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INFLUENCE OF WASHING CONDITIONS ON EFFECTIVE COMPONENTS OF PROTHROMBIN COMPLEX CONCENTRATES a
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Haijun Cao , Changqing Li , Yun Huang , Shengliang Ye , Bin Liu a
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, Zongkui Wang , Xi Du , Xuejun Zhang & Fangzhao Lin
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Institute of Blood Transfusion , Chinese Academy of Medical Sciences & Peking Union Medical College , Chengdu , China b
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School of Materials Science and Engineering , Southwest Petroleum University , Chengdu , China Accepted author version posted online: 14 Jun 2013.Published online: 23 Oct 2013.
To cite this article: Haijun Cao , Changqing Li , Yun Huang , Shengliang Ye , Bin Liu , Zongkui Wang , Xi Du , Xuejun Zhang & Fangzhao Lin (2014) INFLUENCE OF WASHING CONDITIONS ON EFFECTIVE COMPONENTS OF PROTHROMBIN COMPLEX CONCENTRATES, Preparative Biochemistry and Biotechnology, 44:2, 164-181, DOI: 10.1080/10826068.2013.803479 To link to this article: http://dx.doi.org/10.1080/10826068.2013.803479
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Preparative Biochemistry & Biotechnology, 44:164–181, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.803479
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INFLUENCE OF WASHING CONDITIONS ON EFFECTIVE COMPONENTS OF PROTHROMBIN COMPLEX CONCENTRATES
Haijun Cao,1 Changqing Li,1 Yun Huang,2 Shengliang Ye,1 Bin Liu,1 Zongkui Wang,1 Xi Du,1 Xuejun Zhang,1 and Fangzhao Lin1 1 Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, Chengdu, China 2 School of Materials Science and Engineering, Southwest Petroleum University, Chengdu, China
& In order to increase the yield of prothrombin complex concentrates (PCCs) and to reduce their associated thrombotic risks, the influence of washing conditions on the yield, purity, and balance of coagulation factors (FII, FVII, FIX, and FX), and inhibitor proteins (PC, PS, PZ, and AT [antithrombin]) in PCCs was investigated by orthogonal testing, in which three variables (sodium citrate, NaCl, and pH) and their three levels were selected. It was found that AT yield and purity were extraordinarily low, and at lower NaCl content, the general yield, purity, and balance were higher, lower, and better, respectively; however, the results became contrary at higher NaCl. Moreover, within the investigated levels, NaCl was the first determinant for the yield except AT and the purity except FVII, PC, PS, and AT. Sodium citrate was the first determinant for AT yield and FVII, PS, and AT purity. The yield except FII, PS, and AT decreased and the purity except PC increased with increase of sodium citrate content. Just for PC purity, pH was the first determinant. The effect with pH fluctuation on the yield and purity was characteristically unobvious. The outcome undoubtedly supplies the guidance to further improve PCCs. Keywords coagulation factor, coagulation inhibitor, prothrombin complex concentrates, purity, washing condition, yield
INTRODUCTION Prothrombin complex concentrates (PCCs), containing numerous vitamin K-dependent proteins, are the intermediate purity products prepared from pooled plasma.[1,2] Although originally introduced for treatment of hemophilia B, which results from coagulation factor (F) IX Address correspondence to Changqing Li, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, 610052 Chengdu, China. E-mail: lichangqing268@ 163.com. Address correspondence also to Yun Huang, School of Materials Science and Engineering, Southwest Petroleum University, 610500 Chengdu, China. E-mail: [email protected]
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deficiency, PCCs have been rather widely used for treating genetic and acquired deficiencies of other vitamin K-dependent coagulation factors such as FII, FVII, and FX, of protein C (PC) and protein S (PS), and liver disease and urgent reversal of coumarin-induced anticoagulation.[3–6] In recent years, the number of patients receiving oral anticoagulants has increased,[7] and PCCs are increasingly recognized as the treatment of important choice for anticoagulant overdose.[8–11] Since PCCs were applied to clinical use, many thrombotic complications after PCCs administration have been reported.[12–15] The thrombotic complications include deep vein thrombosis (DVT), arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, and disseminated intravascular coagulation (DIC).[15–18] Today’s PCCs have improved safety compared with previously,[15] but the complications induced by PCCs, such as deep vein thrombosis (DVT), ischemic stroke, pulmonary embolism, and myocardial infarction still occurred.[19–21] Though the potential causes of thrombosis with PCCs remain a subject for debate,[19] PCCs’ composition is one of important risk factors for associated thromboembolism.[20] In the 1990s, a cluster of five fatal thrombotic events associated with PCCs in German patients undergoing surgery was reported.[14] Compared with other PCCs, the PCCs had high levels of FII and FX and activated FVII, low levels of PS, and no free antithrombin (AT) activity, although heparin levels were relatively high.[14,22] More and more studies showed avoidance of activated factors, greater purity, and a well-balanced content of coagulation factors and inhibitors, especially avoiding excessive levels of FII and FX and improving inhibitors PC, as well as PS in its functionally active form, in PCCs could contribute to reduced potential of causing thromboembolic events.[2,3,4,15,19,20] In 1990, seven different PCCs were analyzed and the results displayed that the potency of coagulation factors and inhibitors varied considerably. Most PCCs contained excessive FII and FX compared with FIX, while two preparations did not contain PS, and additionally one of the preceding two preparations did not contain PC.[24] In 2008, a series of studies was performed to compare biochemical properties of seven PCCs and the data demonstrated all PCCs were negative for activated coagulation factors, but all PCCs contained a considerable concentration of FII.[7,19] In addition, the commercially available PCCs in China had higher FII=FIX and FX=FIX ratio, and great variation in coagulation inhibitors content according to our present investigation. DEAE-Sephadex as an ionic exchanger was firstly introduced for PCCs production in 1969.[25] The DEAE-Sephadex resin has a strong capacity for binding vitamin K-dependent plasma proteins, and DEAE-Sephadex A-50 (A-50) has been extensively applied to purify PCCs at present.[23,26–28] However, the mechanical strength of the swollen A-50 resin is very poor, and it
