Compositional Differences in Commercially Available Prothrombin Complex Concentrates

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1 Original Article Compositional Differences in Commercially Available Prothrombin Complex Concentrates Clinical and Applied Thrombosis/Hemostasis 2014, Vol. 20(3) ª The Author(s) 2013 Reprints and permission: sagepub.com/journalspermissions.nav DOI: / cat.sagepub.com Nasir Sadeghi, MSc 1, Daniel Kahn, BSc 1, Daneyal Sayed, BSc 1, Debra Hoppenstadt, PhD 1, Walter Jeske, PhD 1, Job Harenberg, MD, PhD 2, Phillip DeChristopher, MD, PhD 1, and Jawed Fareed, PhD 1 Abstract Prothrombin complex concentrates (PCCs) are used in the management of bleeding complications with conventional oral anticoagulant drugs. Since the activation of these complexes results in the generation of factor Xa and IIa, these agents may potentially neutralize the newly developed Xa and thrombin inhibitors. Although the potency of these agents is defined in units that represent the level of factor IX (FIX), other factors including factor II, VII, and X are also present. Protein C, protein S, and protein Z are also present. The purpose of this study is to compare the compositional differences in the currently available PCCs along with the older agents. Measurement of compositional parameters including protein and FIX, mass spectrometric analysis of the native and activated PCCs, Western blotting studies on the native and activated PCCs for the activation products including thrombin, and their effect on blood and plasma coagulation parameters were carried out. Keywords prothrombin complex concentrates, vitamin K antagonist, tissue factors, Innovin, recombiplastin 2G Introduction Prothrombin complex concentrates (PCCs) are purified plasma-derived concentrates with hemostatic activity containing 4 vitamin K-dependent coagulation factors II, VII, IX, and X. 1 These agents were originally developed as a source of factor IX (FIX) for the treatment of hemophilia B. 2 The usage of these concentrates in this disorder has decreased due to the availability of high-purity, plasma-derived FIX, recombinant FIX, and bypassing agents such as activated PCCs and recombinant activated factor VII. Currently, PCCs are mainly used for the emergency reversal of oral vitamin K antagonist therapy, 3,4 because bleeding is the most common complication of oral anticoagulants leading to morbidity and mortality. 5 Moreover, the use of PCCs has been suggested in several liver diseases and in preoperative and trauma-related bleeding. 6-8 Commercially available PCCs exhibit compositional differences and are divided into 3-factor and 4-factor containing products. Four-factor concentrates such as Beriplex and Octaplex contain adequate amounts of vitamin K-dependent factors, while 3-factor concentrate such as Profilnine SD, contain significantly lower amount of factor VII. 9,10 A previous study compared the composition of 7 commercially available PCCs and found differences among these products. 11 Furthermore, differences were also noted between the determined and the label values for various clotting factors. Besides these coagulation factors, PCCs may contain small quantities of anticoagulants like protein C, protein S, and protein Z. Heparin and antithrombin III (AT) are also added to reduce thrombogenicity and prevent activation of coagulation factors by endogenous processes Therefore, these agents may be useful for the rapid reversal of coagulopathy by replacing vitamin K-dependent clotting factors and restoring normal hemostasis in a condition of overanticoagulation induced by vitamin K antagonist. 15 However, their hemostatic potential may differ. The composition of PCCs has been standardized by European Pharmacopoeia guidelines, which state that a PCC should have a FIX potency of at least 20 IU/mLand the relative potencies of clotting factors II and X should be close to FIX but not exceed it by more than 20%. 16,17 The overall clotting factor concentration in PCCs is almost 25 times higher than that in plasma. 9 The 4 clotting factors differ widely in terms of half-life. For instance, factor II has a longer half-life (60-72 hours) compared to other factors (6-24 hours). 18 The aim of the study was to evaluate the composition of the currently available PCCs such as Profilnine 1 Department of Pathology, Loyola University Medical Center, Maywood, IL, USA 2 Karls University Heidelberg, Germany Corresponding Author: Nasir Sadeghi, MSc, Department of Pathology and Pharmacology, Loyola University Medical Center, 2160 South First Avenue, Maywood, IL 60153, USA. sadeghi.nasir@gmail.com

2 Sadeghi et al 257 SD, Beriplex, Cofact, Octaplex, Prothromplex, along with the older agents such as Konyne, Preconetiv, and FEIBA. Compositional differences in terms of protein content, additives, coagulation factors, mass spectrometric profile, and activation product analysis along with the amount of thrombin generated were investigated. Since FEIBA is an activated PCC, it was included to determine the relative role of activated coagulation factors on various parameters included in the study. Materials and Methods Profilnine SD was purchased from Grifols Biologicals Inc; Cofact obtained from Sanquin Blood Supply Company, the Netherlands; Beriplex was purchased from CSL Behring Canada, Inc, Canada; and Octaplex obtained from Octapharma Pharmaceutical Productions GmbH, Austria, whereas Prothromplex NF 600 and FEIBA were purchased from Baxter Healthcare Corporation. Original batches of Konyne were obtained from former Cutter-Miles Inc (California) and Preconetiv from former Kabi Pharmacia (Stockholm, Sweden). Innovin brand of thromboplastin was obtained from Dade (Behring, Germany) and RecombiPlasTin 2G from Instrumentation Laboratory Company (Bedford, Massachusetts ). Each of the concentrates was diluted with their designated diluent buffer or sterile water as described by the manufacturer. These samples were further diluted with saline to obtain 10, 5, 2.5, 5, and 1U/mL solutions. Applicable statistical methods were used for data analysis. All results are presented in terms of mean of 2 determination protein content, prothrombin time (PT), activated partial thromboplastin time (aptt), thrombin generation, AT levels, and FIX antigen levels. The heparin level represents the mean 3 to 5 determination and +1 standard deviation. Protein Quantitation Protein content was quantitated in each PCC at a concentration of 10 U/mL using the modified Lowry method. 