the investigator, based on clinical presentation or on the prior

Á. Altorjay et al. / Thrombosis Research 135 (2015) 485–491

INR results. Treatment with whole blood, FFP, and/or platelets was not permitted within 6 hours prior to study enrollment.

487

Table 1 Subjects’ demographics and baseline characteristics. SA dataset (n = 61)

Assessments Blood samples were drawn for determination of INR, PT, additional coagulation markers (i.e., fibrinogen, D-dimer, protein C activity, and antithrombin activity), and hematology (i.e., hemoglobin, hematocrit, erythrocytes, platelets, and leukocytes) before and after administration of PCC at 15 ± 5 min, 30 ± 5 min, 1 hr ± 10 min, 3 hr ± 30 min, 6 hr ± 60 min, 12 hr ± 60 min, 24 hr ± 120 min, 48 hr ± 120 min, and 72 hr ± 4 hr. Blood samples for clinical chemistry were collected at screening. The clinical significance of abnormal laboratory findings in coagulation markers, hematology, and clinical chemistry was assessed by the investigators based on the compendium of normal values for the reference laboratory. Vital signs were assessed at screening, prior to (within 30 minutes), and immediately after (within 10 minutes) infusion. In-vivo recovery (also commonly referred to as incremental recovery) was calculated based on the levels of coagulation factors II, VII, IX, and X measured in blood samples drawn within 30 minutes prior to and 30 ± 5 min after infusion with PCC. The occurrence of AEs was monitored during the 15-day safety follow-up period. Statistical Analysis The planned sample size of 50 subjects was chosen to allow for calculation of a 95% two-sided, exact (Clopper-Pearson) confidence interval (CI) with a width of ≤ 20% assuming a success rate for the primary endpoint between 88% and 92%. The proportion of subjects achieving an INR of ≤1.3 within 30 ± 5 minutes after administration of PCC (primary endpoint) was analyzed by point estimate and 95% CI. Demographics, baseline characteristics, INR, PT, in-vivo recovery, coagulation factor levels, and laboratory values were summarized by descriptive statistics.

Age, median (range) in years Height, median (range) in cm Weight, median (range) in kg BMI, median (range) in kg/m2 Gender, n (%) Female Male

73.0 (25–93) 172.0 (151–185) 82.00 (54.0-115.3) 27.55 (19.9-36.6) 20 (32.8) 41 (67.2)

A large proportion of subjects (44%) were placed on anticoagulant treatment due to atrial fibrillation. The vast majority of subjects were being treated with coumarin-derivates, with far fewer subjects receiving warfarin products (4.9%). The administration of vitamin K during this study was carried out according to local clinical practice and the treating physician’s decision. Vitamin K was prescribed to 5/61 patients. Reasons behind this clinical decision include the fact that the vast majority (N80%) of patients were receiving the short-acting coumarin-derivative, acenocoumarol, rather than the longer-acting phenprocoumon (3.3% of patients), and whether additional interventions or surgery could be excluded at the time of initial treatment. PCC was administered prophylactically to 48 subjects (78.7%) undergoing interventional procedures, subdivided into urgent surgeries in 45 subjects and invasive procedures in 3 subjects, and the remaining 13 subjects (21.3 %) were treated for acute bleeding episodes. IP Infusion Based on the results from the baseline INR measurements, 50 subjects (82.0%) received the lowest intended dose (25 IU/kg), 3 subjects (4.9%) received the middle dose (35 IU/kg), and 8 subjects (13.1%) received the highest dose (50 IU/kg) with a median (range) total of 2400 (1350 to 4200) IU of FIX infused. The median (range) duration of infusion was 40 (13 to 75) minutes.

Results

Normalization of Coagulation Activity

Subjects

This study met its primary endpoint with all 59 subjects (100%) in the FA dataset achieving normalization of INR to ≤ 1.3 within 30 minutes after administration of a single dose of PCC. Most subjects (n = 57/59; 96.6%) reached an INR ≤ 1.3 within 15 minutes after infusion and the majority (47/59; 79.7%) maintained an INR at or below 1.3 for at least 6 hours. The median (range) INR value fell from 4.0 (1.9-7.5) at baseline to 1.0 (1.0-1.3) within 15 minutes (see Fig. 1). Rapid normalization of coagulation activity was confirmed by the PT analysis with a rise in median (range) PT from 15% of normal (2%-46%) at baseline, indicating prolonged PT due to anticoagulant therapy, to