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is not suitable in the column chromatographic technique. Two-step batch separation is adopted to isolate PCCs using the A-50 resin, including two steps of ionic exchange. Batch procedure is carried out by stirring and less automation, which brings great difficulty in study on ionic exchange conditions. Although most of the manufacturers employed the sodium citrate–NaCl solution system for the A-50 resin washing and elution conditions,[23,28–30] so far there are no reports about the correlation between different sodium citrate–NaCl washing system for the A-50 resin and components of PCCs. This study, therefore, was to examine the influence of different sodium citrate–NaCl washing conditions on the effective components (coagulation factors, FII, FVII, FIX, and FX, and inhibitor proteins PC, PS, PZ, and AT) in PCCs, and to obtain the effect regularity and tendencies to further improve PCCs. EXPERIMENTAL Materials Fresh and citrate-stabilized human plasma was obtained from Guanghan plasma apheresis station (Deyang, China) and stored at 60 C until use. The coagulation factor clotting activity kits (FII, VII, IX, and X) were obtained from Chengdu Union (Chengdu, China). NaCl, sodium citrate, and glycine were purchased from Xilong Chemical (Guangzhou, China), Na heparin was from Sunshine Biotech (Nanjing, China), and protein assay reagent was from CWBiotech (Beijing, China). DEAE-Sephadex A-50 was purchased from GE (Uppsala, Sweden), protamine sulfate, sodium dodecyl sulfate, b-mercaptoethanol, and 30% acrylamide=bis solution (29=1) for the electrophoresis were all from Sigma (St. Louis, MO), Pageruler plus prestained protein ladder (protein weight marker) from Fermentas (Glen Burnie, MD), and both Tris and Coomassie brilliant blue R-250 from Amresco (Solon, OH). The PZ kit, PC activator, S2166, calibrator plasma, normal plasma, and abnormal plasma were all purchased from HBM (Andre´sy, France). The AT kit and PS Ac kit were from ADI (Stamford, CT) and Siemens (Tarrytown, NY), respectively. Washing Conditions for Purifying PCCs Three variables (sodium citrate, NaCl, and pH) and three levels (0.01, 0.015, 0.02 mol=L of sodium citrate, 0.10, 0.15, 0.20 mol=L of NaCl, and 6.8, 7.0, 7.2 for pH) were chosen as the washing conditions for PCCs purification. An orthogonal L9(3)3 test was designed and nine kinds of washing conditions were prepared, shown in Table 1. The levels of sodium citrate, NaCl, and pH in washing conditions for PCCs were determined according
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Washing Conditions and Components of PCCs TABLE 1 L9 (3)3 Orthogonal Test Design (Three Variables and Three Levels) Variables and Levels Group Number
(A) Sodium Citrate (mol=L)
(B) NaCl (mol=L)
(C) pH
0.01 0.01 0.01 0.015 0.015 0.015 0.02 0.02 0.02
0.10 0.15 0.20 0.10 0.15 0.20 0.10 0.15 0.20
6.80 7.00 7.20 7.00 7.20 6.80 7.20 6.80 7.00
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1 2 3 4 5 6 7 8 9
to the previous research[29,31] and the present production technologies for PCCs in China.[32] Preparation of PCCs The purification steps have been described before.[29,32] The plasma was thawed at 0–2 C, then centrifugated to remove the cryoprecipitate. The cryo-poor plasma was recovered the starting material. To the cryo-poor plasma was added heparin of 0.2 IU=mL, and this was stored at 4 C for less than 4 hr before use. The A-50 resin powder was allowed to swell and equilibrate in sodium citrate buffer (0.02 mol=L, pH 6.8) with 0.05 mol=L NaCl and decanted 6–8 times until pH of the supernatant was approximately 6.8. The equilibrated A-50 resin, in the proportion of 1.67 g resin powder to 1 L plasma, was mixed with the cryo-poor plasma containing heparin to adsorb PCCs for 50 min, at 4 C. After that, the A-50 resin was washed two times with the corresponding washing condition listed in Table 1. At last, PCCs were eluted two times with sodium citrate buffer (0.015 mol=L, pH 6.9) containing 2 mol=L NaCl. Nine test groups for preparation of PCCs were performed simultaneously according to the preceding methods and repeated three times. Determination of Effective Components in PCCs Coagulation Factors Potency The potency of FII, VII, IX, or X was determined using an automatic coagulometer (CA1500, Sysmex, Japan) with a one-stage clotting assay.[33] The fresh PCCs were diluted 15-fold with ultrapure water for further measure, and to eliminate interference of high content of salt ion on determination of coagulation factor activity.
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The calibrator plasma was used to create calibration curve and the normal plasma was used to control measure. FII, VII, or X was measured by using PT reagent and respective corresponding factor-deficient substrate plasma.[7] In this assay, 50 ml sample was mixed with 50 ml factor-deficient plasma, and then incubated for 180 s at 37 C. The coagulation reaction was initiated by adding 100 ml PT reagent. FIX was determined by APPT reagent and FIX-deficient plasma.[7] Heparin in cryo-poor plasma was neutralized by protamin sulfate. For this assay, 50 ml sample was mixed with 50 ml deficient plasma, and incubated for 60 s at 37 C, and then 50 ml APPT was added and incubated for another 240 s at 37 C. The coagulation reaction was initiated after the addition of 50 ml CaCl2(0.025 mol=L).
Coagulation Inhibitors Activity or Antigen PCCs samples analyzed for coagulation inhibitors were stored at 4 C less than 2 hr and diluted to at least 1=10. The activity of PC and AT was measured by chromogenic assay, as described previously.[34,35] The activity assay for PS was performed by a coagulation method.[36,37] The assays were all standardized with calibrator plasma and controlled by the normal and abnormal plasma. A one-step immunoassay was performed to measure PZ antigen. PC activity was measured by PC activator (prota C) and activated PC substrate (S2166).[34] The 50 ml prediluted sample and 50 ml of 0.05 IU= mL prota C were mixed and incubated in water bath at 37 C for 9 min, and then 50 ml of 1.0 mmol=mL S2166 was added and uniformly mixed. The color reaction was stopped by 50% acetic acid (v=v) followed 10 min of incubation at 37 C, and then the adsorption was immediately measured at optical density (OD) 405 nm using microplate readers (SpectraMax M2e, Molecular Devices, USA). The assay for AT activity was carried out with the AT kit and the assay process included two stages.[35] First, 50 ml sample diluted by dilution buffer was added to a microplate and incubated at 37 C for 3 min, and then 50 ml Thrombin reagent (thrombin) was introduced and continuously incubated for 1 min. Second, 50 ml Spectrozyme TH (thrombin substrate) was added, mixed, and incubated for 1 min at 37 C. At last, the reaction was quenched by acetic acid. The rate of change of absorbance at OD 405 nm was detected. PS activity was measured by the PS Ac kit on the automatic coagulometer. In this analysis, first, 16 ml sample was mixed with 95 ml PS-deficient plasma. Second, the 16-ml resulting mixture was added to 47 ml PS-deficient plasma and then the whole system was incubated at 37 C for 20 s. Third, 58 ml APC Reagent (activated protein C) was added and incubated for
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110 s at 37 C. At last, the coagulation time, which was directly proportional to PS activity, was determined after addition of 145 ml Starting Reagent (activator). PZ antigen was assayed using the protein Z kit. The specific polyclonal antibody coupled with horseradish peroxidase was used to capture PZ. The concentration of PZ was detected according to the amount of color developed by hydrogen peroxide and peroxidase substrate. This assay was carried out in accordance with the manufacturer instructions. Total Protein Content The total protein content of PCCs was measured using the protein assay reagent according to the Bradford method[38] and the assay was performed according to manufacturer’s instructions. Yield and Purity of Coagulation Factors and Inhibitors The yield of coagulation factor or inhibitor was calculated based on Eq. (1), Yield% ¼
V p Ua 100% VC UA
ð1Þ
where Vp (mL) is PCCs volume, Ua (IU=mL, mg=mL) is activity or antigen content of coagulation factor or inhibitor per milliliter in PCCs, VC (mL) is cryo-poor plasma volume, and UA (IU=mL, mg=mL) is activity or antigen content of coagulation factor or inhibitor per milliliter in cryo-poor plasma. The kind of coagulation factor or inhibitor of Ua is the same as UA . The purity was calculated using Eq. (2), Purity ðIU=mg; mg=mgÞ ¼