19 Clot-Based Method Prothrombin time and aptt assays were performed in PCCs supplemented with normal human plasma (NHP) at a 1 FIX U equivalent PCCs/mL. These measurements were carried out on the ACL 300 plus instrument (Manufactured by Instrumentation Laboratory, and is available from Diamond Diagnostics in refurbished condition.). For PT measurement RecombiPlasTin 2G and Innovin reagents were used, while Actin FSL (Siemens Healthcare Diagnostics) reagent was used for aptt measurement. Heparin and AT Measurement The BIOPHEN Heparin Anti-Xa kit (Aniara, West Chester, Ohio), a 2-stage chromogenic assay, was used to measure heparin levels. This method is based on the inhibition of a constant amount of Factor Xa (FXa) by the heparinized samples in the presence of exogenous AT (stage 1) and cleavage of FXa-specific chromogenic substrate (stage 2). All PCCs were diluted with saline to provide initial concentration of 10 U/mL. Samples were further diluted 1:10 in buffer and added to a microplate along with AT. FXa was then added in excess (stage 1). Upon incubation, FXa chromogenic substrate was added to the microwells and substrate activity was stopped with the addition of 20% acetic acid. Absorbance was measured at 405 nm. The BIOPHEN Antithrombin III 5 assay was used to measure AT using a chromogenic, Anti-Xa based method. In the presence of heparin, an AT heparin complex was formed which facilitated the inhibition of FXa. By adding excess FXa to a system, followed by the addition of FXa chromogenic substrate, FXa inhibition by AT heparin complex can be measured. All PCCs were diluted with saline to provide an initial concentration of 10 U/mL. Samples were diluted 1:20 with saline and were added to a microplate. Preincubated FXa was added in excess to ensure that AT was the limiting reagent. Upon incubation, a chromogenic substrate measuring FXa was added. Substrate activity was stopped with 20% acetic acid and optical density was measured at 405 nm. Factor IX Antigen Level Measurement All PCCs were diluted with saline to provide an initial concentration of 1 U/mL. Then samples were diluted 1:50 with sample diluent. Diluted sample of 200 ml was added to an enzymelinked immunosorbent assay (ELISA) plate followed by addition of 50 ml of conjugate anti-human FIX horseradish peroxidase (HRP) into each well. Samples were incubated at room temperature for 1 hour. After 5 times of washing, 200 ml of the 3,3 0,5,5 0 -tetramethylbenzidine/h 2 O 2 substrate was added into each well and incubated at room temperature for exactly 5 minutes. Stop solution of 50 ml was added into each sample to stop the reactions. Plate was read on SpectraMax Plus 384 (Molecular Devices LLC, Sunnyvale, California, USA) at wavelength of 450 nm. Factor IX antigen levels were cross-calibrated using appropriate references. Thrombin Generation Assay Tissue factor-mediated thrombin generation by each of the prothrombin complex was studied using a modified fluorometric method (Technothrombin TGA-Technoclone, Vienna, Austria). Each of the PCCs was diluted to 5 U/mL. To 40 mlofdiluted PCCs 10 ml of TGA reagent RC high was added followed by 50 ml of TGA substrate and read in the fluorometer (Biotek Readers) for 90 minutes with 1 minute measurement interval at 37 C. Raw data were exported to the Excel program with the Gen5 format to calculate the thrombin generation in nmol/l. Mass Spectrometric Analysis Mass spectrometric analysis was carried out using a surfaceenhanced laser desorption/ionization (SELDI) method to determine the protein composition of each concentrate. The analysis was carried out utilizing BioRad ProChip mass spectrometer

3 258 Clinical and Applied Thrombosis/Hemostasis 20(3) (PCS 4000; Bio-Rad, Hercules, California). The protein composition of native and activated prothrombin complexes were investigated using SELDI gold chip. Native PCC controls were prepared by mixing 25 ml of 10 U/mL of each of the PCCs with 50 ml saline and 25 ml Tris buffer. Activated samples were prepared as follows: 25 ml PCC mixed with 25 ml Tris buffer (ph 8.5) followed by addition 25 ml saline and 25 ml RecombiPlasTin 2G or Innovin. Controls and activated samples were incubated for 30 minutes and then 25 ml EDTA (2 mg/ml) was added to stop the reaction. Stepwise washing of the gold chip was carried out for 6 minutes in acetone; followed by 6 minutes in 0.01 N HCl; 6 minutes in a 50% methanol and N HCl mixtures; and finally 6 minutes in 100% methanol. The chip was allowed to air dry for at least 10 minutes before the application of the samples. Each of the individual samples was diluted at a 1:10 ratio with Tris-HCl (ph 7.5) buffer. An energy absorbing matrix (EAM) solution was prepared by dissolving sinapinic acid in equal volumes of 100% acetonitrile and 0.1% trifluroacetic acid. The sinapinic acid used allows for a saturated solution that cocrystallizes with the proteins in the sample; this cocrystallization allows for the ionization of the proteins in the sample. Diluted sample of 5 ml was added to 10 ml of EAM and mixed well. From the resulting mixture 5 ml is spotted on the gold chip. The gold chip is allowed to air dry for 1 hour before analysis on the SELDI. The chip was processed at 2 laser power settings. At the 1500 nj setting, data regarding the molecular weight () range between 0 and 20 kda is acquired. At the 3500 nj setting, data from the range between 20 and 150 kda is acquired. Immunoblotting Studies The immunoblotting studies were carried out utilizing immunoglobulin G (IgG) isolated from antisera generated against recombinant human thrombin in rabbits. A commercial lot of Recothrom (ZAC0712A) was obtained from ZymoGenetics Inc (Seattle, Washington). This product was used for immunization of the rabbits and as a reference thrombin for immunoblotting studies. To generate specific antisera in rabbits, Recothrom was provided to Lampire Biologics (Pipersville, Pennsylvania). Individual groups (n ¼ 3) of rabbits were challenged repeatedly with human recombinant thrombin, over a 9-month period. To generate specific antisera in rabbits, Recothrom was conjugated with keyhole lymphocyte hemocyanin as a carrier and was administered intravenously at a dosage of 100 mg using standard immunologic methods. A total of 10 injections were administered during this time, with a 4-week interval between each injection. Preimmune blood and antiserum were collected from each rabbit in serial order as shown previously. 