Sixty-one subjects each received a single infusion of PCC, no subjects were lost to follow-up, and all 61 were included in the safety analysis (SA). Fifty-nine subjects had data for the primary outcome measure, completing the examination at 30 ± 5 minutes after infusion with a valid INR measurement, and were included in the full analysis (FA). The reasons for excluding 2 subjects from the FA dataset were that one subject had the blood draw for the INR assessment (primary endpoint) performed outside the 30 ± 5 minutes time window and the other subject lacked central laboratory assessment for the primary endpoint. Fifty-eight subjects from the FA dataset fulfilled all exclusion/inclusion criteria and were included in the per-protocol (PP) analysis. The reason for excluding one treated subject from the PP dataset was the failure to meet the inclusion criterion of INR ≥2 at screening (the subject had a screening INR of 1.90 and should not have received PCC treatment according to the study protocol). Safety, demographics, hemostatic efficacy, and baseline characteristics are reported for the SA dataset; efficacy is reported for the FA dataset. The results for the PP dataset were similar to the results for the FA dataset and are not included here. A total of 4 subjects were enrolled and treated at 2 sites in Austria, and the remaining 57 were included at 3 sites in Hungary. Demographics and baseline characteristics are summarized in Table 1 and details of surgeries, procedures and bleeds are shown in Table 2. Medical indications for subjects being prescribed oral anticoagulant treatment are shown in Table 3 and the oral anticoagulants prescribed are summarized in Table 4.

Table 2 Reason for treatment with investigational product. Indication

Number of subjects (%)

Acute bleeding episode Gastrointestinal bleeds Subconjunctival bleed

13 (21.3%) 12 1

Invasive procedures Endoscopic operations/biopsies

3 (4.9%) 3

Urgent surgery Hernioplasty Tumor excision and related surgeries Gastrointestinal surgeries Osteosynthesis Cholecystectomy Minor amputations Other abdominal surgeries Cardiovascular surgery

45 (73.8%) 14 7 7 6 6 3 1 1

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7.5

Table 3 Indication for oral anticoagulation therapy. Indication

Number (%) of subjects (n = 61)

Atrial fibrillation (AF) Venous thromboembolism (VTE) AF + VTE Artificial heart valve Other cardiac pathology

27 (44.3%) 15 (24.6%) 8 (13.1%) 4 (6.6%) 7 (11.5%)

5.0 4.0

INR

3.0

83% of normal (60%-150%) at 15 minutes and 84% of normal (59%-150%) at 30 minutes after infusion with PCC. A 28 % decrease in activity was observed between 1 hour and 24 hours after infusion as the PCC was cleared from the body, which was followed by an increase beyond 24 hours after infusion as the endogenous production of vitamin Kdependent coagulation factors recovered. Thirty minutes after infusion with PCC, a marked increase from baseline activities of coagulation factors II, VII, IX, and X was observed (see Table 5), with median in-vivo recoveries of 2.03, 1.76, 1.12, and 1.85 IU/dL:IU/kg, respectively. Additional markers of coagulation measured included antithrombin activity, fibrinogen, D-dimer and protein C activity. Protein C activity followed the same pattern as the coagulation factor activities with a marked rise in median activity from 37% at baseline to 88% at 15 minutes after PCC infusion providing evidence of a post-infusion increase (see Fig. 2a). Median antithrombin activity stayed within 88% and 94% of normal (reference range: 70% to 120%) for the duration of the 72hour follow-up period. The median fibrinogen level was 350 mg/dL at baseline, stayed within reference range (180 to 390 mg/dL) up to 48 hours after infusion and then rose slightly above the reference range beyond 48 hours (460 to 510 mg/dL; see Fig. 2b). The median level of D-dimer was 0.36 μg/dL at baseline, stayed within reference range (0 to 0.49 μg/dl) up to 6 hours after infusion and then rose above reference range beyond 6 hours (0.70 to 1.14 μg/dL; see Fig. 2c). In this study, no major increases in overall D-dimer levels were reported. Two patients who experienced possibly related thrombotic events (an acute myocardial infarction and a DVT) had relatively high Ddimer increases (a maximum of 3.32 μg/dl at 72 h post PCC infusion in the patient with acute myocardial infarction, and 2.24 μg/dl at 48 h post PCC infusion in the patient with DVT), however, other patients reporting no thromboembolic adverse events were determined to have much higher D-dimer levels.

Overall Hemostatic Efficacy Rating The overall hemostatic efficacy of the PCC was assessed in all 61 subjects. Among subjects experiencing acute bleeds, the hemostatic efficacy of PCC treatment was rated as “excellent” in 12 subjects; one subject was assessed by the treating physician as attaining an overall hemostatic efficacy rating of “good” (all bleeds resolved). Among the 48 subjects treated prophylactically before surgery or other interventional procedures, the hemostatic efficacy of the investigational product was rated as “excellent” (no reports of excessive bleeding) in all cases.