Ua Cp
ð2Þ
where Ua (IU=mL, mg=mL) is activity or antigen content of coagulation factor or inhibitor per milliliter in PCCs, and Cp (mg=mL) is total protein content of PCCs per milliliter. Statistical and Orthogonal Test Analysis The experiment results were presented as the mean standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the differences in the yield, purity, and balance of coagulation factors or inhibitors among nine test groups, respectively. The sum of the yield or purity of FII, VII, IX, and X represented the coagulation factors yield or purity of PCCs in each test group, and the sum of the yield or purity of
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PC, PS, PZ, and AT represented the coagulation inhibitors yield or purity of PCCs. The SD of the purity between four factors represented the factors balance in each test group, and the SD of the purity between four inhibitors represented the inhibitors balance. SPSS software was used for these statistical analyses (Version 17.0, SPSS, Inc., USA). Differences are considered statistically significant at p < 0.05. Furthermore, under different washing conditions, the yield, purity, and balance of coagulation factors or inhibitors in PCCs were analyzed by the direct comparison method of orthogonal test.[39] The influence of three variables and their three levels on yield and purity of the factors or inhibitors in PCCs was analyzed by the heuristics analysis method of orthogonal test.[39] SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), using 4% stacking gel and 10% running gel in a Tris–glycine system, was performed to analyze PCCs. The sample was prepared as follows: 200 ml PCCs from three repetitions were gently mingled and diluted by ultrapure water to 1 mol=L NaCl concentration; 80 ml diluted sample was added to 20 ml Tris–glycine buffer with 5% (v=v) b-mercaptoethanol (fivefold concentrate), and then the mixture was boiled at 100 C for 5 min. Protein weight marker was also loaded onto the gels for standards. The electrophoresis was carried out at potential difference of 60 V in stacking gel, and then at 120 V in running gel. After electrophoresis, gel was stained with Coomassie brilliant blue R-250 and destained with a methanol=acetic acid mixture. RESULTS AND DISCUSSION Yield and Purity of Coagulation Factors and Inhibitors in Orthogonal Test The yield and purity of coagulation factors and inhibitors, obtained from in L9(3)3 orthogonal test, were shown in Figure 1. The yield and purity of coagulation factors showed significant difference among nine groups (p ¼ 0.004 and p ¼ 0.022, respectively). It was also obvious that for coagulation factors, both yield and purity exhibited periodic variations. The yield, especially for FVII, gradually reduced in every three test groups; however, the purity, except FVII in group 3, gradually increased. Significant difference was found in the yield and purity of four inhibitors among nine groups (p ¼ 0.007 and p ¼ 0.019, respectively), but only PS yield and PZ purity showed periodic variations in which PS yield gradually reduced and PZ purity increased, respectively, in all three groups.
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FIGURE 1 The yield of coagulation factors (A), purity of coagulation factors (B), yield of coagulation inhibitors (C), and purity of coagulation inhibitors (D) in the orthogonal test. These data are mean values and the error bars represent SD.
The total yield of four factors was the highest in group 1, and the maximum purity was in group 9 (Figure 1, A and B). The highest yield of four inhibitors was achieved in group 4 (Figure 1C); however, the highest purity of PC, PS, PZ, and AT was distributed in group 3, group 8, group 9, and group 6, respectively (Figure 1D). The discrepancies in yield and purity of coagulation factors and inhibitors of PCCs prepared by different washing conditions were also revealed. Since FVII has weaker absorption with A-50 resin,[40] FVII activity in A-50-prepared PCCs was usually lower.[7,20,24] However, from Figures 1A and 1B, a different but exciting result was exhibited that the yield of FVII was more than that of FII and X in groups 1, 4, and 7, and that the activity of FVII was relatively higher than that of FII and X in the preceding groups. The same character achieved from groups 1, 4, and 7 was 0.1 mol=L NaCl. Therefore, the activity of FVII, II, and X in PCCs can be improved by controlling NaCl content in the washing condition. In addition, both yield and purity of AT were extremely low and even zero in nine groups (Figure 1, C
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and D). The main reason may be that AT was hardly adsorbed by A-50 resin, so most AT remained in the unbound fraction of cryo-poor plasma,[41] which resulted in the content of AT in the A-50-prepared PCCs being very low in any washing conditions. This may be the reason for lots of PCCs with added extra AT.[26,27]
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Balance of Coagulation Factors and Inhibitors in Orthogonal Test The purity of four factors and inhibitors in PCCs was presented in Figures 1B and 1D, and there were significant differences in four factors balance (p ¼ 0.008) as well as four inhibitors balance (p ¼ 0.03) among nine groups. Moreover, it was clearly shown that both the factors and the inhibitors in groups 1 and 4, though there was little and even no AT, had better balance, and in group 1, the SD between four factors and inhibitors was minimum (0.18 and 0.56, respectively), and in group 4, was smaller (0.20 and 0.65 respectively). Meanwhile, the worst balance was in group 9, although it had the highest purity of four factors and PZ, in which the SD between four factors and inhibitors was maximum (0.66 and 1.62, respectively). So the washing conditions caused the great discrepancy in balance of coagulation factors and inhibitors, respectively. The lower levels TABLE 2
Results Analysis of L9 (3)3 Orthogonal Test for Four Coagulation Factors Yield
Coagulation Factor FII
FVII
FIX
FX
k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R
Purity
A
B
C
A
B
C
60.06 0.10a 60.57 1.64 57.41 0.67 3.16b 58.72 3.70 55.04 3.74 49.65 2.57 9.07 74.03 1.43 69.16 2.22 61.32 1.64 12.71 58.08 1.28 55.35 2.23 48.12 1.51 9.96
64.27 0.60 59.46 0.79 54.30 0.58 9.97 71.32 1.47 53.21 1.16 38.88 0.51 32.44 76.06 1.43 68.91 1.05 59.54 1.74 16.52 60.54 1.34 55.83 0.80 45.18 1.41 15.36
57.94 1.45 59.47 1.26 60.62 0.70 2.68 53.55 4.36 56.35 3.50 53.51 2.28 2.84 67.37 2.67 67.11 2.66 70.04 0.48 2.93 53.24 2.27 53.16 2.51 55.14 0.77 1.98
1.46 0.07 1.62 0.09 2.10 0.14 0.64 0.99 0.01 1.03 0.01 1.29 0.04 0.30 1.08 0.06 1.14 0.05 1.33 0.06 0.25 1.20 0.05 1.33 0.05 1.56 0.07 0.36
1.26 0.04 1.68 0.05 2.24 0.11 0.98 1.04 0.02 1.08 0.02 1.19 0.06 0.15 0.90 0.02 1.17 0.03 1.47 0.03 0.57 1.09 0.02 1.34 0.04 1.65 0.05 0.56
1.71 0.11c 1.86 0.18 1.61 0.03 0.25d 1.07 0.02 1.19 0.06 1.05 0.02 0.14 1.18 0.06 1.18 0.08 1.19 0.04 0.01 1.36 0.07 1.38 0.10 1.35 0.03 0.03
ki ¼ R3i SD, where Ri is the amount of the yield at Ai. R ¼ maxfkiA g minfkiA g, and the SD is not involved in the calculation of R value. c C ki ¼ R3i SD, where Ri is the amount of the purity at Ci. d R ¼ maxfkiC g minfkiC g, and the SD is not involved in the calculation of R value. a A b
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of NaCl and sodium citrate were in groups 1 and 4, so it was concluded that the lower content of NaCl and sodium citrate had the possibility to produce a contribution to the better balance of effective components in PCCs.