20 The antiserum from each rabbit and the pooled antisera obtained from 3 individual rabbits in each treatment group were stored at 80 C prior to IgG isolation. HiTrap Protein G HP 1 ml columns (GE Healthcare Bio- Science Crop, Piscataway, New Jersey) were used to isolate Table 1. Protein Content of Various PCCs as Measured by Lowry Method. Prothrombin Complex Concentrate (10 U/mL) Protein Content (mg/10 U FIX activity) Beriplex 3.0 Cofact 8.7 FEIBA 9.3 Konyne 1 Octaplex 9.9 Preconetiv 5.8 Profilnine SD 1.8 Prothromplex 6.6 Abbreviations: FIX, factor IX; PCC, prothrombin complex concentrate. IgG from rabbit anti-recothrom antisera. The eluted IgG fractions were carefully collected and the concentration of harvested IgGs was adjusted with saline to 1 mg/ml based on the protein concentration determined using a modified Lowry assay. All isolated IgG samples were aliquoted and stored at 80 C. Gel electrophoresis and Western blotting were carried out as described previously. 20 In brief, samples were denatured and subjected to electrophoresis through 4% to 20% gradient Tris- N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid sodium dodecyl sulfate (SDS) polyacrylamide mini gels (Pierce Biotechnology, Rockford, Illinois) and then electrotransferred onto nitrocellulose membrane overnight at 4 C. Precision Dual Color Plus protein standards (Bio-Rad Laboratories) were included in each gel. After electrotransfer, the nitrocellulose membranes were blocked with 5% milk/tris-buffer saline (TBST; ph 7.6, 0.1% Tween-20) for 1 hour with gentle shaking, briefly washed in TBST, and incubated overnight with anti-recothrom IgG (0.01 mg/ml in 5% milk/tbst). Blots were then washed and incubated for 1 hour with 1: dilutions in 5% milk/tbst of HRP-conjugated donkey anti-rabbit IgG (H þ L; Thermo Scientific, Rockford, Illinois). The blots were then extensively washed and immunoreactive bands were detected with Super- Signal West Pico Chemiluminescent Substrate (Pierce Biotechnology) followed by film exposure. Results Protein Content, Prothrombin Time, Activated Partial Thromboplastin Time This study was designed to compare compositional differences among various PCCs. The protein content of these PCCs ranged from 3 to 1 mg/10u as measured by Lowry method (Table 1). The protein content of these PCCs showed wide variations. FEIBA, Konyne, Octaplex, and Cofact showed relatively high protein content in comparison to other PCCs. Profilnine and Beriplex contain lesser amounts of protein. All PCCs were supplemented with NHP at a 1 U/mL concentration and clot-based PT and aptt was measured as shown in Table 2. RecombiPlasTin 2G or Innovin reagents were

4 Sadeghi et al 259 Table 2. Effect of Various PCCs on PT and aptt in Normal Human Plasma. Prothrombin Complex Concentrate, 1 U/mL PT (Innovin), Seconds PT (Recombi PlasTin), Seconds aptt (FSL), Seconds Beriplex Cofact FEIBA Konyne Octaplex Preconetiv Profilnine SD Prothromplex NHP Abbreviations: aptt, activated partial thromboplastin time; NHP, normal human plasma; PT, prothrombin time; PCC, prothrombin complex concentrate. utilized for PT measurement and no difference were observed between these 2 reagents. The PT values ranged from 8 to 10 seconds with the exception of FEIBA where the PT value was relatively shorter ( seconds). The aptt values ranged from 34.7 to 95.4 seconds. Prothromplex produced the strongest anticoagulant effect whereas Beriplex, Cofact, Preconetiv, and Profilnine had no effect. Heparin and Antithrombin Level Figure 1 represents the variation in heparin content of various PCCs as measured by Biophen heparin anti-xa colorimetric method. Octaplex and Prothromplex exhibited higher concentration of heparin compared to other PCCs. The heparin content ranged from 0.05 to U/10 U/mL of PCCs. Antithrombin III level was measured by colorimetric method and the results are shown in Figure 2. Antithrombin III levels also ranged widely from 0 to 3 U/10 U/mL PCCs. The AT level was highest in Prothromplex followed by Beriplex. Cofact, FEIBA, Konyne, and Octaplex exhibited lower amount of AT. No AT was observed in Preconetiv and Profilnine. Factor IX antigen Level and Functional Thrombin Generation Factor IX antigen levels of all PCCs were measured using an ELISA method. These levels ranged from 82% to 109% as shown in Figure 3. Konyne had the highest level of FIX antigen (109%) followed by FEIBA, Preconetiv, and Beriplex. Level of FIX antigen was lower in both Profilnine (84%) and Cofact (82%). Table 3 shows the results obtain on the tissue factormediated generation of thrombin in various PCCs. The functional thrombin generation by each prothrombin complex was concentration dependent. At a 5 U/mL, level it ranged from 37 to 1044 nmol/l in various PCCs. Octaplex and Cofact Heparin Concentration (U/ml)* Beriplex Cofact Octaplex Profilnine Prothromplex Feiba Konyne Preconativ Figure 1. Variation in heparin levels in various PCCs. Heparin concentration of various PCCs as measured by chromogenic assay. All concentrations expressed in the reconstituted mixture at a final concentration of 10 U/mL. Octaplex and Prothromplex contained highest amount of heparin. PCC indicates prothrombin complex concentrate. AT ( U/100ml) Beriplex Cofact Octaplex Profilnine Prothromplex FEIBA Konyne Preconetiv Figure 2. Variation in AT levels of various PCCs. Antithrombin III level of various PCCs as measured by chromogenic assay. Although Preconetiv and Profilnine had no AT, Prothromplex and Beriplex contained higher amount of AT. AT indicates antithrombin III; PCC, prothrombin complex concentrate. produced the strongest responses whereas Beriplex and Prothromplex produced the weakest effects. Surface-Enhanced Laser Desorption/Ionization Analysis The composite data on the SELDI analysis of native and activated PCCs with 2 tissue factors, namely, Innovin and RecombiPlasTin 2G is given in Figures 4 to 7. As shown in Figure 4A, in the spectra of native Beriplex the prominent peaks were found to be at and. The 71-kDa peak represents prothrombin whereas the 66-kDa peak represents albumin. Upon activation with RecombiPlasTin 2G, the peak at