Table 4 Anticoagulants administered to subjects prior to receiving Prothromplex Total. Anticoagulant

Number (%) of subjects (n = 61)

Acenocoumarol Phenprocoumon Warfarin Acenocoumarol, followed by Warfarin Not specified

49 (80.3%) 2 (3.3%) 3 (4.9%) 2 (3.3%) 5 (8.2%)

2.0

1.5

1.0 BL Time post infusion [h] Fig. 1. INR values over time after infusion with PCC. The bottom and top of the boxes are the first and third quartiles, respectively, and the band inside the box is the median. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Data points outside of the whiskers are shown as dots.

Safety Of the 61 subjects infused with PCC, 24 subjects (39.3%) experienced a total of 66 AEs (33 mild, 28 moderate, and 5 severe) during the 15-day safety follow-up period. Of these 66 adverse events, 8 were classed as serious (SAEs) and occurred in 3 subjects (4.9%). There were 2 deaths during the study (one 89 year old subject died of septic shock [following perforation of the sigmoid colon and subsequent peritonitis] 13 days after the PCC infusion; and one 83 year old subject died of ventricular fibrillation 14 days after the PCC infusion); neither of these deaths was considered treatment-related by the treating physician. Hospitalization was not prolonged beyond the expected duration of admission following treatment with PCC and no reoperations were reported. Of the 66 AEs, a total of 3 adverse drug reactions (i.e. AEs considered possibly treatment-related) were reported, 2 of which were SAEs (acute myocardial infarction and deep vein thrombosis, respectively), and 1 of which was a non-serious AE of pyrexia occurring 2 hours after infusion. The first possibly treatment-related SAE of acute myocardial infarction occurred 1 day after infusion with PCC (25 IU/kg dose) in an 83-yearold female subject with a history of diverse cardiovascular pathologies including myocardial infarction, ischemic heart disease, hypertension, and atrial fibrillation. This subject later died following ventricular fibrillation as described above. The second possibly treatment-related SAE of deep vein thrombosis in the left upper limb occurred 5 days after

Table 5 Descriptive statistics and in-vivo recovery of coagulation factors II, IV, IX, and X. Samples were taken with 30 minutes before infusion and within 30 ± 5 minutes after infusion. Factor activity % of normal

In-vivo recovery IU/dL:IU/kg

Median (range)

Pre-infusion

Post-infusion

FII FVII FIX FX

20.0 (1–46) 16.0 (2–150) 34.0 (3–146) 10.0 (2–32)

82.0 (54–150) 65.0 (44–276) 66.0 (28–149) 64.5 (48–116)

2.03 (0.9-3.1) 1.76 (0.5-6.9) 1.12 (0.4-2.0) 1.85 (0.8-2.7)

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infusion (25 IU/kg dose) in an 82-year-old male with a history of previous thrombotic episodes (having experienced DVTs in the lower limbs in 1979 and in 2006, respectively).

489

None of the clinically significant laboratory findings were considered to be related to treatment. Discussion

a

Protein C Activity [%]

150

100

50

0 BL

0.5 Time post infusion [h]

b 1000

Fibrinogen [mg/dL]

800

600

400

200

0 BL

0.5 Time post infusion [h]

c 100.0 50.0

D−Dimer [µg/dL]