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Influence of Washing Conditions on Four Coagulation Factors The orthogonal test results of four factors in PCCs were analyzed and are listed in Table 2. Obviously, according to R values the same influence order of three variables on the yield and purity of four factors was B > A > C, except that the influence order on FVII purity was A > B > C. Thus, the minimum effect on yield and purity was from pH (C), and NaCl (B) was the most important determinant, while sodium citrate (A) was only the primary determinant for FVII purity. The influence of levels of three variables on these factors yield was further investigated and the results are displayed in Figure 2. It was perceived that influence of NaCl on the yield was the same, in which the yield
FIGURE 2 Influence of different levels of sodium citrate, NaCl, and pH on the yield of FII (A), FVII (B), FIX (C), and FX (D). These data are mean values and the error bars represent SD.
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decreased with increase of NaCl content, and that the influence of sodium citrate was not completely consistent. The yield of FVII, IX, and X decreased with increase of sodium citrate; however, that of FII increased when sodium citrate concentrate increased from 0.01 mol=L to 0.015 mol=L, and then decreased when sodium citrate increased to 0.02 mol=L. The influence of pH on yield was very inconsistent. FII yield increased with pH, and FVII yield increased when pH increased from 6.8 to 7.0, and then decreased when pH increased to 7.2. The influence of pH on the yield of FIX and X was completely different from FII and VII, and was just opposite to FVII. In Figure 3, the influence of different levels on four factors purity emerges. Obviously, the influence of levels of NaCl on the factors purity was consistent with that of sodium citrate, in which the purity increased with their concentrations increasing. However, the influence of pH on purity was not wholly uniform. For purity of FII, VII, and X, the influence was consistent, in which the purity increased for pH 6.8–7.0, and then
FIGURE 3 Influence of different levels of sodium citrate, NaCl, and pH on the purity of FII (A), FVII (B), FIX (C), and FX (D). These data are mean values and the error bars represent SD.
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decreased for pH 7.0–7.2 (Figure 3, A, B, and D). As seen from Figure 3C, the effect of pH on FIX purity was not the same as on the other three factors, and FIX purity was a little lower at pH 7.0.
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Influence of Washing Conditions on Four Coagulation Inhibitors The orthogonal test results for four inhibitors of PCCs were analyzed and are listed in Table 3. Based on R value, the influence orders of the three variables on yield of the four inhibitors were not exactly the same. The order for PC yield was B > A > C, as well as for PS, but for PZ, B > C > A, and for AT, A > B > C. The influence orders on purity of PC, PS, PZ, and AT were C > B > A, A > B > C, B > A > C, and A > B > C, respectively. From the preceding, it was demonstrated that NaCl had a stronger effect on the yield of four inhibitors, but for purity, three variables showed different effects. The influence of the levels on yield of four inhibitors is presented in Figure 4. The influence of NaCl on the yield of PC, PS, and PZ exhibited the same tendency, and in the range of 0.1–0.2 mol=L NaCl, the higher the concentrations, the lower is the yield of PC, PS, and PZ. The influence of NaCl on AT yield was different from this. When NaCl was 0.15 mol=L, AT TABLE 3
Results Analysis of L9 (3)3 Orthogonal Test for Four Coagulation Inhibitors Yield
Coagulation Inhibitor PC
PS
PZ
AT
a A
k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R
A
B
24.59 0.63a 21.80 2.38 13.95 2.12 10.64b 20.43 1.26 21.19 1.89 16.72 1.57 4.47 48.92 0.23 48.15 1.83 44.90 0.45 4.02 0.03 0.02 3.48 0.08 3.13 0.02 3.45
25.00 1.11 22.43 0.86 12.92 2.71 12.08 26.28 0.60 20.81 0.56 11.25 0.68 15.03 50.20 0.80 48.00 0.81 43.82 1.25 6.38 2.27 0.40 2.13 0.38 2.24 0.38 0.14
ki calculated according to Table 2. R calculated according to Table 2. c C ki calculated according to Table 2. d R calculated according to Table 2. b
Purity C
A
16.00 1.34 0.62 0.05 19.30 3.10 0.61 0.03 25.05 0.58 0.47 0.06 9.05 0.15 19.00 1.65 0.46 0.01 18.81 2.11 0.56 0.02 20.58 1.04 0.59 0.02 1.77 0.13 43.46 1.09 2.17 0.15 48.68 1.16 2.29 0.09 49.83 0.46 2.92 0.20 6.37 0.75 2.23 0.40 0.001 0.001 2.24 0.40 0.09 0.007 2.17 0.37 0.10 0.006 0.07 0.10
B
C
0.54 0.03 0.68 0.02 0.47 0.07 0.21 0.57 0.01 0.57 0.03 0.47 0.003 0.10 1.79 0.06 2.37 0.06 3.21 0.14 1.42 0.04 0.008 0.07 0.01 0.09 0.01 0.05
0.51 0.03c 0.46 0.06 0.72 0.30 0.26d 0.54 0.02 0.50 0.01 0.57 0.02 0.07 2.29 0.15 2.63 0.24 2.45 0.09 0.34 0.08 0.01 0.06 0.01 0.05 0.008 0.03
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FIGURE 4 Influence of different levels of sodium citrate, NaCl, and pH on the yield of PC (a), PS (b), PZ (c), and AT (d). These data are mean values and the error bars represent SD.
yield was the minimum. The influence of sodium citrate on PC and PZ did not accord with PS and AT. Within 0.01–0.02 mol=L of sodium citrate, the higher the concentrations, the lower is the yield of PC and PZ; however the yield of PS and AT increased when sodium citrate increased from 0.01 mol=L to 0.015 mol=L, and then decreased when sodium citrate increased to 0.02 mol=L. The influence of pH on yield was not still accordant. In the range of pH 6.8–7.2, the higher pH, the higher was the yield of PC and PZ, but PS yield decreased when pH increased from 6.8 to 7.0, and then increased when pH increased to 7.2. The variation tendency of AT yield with levels of pH was exactly contrary to that of PS. The influence of different levels of three variables on four inhibitors purity is shown in Figure 5. The results display that the NaCl influence on purity of PC and PS was distinctly different from that on purity of PZ and AT. For PC and PS, the purity increased when NaCl increased from 0.1 mol=L to 0.15 mol=L, and then decreased when NaCl increased to 0.2 mol=L; nevertheless, PZ and AT purity constantly increased with NaCl.