5 260 Clinical and Applied Thrombosis/Hemostasis 20(3) % Factor IX Ag levels / 1 U/ml PCCs Beriplex Cofact Octaplex Profilnine Prothromplex FEIBA Konyne Preconetiv Figure 3. Factor IX antigen levels in various PCCs. Factor IX antigen level of 8 PCCs determined by an enzyme-linked immunosorbent assay (ELISA) method. Konyne exhibited highest level of factor IX. Factor IX antigen level ranged from 80% to 109%/1 U/mL. PCC indicates prothrombin complex concentrate. Table 3. Functional Thrombin Generation All PCCs Tested at 5 U/mL. Prothrombin Complex Concentrate Thrombin, nmol/l Beriplex Cofact FEIBA Konyne Octaplex Preconetiv Profilnine SD Prothromplex 37.0 Abbreviation: PCC, prothrombin complex concentrate. was no longer detectable and a new peak at was generated. This peak represents thrombin. Upon activation with Innovin there was similar generation of a new peak at and a decrease in the 71-kDa peak. A distinct peak at 62 kda represents the albumin component in the tissue factor reagent. Figure 4B represents composite analysis of native and activated Cofact. The prominent peak of native spectra was found to be at ; this peak represents prothrombin. Upon activation with RecombiPlasTin 2G, the peak at was no longer detectable and the generation of thrombin was noted at. Upon activation with Innovin, there was a similar generation of peak at and decrease in the 71-kDa peak. A distinct peak at 62 kda represents the albumin which is a component in the tissue factor. Figure 5A depicts SELDI analysis of native and activated Octaplex which shows that the native spectra have a prominent peak, prothrombin, which is located at. Upon activation with RecombiPlasTin 2G, the peak at is no longer detectable and a new peak at was generated. This peak represents thrombin. Upon activation with Innovin there is similar generation of a new peak at and a decrease in the 71-kDa peak. A distinct peak at represents the albumin, a component in the tissue factor. The composite data on the SELDI analysis of native and activated Profilnine SD with RecombiPlasTin 2G and Innovin is given in Figure 5B. In the Profilnine SD, native spectra exhibits the prominent peak which is located at. This peak is representative of prothrombin. Upon activation with RecombiPlasTin 2G, the peak at is no longer detectable and a new peak at was generated. This peak represents thrombin. Upon activation with Innovin, there is similar generation of a new peak at and a decrease in the 71-kDa peak. A distinct peak at represents albumin, a component in the tissue factor. In addition to the thrombin peak, an additional peak around 50 kda was generated which represents prethrombin. In the case of Profilnine SD, the majority of the prothrombin has been converted into prethrombin and not fully converted into thrombin. Surface-enhanced laser desorption/ionization profile of native and activated Prothromplex is shown in Figure 6A. Native spectra of Prothromplex exhibited the prominent peaks detected at and. These peaks are prothrombin and albumin, respectively. Upon activation with RecombiPlas- Tin 2G, the peak at is no longer visible, while the 66 kda peak remained intact. Moreover, a new peak at was generated which represented thrombin. Upon activation with Innovin, there is similar generation of a new peak at and decrease in the 71-kDa peak. A distinct peak at represents albumin, a component in the tissue factor. Figure 6B shows SELDI profile of native and activated FEIBA. In the native spectra, the prominent peaks are located at and. These peaks are prothrombin and albumin, respectively. By activation with RecombiPlasTin 2G, the peak at 71 kda is no longer detectable, while the 66-kDa peak remained intact. Furthermore, a new peak at was generated which represented thrombin. Upon activation with Innovin, there is similar generation of a new peak at and decrease in the 71-kDa peak. A distinct peak at 62 kda represents the albumin a component in the tissue factor. Similarly, composite data on the SELDI analysis of native Konyne and the activated Konyne with RecombiPlasTin 2G and Innovin is given in Figure 7A. In the native spectra, the prominent prothrombin peak is located at and another peak is located at 58 kda. Upon activation with RecombiPlas- Tin 2G the peak at was no longer detectable and a new peak at was generated. This peak represents thrombin. Also, a 58-kDa peak was also decreased. Upon activation with Innovin, there was similar generation of a new peak at and decrease in the 71-kDa peak. A distinct peak at represents the albumin a component in the tissue factor. Furthermore, a 58-kDa peak was decreased using Innovin which had similar effect by utilizing RecombiPlasTin 2G to activate Konyne. Figure 7B depicts the effect of tissue factors on native Preconetiv. A native spectrum of Preconetiv showed the prominent peak of prothrombin which was located at. Native spectrum also showed a 58-kDa peak similar to native Konyne with lower amount. Upon activation with RecombiPlasTin 2G,

6 Sadeghi et al 261 A Beriplex Beriplex + Innovin Beriplex + RecombiPlasTin 2G Native Beriplex B Cofact Cofact + Innovin Cofact + RecombiPlasTin 2G Native Cofact Figure 4. Composite data on surface-enhanced laser desorption/ionization (SELDI) analysis of native PCCs and activated PCCs with various tissue factors. Mass spectrometer analysis of native and activated Beriplex and Cofact. Native Beriplex and Cofact exhibit a major peak at, whereas upon activation with both RecombiPlasTin 2G and Innovin this peak diminished and peak is generated indicating formation of thrombin. X-axis represents molecular weight () and y-axis peak intensity (PI). PCC indicates prothrombin complex concentrate.