10.0 5.0

1.0 0.5

0.1 BL

0.5 Time post infusion [h]

The results from this prospective study demonstrated that Prothromplex Total was safe and effective in reversal of oral anticoagulation in 48 subjects who were treated prophylactically with PCC before undergoing interventional procedures and in 13 subjects (11 of whom were included in the FA dataset) who received PCC to stop acute bleeding episodes. With pre-treatment INRs ranging from 1.9 to 7.5, a single infusion of PCC rapidly normalized INR to ≤ 1.3 in all subjects. No subjects required a second dose of PCC. The rapid normalization of INR was accompanied by excellent overall hemostatic efficacy in 60/61 (98.4%) subjects, as assessed by the treating physicians. INR and PT measurements at later times after infusion show that almost all subjects achieved normalization of coagulation activity within 15 minutes and maintained normal activity for at least 6 hours. The precision of Prothromplex Total in achieving normalization of INR in this study is superior to the standard FFP treatment protocol described in two reports, [8,16] which involved an 800 ml infusion volume to reduce INR to 2.3 after 15 minutes. Our results are comparable to observations made in similar studies of other PCCs, where INRs of ≤ 1.3 were achieved within 10–30 minutes after infusion [17–19]. All 12 subjects treated with PCC for acute bleeding episodes had satisfactory resolution of bleeding accompanied by a hemostatic efficacy rating of excellent, which is similar to what has been observed with other products [17,19]. PCCs can be administered as a standard fixed dose or the dose can be individualized based on factors such as pre-treatment INR, target posttreatment INR, and bodyweight. Various PCC dosing regimens have been described in previous studies [20–23]. We chose the INR-based method utilized in this study to provide clinicians with a simple and objective means of assessing the dose of PCC treatment needed. All subjects’ blood samples were regularly tested for INR levels and this ensured that clinicians were able to assess the effectiveness of anticoagulant reversal treatment and adjust dosage as required. In a previous study, using a standard fixed dose of another PCC for warfarin reversal in 19 subjects suffering from acute bleeds, the range of post-treatment INRs was 1.2 to 2.2 [20]. In this study of Prothromplex Total, where subjects were assigned to 1 of 3 doses based on pre-treatment INR, the range of post-treatment INRs was 1.0 to 1.3 at 30 minutes after infusion. The narrow range of post-treatment INRs observed in this study justifies the dosing strategy with individualized dosing based on pre-treatment INR. The INR-based dosing strategy is also supported by the results from a randomized controlled trial of another PCC in the reversal of oral anticoagulant therapy where individualized dosing based on INR proved significantly more effective in reaching the target INR than a standard fixed dose [21]. It should be noted that this study included a relatively low number of subjects with acute bleedings (21.3%) compared to other studies on PCCs [19]. However, the majority of patients (78.7%) in the study underwent interventional procedures. Recently, controlled studies in patients with acute bleeds and invasive procedures have been completed with another PCC containing 4 coagulation factors (Sarode and colleagues [10]; and ClinicalTrials.gov registered study NCT00803101: not yet published). Efficacy with respect to INR was shown to be slightly lower with the other PCC tested by Sarode and colleagues, with 61/98 (62.2%) of subjects reaching an INR ≤1.3 within 30 minutes compared to 100% (59/59) of subjects in the current study [10].

Fig. 2. Changes in coagulation markers: (a) protein C activity, (b) fibrinogen and (c) D-dimers over time after infusion with PCC. The bottom and top of the boxes are the first and third quartiles, respectively, and the band inside the box is the median. The whiskers extend to the most extreme data point, which is no more than 1.5 times the interquartile range from the box. Data points outside of the whiskers are shown as dots.

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Over the 15-day safety follow-up period, approximately 60% of subjects treated with Prothromplex Total did not experience any AEs. Out of 66 AEs occurring in 24 subjects, the majority were mild, non-serious events; 8 were SAEs (2 fatal, neither of which was treatment-related) and 2 of these SAEs (acute myocardial infarction and deep vein thrombosis) were considered possibly related to treatment. The 2 subjects that experienced the two thrombotic SAEs both had several comorbidities and potential risk factors. An increased risk of thromboembolic events is the underlying reason for this patient population to undergo oral anticoagulation treatment and therefore the reversal needs to be carefully considered based on the physician’s assessment of perceived thrombotic and bleeding risks. In order to detect coagulation activation in the present study, fibrinogen, protein C, antithrombin and D-dimer levels were analyzed. High D-dimer levels suggest the degradation of cross-linked fibrin and may imply the existence of pulmonary embolism, deep vein thrombosis, DIC, or may generally occur postoperatively [24]. On the other hand, decreased levels of antithrombin are associated with an increased risk of both arterial and venous thrombosis and are seen in individuals with active thrombosis and DIC [25]. Over the whole study population, antithrombin levels remained relatively constant throughout. D-dimer levels followed a similar trend to that seen in other PCC studies [19]. Due to a high level of variability in fibrinogen and Ddimers at baseline, and the fact that the study was not powered to test statistical hypotheses on these secondary endpoints, it was difficult to assess post-infusion changes compared to baseline values. The delayed rise in D-dimer, 6 hours after infusion, could potentially be indicative of increased coagulation tendency after an invasive procedure/surgery. The delayed rise in fibrinogen 48 hours after infusion is potentially associated with an acute phase response; a decrease would be of more concern as this could indicate DIC. It was considered that D-dimer levels could possibly predict which patients would experience thromboembolic adverse events, however the results of the current study were inconclusive in this respect. While the two subjects who experienced thromboembolic events did have elevated D-dimer levels (namely a maximum of 3.32 μg/dL at 72 h post PCC infusion in the patient with acute myocardial infarction, and 2.24 μg/dL at 48 h post PCC infusion in the patient with DVT [upper limit of normal: 0.49 μg/dL]), other patients had much higher levels ranging from 12 to 100 μg/dl and yet did not experience any such adverse events. It is not yet known whether an increase in D-dimer levels also reflects a higher risk for thrombosis in patients receiving a PCC concentrate as such studies have not yet been performed. However, the numbers of patients experiencing thromboembolic events after PCC treatment are generally small (approximately 1-3% in clinical studies [10,19,26]). The observed possibly related serious thrombotic adverse events (2/61, 3.3%) reported in this study are comparable to rates observed in other studies on PCC products. Pabinger and colleagues reported on 43 patients administered a PCC product of whom 1 (2.3%) patient experienced a serious (and fatal) thrombotic event and another patient experienced a peripheral arterial embolism which was classed as unrelated to treatment [19]. In a Spanish study on patients undergoing urgent surgery, invasive procedures or intracranial bleeds, only one (1.0%) thromboembolic event was reported among 98 patients on VKA and treated with Prothromplex Total, the PCC product investigated here [26]. In a study assessing acute bleeds, Sarode and colleagues reported 8 patients with thromboembolic events among 103 patients (7.8%) receiving a PCC product, of which four (3.9%) were considered to be related to the investigational product. Furthermore, in Sarode’s study, a similar rate (2.8%) of related thromboembolic events was also reported among patients treated with FFP [10]. The administration of vitamin K during this study was carried out according to local clinical practice and the treating physician’s decision. Vitamin K was prescribed to 5/61 patients. Reasons behind this clinical decision include the fact that the vast majority (N80%) of patients were receiving the short-acting coumarin-derivative, acenocoumarol,