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FIGURE 5 Influence of different levels of sodium citrate, NaCl, and pH on the purity of PC (a), PS (b), PZ (c), and AT (d). These data are mean values and the error bars represent SD.
The influence of sodium citrate on PC was different from that on PS, PZ, and AT. For 0.01–0.02 mol=L of sodium citrate, PS, PZ, and AT purity gradually increased with sodium citrate, but PC was just contrary. The influence of pH on purity was as follows: When pH increased from 6.8 to 7.0, PC and PS purity decreased but PZ purity increased, and when pH increased to 7.2, PC and PS purity increased but PZ purity decreased. At pH 6.8–7.2, AT purity decreased with increase of pH.
SDS-PAGE The purity of PCCs in the nine test groups was reflected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (Figure 6). The molecular masses of coagulation factors and inhibitors were about 50 and 70 kD, respectively. As shown in Figure 6, there was a regular variation in PCCs from group 1 to group 9. PCCs from groups 3 (lane 4), 6 (lane 7), and 9 (lane 10) were purer than those from their
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FIGURE 6 SDS-PAGE analysis for PCCs in the orthogonal test. Lanes: 1, Protein weight marker; 2, PCCs from group 1; 3, PCCs from group 2; 4, PCCs from group 3; 5, PCCs from group 4; 6, PCCs from group 5; 7, PCCs from group 6; 8, PCCs from group 7; 9, PCCs from group 8; 10, PCCs from group 9. PCCs were separated under reduced conditions (color figure available online).
preceding two groups, respectively, and PCCs from group 9 (lane 10) were the purest. These results displayed by SDS-PAGE were consistent with that in Figure 2B.
CONCLUSION The present research first in the field demonstrated the influence regularity of washing conditions on the yield, purity, and balance of coagulation factors and inhibitors in PCCs. It was found that the different washing conditions produced a significant discrepancy in their yield, purity, and balance. In three variables of sodium citrate, NaCl, and pH, for most coagulation factors and inhibitors, NaCl was the first and main determinant for the yield and purity and for the balance, respectively, and sodium citrate was the first determinant for both the yield of AT and the purity of FVII, PS, and AT, and pH was the first determinant for only the PC purity. In different levels of three variables, the influence of NaCl on the yield and purity was generally consistent with that of sodium citrate, while with the increase of NaCl and sodium citrate, the yield of coagulation factors and inhibitors except FII, PS, and AT decreased, and the purity except PC and PS increased. However, the influence of pH showed an inconsistent tendency. According to the regularity already described, the yield, purity, and balance of coagulation factors and inhibitors can be improved by adjusting washing conditions in different batch procedures. Furthermore, for certain factors or inhibitors, there were some inconsistencies of influence of sodium citrate, NaCl, and pH on the yield or purity, and those can be used to further improve the yield or purity of specific factor or inhibitor.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge financial support from the Public Welfare Industry of Health of China (200902008), the National High Technology Research and Development Program of China (863 Program; 2012AA021903), the Open Fund (PLN1111) of State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation of Southwest Petroleum University, and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20115121120005).
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38. Bradford, M.M. A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein–Dye Binding. Anal. Biochem. 1976, 72, 248–254. 39. Yiming, S.; Wei, X.; Chaoying, Q.; Yong, X. Mathematical Statistics. 3rd ed.; Science Press: Beijing, China, 2009; pp. 172–186. 40. Burnouf, T. Chromatography in Plasma Fractionation: Benefits and Future Trends. J. Chromatogr. B Biomed. Appl. 1995, 664(1), 3–15. 41. Burnouf, T. Modern Plasma Fractionation. Transfus. Med. Rev. 2007, 21(2), 101–117.
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INFLUENCE OF WASHING CONDITIONS ON EFFECTIVE COMPONENTS OF PROTHROMBIN COMPLEX CONCENTRATES a
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Haijun Cao , Changqing Li , Yun Huang , Shengliang Ye , Bin Liu a
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, Zongkui Wang , Xi Du , Xuejun Zhang & Fangzhao Lin
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Institute of Blood Transfusion , Chinese Academy of Medical Sciences & Peking Union Medical College , Chengdu , China b
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School of Materials Science and Engineering , Southwest Petroleum University , Chengdu , China Accepted author version posted online: 14 Jun 2013.Published online: 23 Oct 2013.
To cite this article: Haijun Cao , Changqing Li , Yun Huang , Shengliang Ye , Bin Liu , Zongkui Wang , Xi Du , Xuejun Zhang & Fangzhao Lin (2014) INFLUENCE OF WASHING CONDITIONS ON EFFECTIVE COMPONENTS OF PROTHROMBIN COMPLEX CONCENTRATES, Preparative Biochemistry and Biotechnology, 44:2, 164-181, DOI: 10.1080/10826068.2013.803479 To link to this article: http://dx.doi.org/10.1080/10826068.2013.803479
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Preparative Biochemistry & Biotechnology, 44:164–181, 2014 Copyright # Taylor & Francis Group, LLC ISSN: 1082-6068 print/1532-2297 online DOI: 10.1080/10826068.2013.803479
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INFLUENCE OF WASHING CONDITIONS ON EFFECTIVE COMPONENTS OF PROTHROMBIN COMPLEX CONCENTRATES
Haijun Cao,1 Changqing Li,1 Yun Huang,2 Shengliang Ye,1 Bin Liu,1 Zongkui Wang,1 Xi Du,1 Xuejun Zhang,1 and Fangzhao Lin1 1 Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, Chengdu, China 2 School of Materials Science and Engineering, Southwest Petroleum University, Chengdu, China
& In order to increase the yield of prothrombin complex concentrates (PCCs) and to reduce their associated thrombotic risks, the influence of washing conditions on the yield, purity, and balance of coagulation factors (FII, FVII, FIX, and FX), and inhibitor proteins (PC, PS, PZ, and AT [antithrombin]) in PCCs was investigated by orthogonal testing, in which three variables (sodium citrate, NaCl, and pH) and their three levels were selected. It was found that AT yield and purity were extraordinarily low, and at lower NaCl content, the general yield, purity, and balance were higher, lower, and better, respectively; however, the results became contrary at higher NaCl. Moreover, within the investigated levels, NaCl was the first determinant for the yield except AT and the purity except FVII, PC, PS, and AT. Sodium citrate was the first determinant for AT yield and FVII, PS, and AT purity. The yield except FII, PS, and AT decreased and the purity except PC increased with increase of sodium citrate content. Just for PC purity, pH was the first determinant. The effect with pH fluctuation on the yield and purity was characteristically unobvious. The outcome undoubtedly supplies the guidance to further improve PCCs. Keywords coagulation factor, coagulation inhibitor, prothrombin complex concentrates, purity, washing condition, yield
INTRODUCTION Prothrombin complex concentrates (PCCs), containing numerous vitamin K-dependent proteins, are the intermediate purity products prepared from pooled plasma.[1,2] Although originally introduced for treatment of hemophilia B, which results from coagulation factor (F) IX Address correspondence to Changqing Li, Institute of Blood Transfusion, Chinese Academy of Medical Sciences & Peking Union Medical College, 610052 Chengdu, China. E-mail: lichangqing268@ 163.com. Address correspondence also to Yun Huang, School of Materials Science and Engineering, Southwest Petroleum University, 610500 Chengdu, China. E-mail: [email protected]