7 262 Clinical and Applied Thrombosis/Hemostasis 20(3) A B Octaplex Profilnine 50 kda Octaplex + Innovin Octaplex + RecombiPlasTin2G Profilnine + Innovin Profilnine + RecombiPlasTin 2G Native Octaplex Native Profilnine Figure 5. Composite data on surface-enhanced laser desorption/ionization (SELDI) analysis of native PCCs and activated PCCs with various tissue factors. Mass spectrometer analysis of native and activated Octaplex and Profilnine. Native Octaplex and Profilnine exhibited a major peak at, whereas upon activation with both RecombiPlasTin 2G and Innovin this peak diminished and peak is generated indicating the formation of thrombin. Activated Profilnine exhibited an additional 50 kda peak. X-axis represents molecular weight () and y-axis peak intensity (PI). PCC indicates prothrombin complex concentrate.

8 Sadeghi et al 263 A B Prothromplex FEIBA Prothromplex + Innovin Prothromplex + RecombiPlasTin 2G FEIBA + Innovin FEIBA + RecombiPlasTin 2G Native Prothromplex Native FEIBA Figure 6. Composite data on surface-enhanced laser desorption/ionization (SELDI) analysis of native PCCs and activated PCCs with various tissue factors. Mass spectrometer analysis of native and activated Prothromplex and FEIBA. Native Prothromplex and FEIBA exhibited a major peak at, whereas upon activation with both RecombiPlasTin 2G and Innovin this peak diminished and 36-kDa peak is generated indicating formation of thrombin. The x-axis represents molecular weight () and y-axis peak intensity (PI). PCC indicates prothrombin complex concentrate.

9 264 Clinical and Applied Thrombosis/Hemostasis 20(3) A B Konyne Preconativ 50 kda Konyne + Innovin Konyne + RecombiPlasTin 2G 58 kda Preconativ + Innovin Preconativ + RecombiPlasTin 2G 58 kda Native Konyne Native Preconativ Figure 7. Composite data on surface-enhanced laser desorption/ionization (SELDI) analysis of native prothrombin complex concentrates (PCCs) and activated PCCs with various tissue factors. Mass spectrometer analysis of native and activated Konyne and Preconetiv. Native Konyne and Preconetiv exhibited a major peak at, whereas upon activation with both RecombiPlasTin 2G and Innovin this peak diminished and 36-kDa peak is generated indicating formation of thrombin. Activated Preconetiv exhibited an additional 50-kDa peak. The x-axis represents molecular weight () and y-axis peak intensity (PI).

10 Sadeghi et al 265 Profilnine Beriplex kda Prothrombin Prethrombin KDa Thrombin Figure 8. Native prothrombin complex concentrates (PCCs). Gel electrophoresis analysis of various PCCs mixtures. Coomassie bluestained gel showed molecular weight markers (lane 1), Profilnine (lane 2), Cofact (lane 3), Beriplex (lane 4), Octaplex (lane 5), Prothromplex (lane 6), Feiba (lane7), Konyne (lane 8), Preconetiv (lane 9), and recombinant thrombin (lane 10). the peak at was no longer detectable and a new peak at was generated. This peak represents thrombin. Similarly, upon activation of Preconetiv with Innovin, there was similar generation of a new peak at and decrease in the peak. A distinct peak at represents albumin, a component in the tissue factor. In addition to the thrombin peak an additional prethrombin peak around 50 kda was generated. The use of Innovin converted the majority of the prothrombin into prethrombin and not fully into thrombin. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis Analysis To further analyze the compositional profile of various PCCs, SDS polyacrylamide gel electrophoresis (PAGE) analysis was performed in the native and activated PCCs with different tissue factors. Figure 8 shows a composite analysis of the native Profilnine SD (lane 2), native Cofact (lane 3), native Beriplex (lane 4), native Octaplex (lane 5), native Prothromplex (lane 6), native FEIBA (lane 7), native Konyne (lane 8), native Preconetiv (lane 9), and control human a thrombin (lane 10). As can be seen in this figure, Profilnine SD exhibited an intense 75-kDa band, which may indicate prothrombin and two unidentified faint bands at 250 kda and 140 kda. Similar to Profilnine SD, Beriplex showed an intense band at 75 kda and an extra band at which may represent albumin. Cofact and Octaplex exhibited similar profile as both exhibited bands at 250 kda, a doublet band at 100 to 140 kda region, 80-kDa band, an intense 75-kDa prothrombin band, singlet 36-kDa band, a doublet 18- to 20-kDa band, and 15-kDa band. Prothromplex, FEIBA, and Konyne exhibited similar protein profile with minor differences. All contained bands at 250 kda and 100 kda, an intense prothrombin band at 75 kda, 66-kDa band, faint 25-kDa band, singlet 36-kDa band, and 15-kDa band. Preconetiv showed a lesser protein content when compared to Prothromplex, FEIBA, and Konyne by showing bands at 250 kda, 100 kda, 75 kda, 20 kda, and 15 kda, respectively. The Figure 9. Gel electrophoretic profile of various prothrombin complex concentrates (PCCs) upon activation by Innovin. Gel electrophoresis analysis of various PCCs mixtures. Coomassie blue-stained gel showed molecular weight markers (lane 1), activated Profilnine (lane 2), activated Cofact (lane 3), activated Beriplex (lane 4), activated Octaplex (lane 5), activated Prothromplex (lane 6), activated Feiba (lane7), activated Konyne (lane 8), activated Preconetiv (lane 9), and recombinant thrombin (lane 10). SDS-PAGE profile of these complexes similar to SELDI analysis showed multiple protein bands ranging from 15 to 250 kda. While Beriplex and Profilnine SD showed a much lesser prevalence of these bands. Profilnine SD mainly comprised 250, 140, 75 kda bands and Beriplex mainly consisted of 75 and bands. The other complexes contain multiple additional bands in the range of 15 to, representing albumin and other degradation products. To evaluate the effect of tissue factor on PCCs additional SDS-PAGE analysis was carried out. Figure 9 exhibits composite protein profile analysis of PCCs mixture incubated with