rather than the longer-acting phenprocoumon (3.3% of patients), and whether additional interventions or surgery could be excluded at the time of initial treatment. In patients presenting with significant VKA-associated bleeding, the benefit of treatment with PCC must be weighed against the thrombotic risk. Previous studies have demonstrated that maximum INR correction occurs within 30 minutes after administration of PCC [10,19], and comparative studies have also shown that this correction can take up to 24 h following vitamin K administration only [22,23]. In this study, physicians were required to enroll only patients requiring urgent surgery or experiencing active bleeding. As each patient in this study was also premedicated with oral anticoagulants, they all had an increased risk of thrombosis compared to the general healthy population. The clinical decision to administer PCC was made by the treating physicians based on the clinical situation and the patient’s best interests. While there is a risk of thromboembolic events occurring following treatment with PCC products, anticoagulant reversal with the slower acting vitamin K alone also puts a patient at risk of such complications and may cause additional delays in surgery. The number of thromboembolic events reported in this study was low and could conceivably have occurred even without PCC treatment. PCC should therefore be administered judiciously to patients in whom the hemostatic benefit clearly outweighs the thrombotic risk Conclusion Prothromplex Total produces rapid normalization of INR coupled with hemostatic efficacy, immediately reversing the effects of oral anticoagulant therapy and restoring levels of vitamin K dependent procoagulants. Excessive bleeding was effectively prevented in subjects undergoing interventional procedures and bleeding was decreased or stopped in subjects presenting with acute bleeding episodes. Furthermore, the well-established safety profile of Prothromplex Total was confirmed, with no new safety concerns raised. Authorship All authors approved the final version of the manuscript. Leock Y. Ngo and David M. Gelmont contributed to the conception and design of the study, and the interpretation of the data. Werner Engl contributed to the design of the study and carried out the statistical analysis. Erik R. Ahlstrom and Clair L. Firth interpreted the data, wrote, and revised the manuscript. Áron Altorjay, Éva Szabó, Zoltán Boda, Ludwig Kramer, and Ingrid Pabinger enrolled patients, administered PCC, collected data, and reviewed the draft manuscript. Conflict of Interest Statement Leock Y. Ngo, Werner Engl, Clair L. Firth, Erik R. Ahlstrom and David M. Gelmont were all employed by Baxter Healthcare Corporation at the time of the study. Leock Y. Ngo and David M. Gelmont own stocks or shares in Baxter Healthcare Corporation. David M. Gelmont holds a patent in Baxter Healthcare Corporation. Both the study and the manuscript were funded in their entirety by Baxter Healthcare Corporation. Zoltán Boda has received remuneration for lectures given as a member of the Hungarian Baxter Advisory Board. The clinical institutions of Áron Altorjay, Éva Szabó, Zoltán Boda, Ludwig M. Kramer, and Ingrid Pabinger received funding from Baxter Healthcare Corporation to carry out the study. Acknowledgements The authors would like to thank all clinical personnel and patients involved with this study. Furthermore, we would like to thank Baxter Healthcare Corporation for funding this study in full.

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