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deficiency, PCCs have been rather widely used for treating genetic and acquired deficiencies of other vitamin K-dependent coagulation factors such as FII, FVII, and FX, of protein C (PC) and protein S (PS), and liver disease and urgent reversal of coumarin-induced anticoagulation.[3–6] In recent years, the number of patients receiving oral anticoagulants has increased,[7] and PCCs are increasingly recognized as the treatment of important choice for anticoagulant overdose.[8–11] Since PCCs were applied to clinical use, many thrombotic complications after PCCs administration have been reported.[12–15] The thrombotic complications include deep vein thrombosis (DVT), arterial thrombosis, ischemic stroke, pulmonary embolism, myocardial infarction, and disseminated intravascular coagulation (DIC).[15–18] Today’s PCCs have improved safety compared with previously,[15] but the complications induced by PCCs, such as deep vein thrombosis (DVT), ischemic stroke, pulmonary embolism, and myocardial infarction still occurred.[19–21] Though the potential causes of thrombosis with PCCs remain a subject for debate,[19] PCCs’ composition is one of important risk factors for associated thromboembolism.[20] In the 1990s, a cluster of five fatal thrombotic events associated with PCCs in German patients undergoing surgery was reported.[14] Compared with other PCCs, the PCCs had high levels of FII and FX and activated FVII, low levels of PS, and no free antithrombin (AT) activity, although heparin levels were relatively high.[14,22] More and more studies showed avoidance of activated factors, greater purity, and a well-balanced content of coagulation factors and inhibitors, especially avoiding excessive levels of FII and FX and improving inhibitors PC, as well as PS in its functionally active form, in PCCs could contribute to reduced potential of causing thromboembolic events.[2,3,4,15,19,20] In 1990, seven different PCCs were analyzed and the results displayed that the potency of coagulation factors and inhibitors varied considerably. Most PCCs contained excessive FII and FX compared with FIX, while two preparations did not contain PS, and additionally one of the preceding two preparations did not contain PC.[24] In 2008, a series of studies was performed to compare biochemical properties of seven PCCs and the data demonstrated all PCCs were negative for activated coagulation factors, but all PCCs contained a considerable concentration of FII.[7,19] In addition, the commercially available PCCs in China had higher FII=FIX and FX=FIX ratio, and great variation in coagulation inhibitors content according to our present investigation. DEAE-Sephadex as an ionic exchanger was firstly introduced for PCCs production in 1969.[25] The DEAE-Sephadex resin has a strong capacity for binding vitamin K-dependent plasma proteins, and DEAE-Sephadex A-50 (A-50) has been extensively applied to purify PCCs at present.[23,26–28] However, the mechanical strength of the swollen A-50 resin is very poor, and it
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is not suitable in the column chromatographic technique. Two-step batch separation is adopted to isolate PCCs using the A-50 resin, including two steps of ionic exchange. Batch procedure is carried out by stirring and less automation, which brings great difficulty in study on ionic exchange conditions. Although most of the manufacturers employed the sodium citrate–NaCl solution system for the A-50 resin washing and elution conditions,[23,28–30] so far there are no reports about the correlation between different sodium citrate–NaCl washing system for the A-50 resin and components of PCCs. This study, therefore, was to examine the influence of different sodium citrate–NaCl washing conditions on the effective components (coagulation factors, FII, FVII, FIX, and FX, and inhibitor proteins PC, PS, PZ, and AT) in PCCs, and to obtain the effect regularity and tendencies to further improve PCCs. EXPERIMENTAL Materials Fresh and citrate-stabilized human plasma was obtained from Guanghan plasma apheresis station (Deyang, China) and stored at 60 C until use. The coagulation factor clotting activity kits (FII, VII, IX, and X) were obtained from Chengdu Union (Chengdu, China). NaCl, sodium citrate, and glycine were purchased from Xilong Chemical (Guangzhou, China), Na heparin was from Sunshine Biotech (Nanjing, China), and protein assay reagent was from CWBiotech (Beijing, China). DEAE-Sephadex A-50 was purchased from GE (Uppsala, Sweden), protamine sulfate, sodium dodecyl sulfate, b-mercaptoethanol, and 30% acrylamide=bis solution (29=1) for the electrophoresis were all from Sigma (St. Louis, MO), Pageruler plus prestained protein ladder (protein weight marker) from Fermentas (Glen Burnie, MD), and both Tris and Coomassie brilliant blue R-250 from Amresco (Solon, OH). The PZ kit, PC activator, S2166, calibrator plasma, normal plasma, and abnormal plasma were all purchased from HBM (Andre´sy, France). The AT kit and PS Ac kit were from ADI (Stamford, CT) and Siemens (Tarrytown, NY), respectively. Washing Conditions for Purifying PCCs Three variables (sodium citrate, NaCl, and pH) and three levels (0.01, 0.015, 0.02 mol=L of sodium citrate, 0.10, 0.15, 0.20 mol=L of NaCl, and 6.8, 7.0, 7.2 for pH) were chosen as the washing conditions for PCCs purification. An orthogonal L9(3)3 test was designed and nine kinds of washing conditions were prepared, shown in Table 1. The levels of sodium citrate, NaCl, and pH in washing conditions for PCCs were determined according
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Washing Conditions and Components of PCCs TABLE 1 L9 (3)3 Orthogonal Test Design (Three Variables and Three Levels) Variables and Levels Group Number
(A) Sodium Citrate (mol=L)
(B) NaCl (mol=L)
(C) pH
0.01 0.01 0.01 0.015 0.015 0.015 0.02 0.02 0.02
0.10 0.15 0.20 0.10 0.15 0.20 0.10 0.15 0.20
6.80 7.00 7.20 7.00 7.20 6.80 7.20 6.80 7.00
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1 2 3 4 5 6 7 8 9
to the previous research[29,31] and the present production technologies for PCCs in China.[32] Preparation of PCCs The purification steps have been described before.[29,32] The plasma was thawed at 0–2 C, then centrifugated to remove the cryoprecipitate. The cryo-poor plasma was recovered the starting material. To the cryo-poor plasma was added heparin of 0.2 IU=mL, and this was stored at 4 C for less than 4 hr before use. The A-50 resin powder was allowed to swell and equilibrate in sodium citrate buffer (0.02 mol=L, pH 6.8) with 0.05 mol=L NaCl and decanted 6–8 times until pH of the supernatant was approximately 6.8. The equilibrated A-50 resin, in the proportion of 1.67 g resin powder to 1 L plasma, was mixed with the cryo-poor plasma containing heparin to adsorb PCCs for 50 min, at 4 C. After that, the A-50 resin was washed two times with the corresponding washing condition listed in Table 1. At last, PCCs were eluted two times with sodium citrate buffer (0.015 mol=L, pH 6.9) containing 2 mol=L NaCl. Nine test groups for preparation of PCCs were performed simultaneously according to the preceding methods and repeated three times. Determination of Effective Components in PCCs Coagulation Factors Potency The potency of FII, VII, IX, or X was determined using an automatic coagulometer (CA1500, Sysmex, Japan) with a one-stage clotting assay.[33] The fresh PCCs were diluted 15-fold with ultrapure water for further measure, and to eliminate interference of high content of salt ion on determination of coagulation factor activity.