Innovin utilizing SDS-PAGE, activated Profilnine SD (lane 2), activated Cofact (lane 3), activated Beriplex (lane 4), activated Octaplex (lane 5), activated Prothromplex (lane 6), activated FEIBA (lane 7), activated Konyne (lane 8), activated Preconetiv (lane 9), and control human a thrombin (lane 10). Distinct thrombin bands were noted upon activation of PCCs by Innovin. All PCCs generated a doublet band at 36 to 37 kda which was comparable to the control thrombin. It seems that Innovin contained albumin as evident by prominent peak at, which made it difficult to analyze conversion of prothrombin to thrombin upon activation. The SDS-PAGE analysis was also carried out in a mixture of PCCs with RecombiPlasTin 2G in order to compare the effect of two tissue factors for the activation of PCCs. Figure 10 shows the protein profile analysis of activated Beriplex (lane 2), activated Cofact (lane 3), activated Octaplex (lane 4), activated Profilnine SD (lane 5), activated Prothromplex (lane 6), activated FEIBA (lane 7), activated Konyne (lane 8), activated Preconetiv (lane 9), and control human a thrombin (lane 10). Similar to activation profile of Innovin on PCCs, a doublet band at 36 to 37 kda was observed in all PCCs by Recombi- PlasTin 2G. This tissue factor was free of albumin, so the band at does not appear in this analysis. The conversion of prothrombin to thrombin in Profilnine SD is evident as prothrombin band at 75 kda disappeared. RecombiPlasTin 2G

11 266 Clinical and Applied Thrombosis/Hemostasis 20(3) KDa Thrombin kda Feiba Prothrombin Prethrombin Thrombin Figure 10. Gel electrophoretic profile of various prothrombin complex concentrate (PCCs) upon activation by RecombiPlasTin 2G. Gel electrophoresis analysis of various PCCs mixtures. Coomassie blue-stained gel showed molecular weight markers (lane 1), activated Beriplex (lane 2), activated Cofact (lane 3), activated Octaplex (lane 4), activated Profilnine (lane 5) activated, Prothromplex (lane 6), activated Feiba (lane7), activated Konyne (lane 8), activated Preconetiv (lane 9), and recombinant thrombin (lane 10). may more effective with a greater extent for both Beriplex and Preconetiv as compared to Cofact, Octaplex, Prothromplex, FEIBA, and Konyne in conversion of prothrombin. This is observed as a much lesser band at 75 kda. The activated Cofact, Octaplex, Prothromplex, FEIBA, and Konyne contain multiple additional bands in the range of 10 to, representing albumin and other degradation products. Immunoblotting Studies The immunoblotting studies were performed to evaluate the presence of thrombin in both native and activated PCCs. Figure 11 shows Western blot analysis of native Profilnine SD (lane 2), native Cofact (lane 3), native Beriplex (lane 4), native Octaplex (lane 5), native Prothromplex (lane 6), native FEIBA (lane 7), native Konyne (lane 8), native Preconetiv (lane 9), and control human a thrombin (lane 10). All PCCs contain major prothrombin bands, 75-kDa band along with a 50-kDa band representing prethrombin. Native FEIBA exhibited a distinct additional 37-kDa dense band representing thrombin. Similarly, the immunoblotting studies were carried out on activated PCCs utilizing Innovin as a tissue factor. Figure 12 depicts immunoblot analysis of activated Beriplex (lane 2), activated Cofact (lane 3), activated Octaplex (lane 4), activated Profilnine SD (lane 5), activated Prothromplex (lane 6), activated FEIBA (lane 7), activated Konyne (lane 8), activated Preconetiv (lane 9), and control human alpha thrombin (lane 10). Upon activation of PCCs by Innovin, all PCCs were capable of generating a distinct doublet thrombin band (36-37 kda) comparable to the control thrombin as measured by immunoblotting technique. The prothrombin band completely disappears for all PCCs except Preconetiv, representing prothrombin, prethrombin, and thrombin. The relative prevalence of the prethrombin band varied widely and nearly diminishes in Profilnine SD, Beriplex, Octaplex, Prothromplex, and Konyne leading to Figure 11. Western blot on Native PCCs. The determination of cross-reactivity of anti-recothrom IgG with native PCCs. Profilnine (lane 2), Cofact (lane3), Beriplex (lane 4), Octaplex (lane 5), Prothromplex (lane 6), Feiba (lane7), Konyne (lane 8), Preconetiv (lane 9), and Recothrombin (lane 10) were probed with 10 mg/ml of rabbit anti-recothrom IgG collected on day 270. Horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG were used as the secondary antibody at a 1: dilution. Relative molecular weight markers, to the left of panel (lane1), identified the size of immunoreactive bands. IgG indicates immunoglobulin G; PCC, prothrombin complex concentrate. the formation of thrombin. Activation of Cofact and FEIBA by Innovin resulted in the generation of thrombin. All PCCs were also activated with tissue factor Recombi- PlasTin 2G to evaluate thrombin generation using Western blot technique. Figure 13 shows immunoblot analysis of activated Beriplex (lane 2), activated Cofact (lane 3), activated Octaplex (lane 4), activated Profilnine SD (lane 5), activated Prothromplex (lane 6), activated FEIBA (lane 7), activated Konyne (lane 8), activated Preconetiv (lane 9), and control human a thrombin (lane 10). Similar to PCCs activated by Innovin, activation of PCCs by RecombiPlasTin 2G were capable of generating a doublet intense immunoreactive band at 36 to 37 kda comparable to the thrombin control. Upon activation of Beriplex, Octaplex, and Prothromplex by RecombiPlasTin 2G, complete generation of thrombin were observed in contrast to activation of these PCCs by Innovin as prethrombin band (50 kda) was not detected. Preconetiv activated by RecombiPlasTin 2G showed 50 kda and a doublet 36- to 37-kDa bands, while Preconetiv activated by Innovin exhibited an additional prothrombin band along with prethrombin and thrombin bands. Discussion While the commercially available PCCs are standardized in terms of comparable FIX units, these agents have been reported to exhibit compositional differences. 11 Moreover, besides this difference, the types of additives also differ in preparations. Because of these differences in PCCs, the endogenous generation of proteases such as the thrombin and Xa may also differ. Purity profile of PCCs may also have an effect on degree of generation of the activated coagulation factors and thrombin upon activation by tissue factor. These variations may contribute to the potential differences on the hemostatic properties of