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The calibrator plasma was used to create calibration curve and the normal plasma was used to control measure. FII, VII, or X was measured by using PT reagent and respective corresponding factor-deficient substrate plasma.[7] In this assay, 50 ml sample was mixed with 50 ml factor-deficient plasma, and then incubated for 180 s at 37 C. The coagulation reaction was initiated by adding 100 ml PT reagent. FIX was determined by APPT reagent and FIX-deficient plasma.[7] Heparin in cryo-poor plasma was neutralized by protamin sulfate. For this assay, 50 ml sample was mixed with 50 ml deficient plasma, and incubated for 60 s at 37 C, and then 50 ml APPT was added and incubated for another 240 s at 37 C. The coagulation reaction was initiated after the addition of 50 ml CaCl2(0.025 mol=L).
Coagulation Inhibitors Activity or Antigen PCCs samples analyzed for coagulation inhibitors were stored at 4 C less than 2 hr and diluted to at least 1=10. The activity of PC and AT was measured by chromogenic assay, as described previously.[34,35] The activity assay for PS was performed by a coagulation method.[36,37] The assays were all standardized with calibrator plasma and controlled by the normal and abnormal plasma. A one-step immunoassay was performed to measure PZ antigen. PC activity was measured by PC activator (prota C) and activated PC substrate (S2166).[34] The 50 ml prediluted sample and 50 ml of 0.05 IU= mL prota C were mixed and incubated in water bath at 37 C for 9 min, and then 50 ml of 1.0 mmol=mL S2166 was added and uniformly mixed. The color reaction was stopped by 50% acetic acid (v=v) followed 10 min of incubation at 37 C, and then the adsorption was immediately measured at optical density (OD) 405 nm using microplate readers (SpectraMax M2e, Molecular Devices, USA). The assay for AT activity was carried out with the AT kit and the assay process included two stages.[35] First, 50 ml sample diluted by dilution buffer was added to a microplate and incubated at 37 C for 3 min, and then 50 ml Thrombin reagent (thrombin) was introduced and continuously incubated for 1 min. Second, 50 ml Spectrozyme TH (thrombin substrate) was added, mixed, and incubated for 1 min at 37 C. At last, the reaction was quenched by acetic acid. The rate of change of absorbance at OD 405 nm was detected. PS activity was measured by the PS Ac kit on the automatic coagulometer. In this analysis, first, 16 ml sample was mixed with 95 ml PS-deficient plasma. Second, the 16-ml resulting mixture was added to 47 ml PS-deficient plasma and then the whole system was incubated at 37 C for 20 s. Third, 58 ml APC Reagent (activated protein C) was added and incubated for
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110 s at 37 C. At last, the coagulation time, which was directly proportional to PS activity, was determined after addition of 145 ml Starting Reagent (activator). PZ antigen was assayed using the protein Z kit. The specific polyclonal antibody coupled with horseradish peroxidase was used to capture PZ. The concentration of PZ was detected according to the amount of color developed by hydrogen peroxide and peroxidase substrate. This assay was carried out in accordance with the manufacturer instructions. Total Protein Content The total protein content of PCCs was measured using the protein assay reagent according to the Bradford method[38] and the assay was performed according to manufacturer’s instructions. Yield and Purity of Coagulation Factors and Inhibitors The yield of coagulation factor or inhibitor was calculated based on Eq. (1), Yield% ¼
V p Ua 100% VC UA
ð1Þ
where Vp (mL) is PCCs volume, Ua (IU=mL, mg=mL) is activity or antigen content of coagulation factor or inhibitor per milliliter in PCCs, VC (mL) is cryo-poor plasma volume, and UA (IU=mL, mg=mL) is activity or antigen content of coagulation factor or inhibitor per milliliter in cryo-poor plasma. The kind of coagulation factor or inhibitor of Ua is the same as UA . The purity was calculated using Eq. (2), Purity ðIU=mg; mg=mgÞ ¼
Ua Cp
ð2Þ
where Ua (IU=mL, mg=mL) is activity or antigen content of coagulation factor or inhibitor per milliliter in PCCs, and Cp (mg=mL) is total protein content of PCCs per milliliter. Statistical and Orthogonal Test Analysis The experiment results were presented as the mean standard deviation (SD). One-way analysis of variance (ANOVA) was used to compare the differences in the yield, purity, and balance of coagulation factors or inhibitors among nine test groups, respectively. The sum of the yield or purity of FII, VII, IX, and X represented the coagulation factors yield or purity of PCCs in each test group, and the sum of the yield or purity of
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PC, PS, PZ, and AT represented the coagulation inhibitors yield or purity of PCCs. The SD of the purity between four factors represented the factors balance in each test group, and the SD of the purity between four inhibitors represented the inhibitors balance. SPSS software was used for these statistical analyses (Version 17.0, SPSS, Inc., USA). Differences are considered statistically significant at p < 0.05. Furthermore, under different washing conditions, the yield, purity, and balance of coagulation factors or inhibitors in PCCs were analyzed by the direct comparison method of orthogonal test.[39] The influence of three variables and their three levels on yield and purity of the factors or inhibitors in PCCs was analyzed by the heuristics analysis method of orthogonal test.[39] SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), using 4% stacking gel and 10% running gel in a Tris–glycine system, was performed to analyze PCCs. The sample was prepared as follows: 200 ml PCCs from three repetitions were gently mingled and diluted by ultrapure water to 1 mol=L NaCl concentration; 80 ml diluted sample was added to 20 ml Tris–glycine buffer with 5% (v=v) b-mercaptoethanol (fivefold concentrate), and then the mixture was boiled at 100 C for 5 min. Protein weight marker was also loaded onto the gels for standards. The electrophoresis was carried out at potential difference of 60 V in stacking gel, and then at 120 V in running gel. After electrophoresis, gel was stained with Coomassie brilliant blue R-250 and destained with a methanol=acetic acid mixture. RESULTS AND DISCUSSION Yield and Purity of Coagulation Factors and Inhibitors in Orthogonal Test The yield and purity of coagulation factors and inhibitors, obtained from in L9(3)3 orthogonal test, were shown in Figure 1. The yield and purity of coagulation factors showed significant difference among nine groups (p ¼ 0.004 and p ¼ 0.022, respectively). It was also obvious that for coagulation factors, both yield and purity exhibited periodic variations. The yield, especially for FVII, gradually reduced in every three test groups; however, the purity, except FVII in group 3, gradually increased. Significant difference was found in the yield and purity of four inhibitors among nine groups (p ¼ 0.007 and p ¼ 0.019, respectively), but only PS yield and PZ purity showed periodic variations in which PS yield gradually reduced and PZ purity increased, respectively, in all three groups.