12 Sadeghi et al 267 kda Prothrombin Prethrombin Thrombin kda Konyne Precone v Profilnine Precone v Figure 12. Western blot analysis of prothrombin complex concentrates (PCCs) activated by Innovin. The determination of cross-reactivity of anti-recothrom IgG with activated PCCs. Profilnine (lane 2), Cofact (lane3), Beriplex (lane 4), Octaplex (lane 5), Prothromplex (lane 6), Feiba (lane7), Konyne (lane 8), Preconetiv (lane 9), and Recothrombin (lane 10) were probed with 10 mg/ml of rabbit anti- Recothrom immunoglobulin G (IgG) collected on day 270. Horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG were used as the secondary antibody at a 1: dilution. Relative molecular weight markers, to the left of panel (lane1), identified the size of immunoreactive bands. various PCCs and the relative risk of thromboembolism. The relative efficacy in neutralizing warfarin or the newly developed oral anticoagulant may also vary. Although the plasma fractionation procedures used for the manufacture of these agents are similar, the relative composition and the protein content widely differ. Moreover, the presence of additives such as heparin and AT also contributes to their functional effects. Therefore, each individual PCC should be considered as a distinct drug and any interchange between the products should be made with caution. This study was carried out on 5 commercially available PCCs. The older products, namely, Preconetiv and Konyne, were also used for comparative purposes. FEIBA which represents an activated prothrombin complex was included in this study to determine extent of preformed thrombin in this complex. Besides the protein content and molecular profile, the activation profiles were studied using 2 distinct tissue factors, namely, RecombiPlasTin 2G and Innovin. While Recombi- PlasTin 2G represents a purer product, the Innovin, a recombinant origin, contained sizable amount of albumin. Differences in activation profile of these 2 agents were also noted. This study was designed to determine the functional generation of the thrombin using different methods. All PCCs exhibited wide variation in protein content; however, the FIX antigen level was comparable. The wide variations in protein content may be due to the process and addition of protein such as albumin. Profilnine and Beriplex contain the least amount of proteins in comparison to other PCCs. Konyne and FEIBA contained high amount of protein. Although the supplementation of various PCCs did not result in any change in the PT/international normalized ratio values, FEIBA produced a shortening of the PT at 1 U/mL. No differences with the other PCCs were noted. The supplementation of Figure 13. Western blot analysis of prothrombin complex concentrates (PCCs) activated by RecombiPlasTin 2G. The determination of cross-reactivity of anti-recothrom immunoglobulin G (IgG) with activated PCCs. Beriplex (lane 2), Cofact (lane3), Octaplex (lane 4), Profilnine (lane 5), Prothromplex (lane 6), Feiba (lane7), Konyne (lane 8), Preconetiv (lane 9), and Recothrombin (lane 10) were probed with 10 mg/ml of rabbit anti-recothrom IgG collected on day 270. Horseradish peroxidase (HRP)-linked donkey anti-rabbit IgG were used as the secondary antibody at a 1: dilution. Relative molecular weight markers, to the left of panel (lane1), identified the size of immunoreactive bands. these PCCs resulted in varying prolongation of the aptt values. Prothromplex, Octaplex, Konyne, and FEIBA produce sizable effects on the aptt, whereas the other PCCs have relatively milder effect on this parameter. The heparin content in Octaplex and Prothromplex was much higher than most of the other PCCs. This study showed that heparin and AT may influence the overall procoagulant/anticoagulant balance in these agents. The AT level in Beriplex and Prothromplex was relatively high. It is interesting to note that despite high AT level and the presence of heparin, Beriplex did not show a prolongation in the aptt values. Preconetiv and Profilnine did not contain any AT. Since FIX content is universally used to standardize the PCCs, FIX antigen levels were measured in all of the PCCs. The FIX antigen level ranged from 80% to 109%/1 U/mL in all of the concentrates. Konyne, FEIBA, and Beriplex had relatively higher FIX antigen levels, whereas the other concentrates were close to one another. This suggests that the standardization procedures practiced by manufactures are uniform. Whether other factors such as II, VII, and X along with protein C and S are also comparable in these products remains an open question. Since the prothrombin complexes endogenously generate thrombin and other activator enzymes, a functional fluorogenic method was used to determine thrombin generation potential of various PCCs. Interestingly, in this study major differences were noted in the ability to generate thrombin among these agents. Thrombin generation varied from 37 to 1044 nmol/l/ 5 FIX unit equivalent. Prothromplex generated the least thrombin whereas Octaplex generated very high amounts of thrombin. Wide variations were noted among these agents. One factor contributing to the differential thrombin generation may be the presence of heparin and AT in these complexes. Other factors such as protein C and S along with protein Z may also contribute to anticoagulant effects observed with these agents.

13 268 Clinical and Applied Thrombosis/Hemostasis 20(3) The functional generation of thrombin by each PCCs was product specific. Although the generation of FXa was not studied in these studies, it is projected that variation in generation of FXa may also contribute the functional heterogeneity of these agents. Therefore, the relative efficacy of each product should individually assess in the vivo setting for the neutralization of anticoagulant effects of newly developed anti Xa and anti IIa agents. The mass spectrometric analysis of all PCCs was carried on the SELDI instrument in the range of 30 to 80 kda as this range covers thrombin and prothrombin. Albumin also falls in this range. Each of the PCCs was analyzed in terms of its native profile, its profile following activation with Innovin, and its profile following activation with RecombiPlasTin 2G. The native prothrombin complexes exhibited prominent peaks at 70 to 72 kda. Upon activation with RecombiPlasTin 2G, the prothrombin peak in this range was diminished in most cases and additional peaks were generated at 50 to 60 kda and 35 to, respectively. Since Innovin contains albumin, there is a distinct peak at in all activation mixtures containing Innovin. Some of the PCCs also contain albumin as an additive and their native spectra also showed the 66-kDa peak. This was prominent in the case of Beriplex, Prothromplex, and FEIBA. Other minor peaks were also observed in the native and activation mixtures. Each of the PCC showed the distinct pattern with a varying level of protein composition. Some of the PCCs such as Profilnine were found that contain a distinct 50-kDa band representing prethrombin. Konyne and Preconetiv exhibited an additional poly component peak in the range of 55 to 60 kda which most likely is a conversion product prior to prethrombin. Upon activation, all PCCs generated varying degrees of thrombin with both activators. It is interesting that while the 58-kDa peak was diminished by activation with thromboplastin in Konyne and Preconetiv, the 50-kDa peak in Preconetiv did not diminish. This suggests the differential proteolysis in these agents. The activation profile by two thromboplastin preparations was also different. Because of the presence of albumin in Innovin, the identification of other component peaks was difficult. However, the RecombiPlasTin 2G-activated preparations showed clear activation process for all PCCs. These studies showed that while the PCCs differ in protein composition, the type of the tissue factor may also influence the activation profile of these agents. The overall generation of thrombin and intermediary products such as prethrombin may be partly due to the presence of other proteins and additives in these agents. The SDS-PAGE profile of all PCCs exhibited distinct molecular profiles in the range of 250 to 10 kda. Profilnine and Beriplex are relatively purer preparations with distinct band in prothrombin region 72 kda; Beriplex contains albumin. Similarly, other prothrombin complexes showed a comparable prothrombin band with varying degree of albumin. Profilnine did not exhibit detectable albumin band. Cofact, Octaplex, Prothromplex, FEIBA, and Konyne showed a distinct bands around 27 to 29 kda, which were comparable to the thrombin doublet in the control. However, this band was absent in Profilnine, Beriplex, and Preconetiv. The levels of prethrombin were also evident in some of the prothrombin complexes. The intensity of various bands in this analysis also differed. All in all, while the protein components in a high range showed a similar pattern, in the lower region these profiles were different. The gel electrophoretic profiles of various PCCs upon activation with Innovin and RecombiPlasTin 2G were comparable; in all preparations thrombin was generated. Because of the presence of the albumin, the Innovin-activated product showed dense band in the albumin region. The proteolytic cleavage profile was more distinct and clear in case of RecombiPlasTin 2G. Moreover, in the low- region (below 25 kda) more generation of fragments was noted. The Western blotting studies of native PCCs showed a distinct prothrombin band in all preparations. In addition, prethrombin was detected in all preparations. In the case of FEIBA, a distinct band representing thrombin was noted. The relative intensity of the prethrombin band differed among these preparations. Upon activation with Innovin, most of the prothrombin bands were diminished. Residual prethrombin peaks were noted in most PCCs with exception of Cofact and FEIBA. In the case of Preconetiv, traces of prothrombin were also noted. When the PCCs were activated with RecombiPlasTin 2G, the prothrombin peak was totally diminished and dense bands representing thrombin were noted. Strong band representing prethrombin was noted in Profilnine, while traces of this protein were noted in Konyne and Preconetiv. In addition, some residual trace proteins were detected in Prothromplex, Konyne, and Preconetiv. These studies utilizing conventional techniques to quantitate protein and utilizing mass spectrometric method along with gel electrophoresis clearly demonstrate the heterogeneity in the composition of the available PCCs. These studies utilized an antibody against human recombinant thrombin to recognize thrombin and related proteins such as prethrombin and prothrombin. It is clear from Western blotting analysis that compositional differences exist in the native and activated PCCs. The SELDI analysis is complementary to Western blotting analysis confirming the presence of prethrombin and other components. Clinical Implications Despite the standardization of PCCs, the composition of these agents differs widely and thus their hemostatic potential is also variable. Moreover, the presence of additives such as heparin and AT may also influence the hemostatic potential of these agents. These studies have demonstrated that the currently available PCCs not only differ in the molecular profile but also in their functional activities such as the generation of thrombin. The presence of heparin and AT may have endogenous modulatory effects on the overall hemostatic effects of these agents. Therefore, the clinical efficacy and safety of these agents may also differ. Thus, each of these products should be consider as a