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FIGURE 1 The yield of coagulation factors (A), purity of coagulation factors (B), yield of coagulation inhibitors (C), and purity of coagulation inhibitors (D) in the orthogonal test. These data are mean values and the error bars represent SD.
The total yield of four factors was the highest in group 1, and the maximum purity was in group 9 (Figure 1, A and B). The highest yield of four inhibitors was achieved in group 4 (Figure 1C); however, the highest purity of PC, PS, PZ, and AT was distributed in group 3, group 8, group 9, and group 6, respectively (Figure 1D). The discrepancies in yield and purity of coagulation factors and inhibitors of PCCs prepared by different washing conditions were also revealed. Since FVII has weaker absorption with A-50 resin,[40] FVII activity in A-50-prepared PCCs was usually lower.[7,20,24] However, from Figures 1A and 1B, a different but exciting result was exhibited that the yield of FVII was more than that of FII and X in groups 1, 4, and 7, and that the activity of FVII was relatively higher than that of FII and X in the preceding groups. The same character achieved from groups 1, 4, and 7 was 0.1 mol=L NaCl. Therefore, the activity of FVII, II, and X in PCCs can be improved by controlling NaCl content in the washing condition. In addition, both yield and purity of AT were extremely low and even zero in nine groups (Figure 1, C
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and D). The main reason may be that AT was hardly adsorbed by A-50 resin, so most AT remained in the unbound fraction of cryo-poor plasma,[41] which resulted in the content of AT in the A-50-prepared PCCs being very low in any washing conditions. This may be the reason for lots of PCCs with added extra AT.[26,27]
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Balance of Coagulation Factors and Inhibitors in Orthogonal Test The purity of four factors and inhibitors in PCCs was presented in Figures 1B and 1D, and there were significant differences in four factors balance (p ¼ 0.008) as well as four inhibitors balance (p ¼ 0.03) among nine groups. Moreover, it was clearly shown that both the factors and the inhibitors in groups 1 and 4, though there was little and even no AT, had better balance, and in group 1, the SD between four factors and inhibitors was minimum (0.18 and 0.56, respectively), and in group 4, was smaller (0.20 and 0.65 respectively). Meanwhile, the worst balance was in group 9, although it had the highest purity of four factors and PZ, in which the SD between four factors and inhibitors was maximum (0.66 and 1.62, respectively). So the washing conditions caused the great discrepancy in balance of coagulation factors and inhibitors, respectively. The lower levels TABLE 2
Results Analysis of L9 (3)3 Orthogonal Test for Four Coagulation Factors Yield
Coagulation Factor FII
FVII
FIX
FX
k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R
Purity
A
B
C
A
B
C
60.06 0.10a 60.57 1.64 57.41 0.67 3.16b 58.72 3.70 55.04 3.74 49.65 2.57 9.07 74.03 1.43 69.16 2.22 61.32 1.64 12.71 58.08 1.28 55.35 2.23 48.12 1.51 9.96
64.27 0.60 59.46 0.79 54.30 0.58 9.97 71.32 1.47 53.21 1.16 38.88 0.51 32.44 76.06 1.43 68.91 1.05 59.54 1.74 16.52 60.54 1.34 55.83 0.80 45.18 1.41 15.36
57.94 1.45 59.47 1.26 60.62 0.70 2.68 53.55 4.36 56.35 3.50 53.51 2.28 2.84 67.37 2.67 67.11 2.66 70.04 0.48 2.93 53.24 2.27 53.16 2.51 55.14 0.77 1.98
1.46 0.07 1.62 0.09 2.10 0.14 0.64 0.99 0.01 1.03 0.01 1.29 0.04 0.30 1.08 0.06 1.14 0.05 1.33 0.06 0.25 1.20 0.05 1.33 0.05 1.56 0.07 0.36
1.26 0.04 1.68 0.05 2.24 0.11 0.98 1.04 0.02 1.08 0.02 1.19 0.06 0.15 0.90 0.02 1.17 0.03 1.47 0.03 0.57 1.09 0.02 1.34 0.04 1.65 0.05 0.56
1.71 0.11c 1.86 0.18 1.61 0.03 0.25d 1.07 0.02 1.19 0.06 1.05 0.02 0.14 1.18 0.06 1.18 0.08 1.19 0.04 0.01 1.36 0.07 1.38 0.10 1.35 0.03 0.03
ki ¼ R3i SD, where Ri is the amount of the yield at Ai. R ¼ maxfkiA g minfkiA g, and the SD is not involved in the calculation of R value. c C ki ¼ R3i SD, where Ri is the amount of the purity at Ci. d R ¼ maxfkiC g minfkiC g, and the SD is not involved in the calculation of R value. a A b
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of NaCl and sodium citrate were in groups 1 and 4, so it was concluded that the lower content of NaCl and sodium citrate had the possibility to produce a contribution to the better balance of effective components in PCCs.
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Influence of Washing Conditions on Four Coagulation Factors The orthogonal test results of four factors in PCCs were analyzed and are listed in Table 2. Obviously, according to R values the same influence order of three variables on the yield and purity of four factors was B > A > C, except that the influence order on FVII purity was A > B > C. Thus, the minimum effect on yield and purity was from pH (C), and NaCl (B) was the most important determinant, while sodium citrate (A) was only the primary determinant for FVII purity. The influence of levels of three variables on these factors yield was further investigated and the results are displayed in Figure 2. It was perceived that influence of NaCl on the yield was the same, in which the yield
FIGURE 2 Influence of different levels of sodium citrate, NaCl, and pH on the yield of FII (A), FVII (B), FIX (C), and FX (D). These data are mean values and the error bars represent SD.
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decreased with increase of NaCl content, and that the influence of sodium citrate was not completely consistent. The yield of FVII, IX, and X decreased with increase of sodium citrate; however, that of FII increased when sodium citrate concentrate increased from 0.01 mol=L to 0.015 mol=L, and then decreased when sodium citrate increased to 0.02 mol=L. The influence of pH on yield was very inconsistent. FII yield increased with pH, and FVII yield increased when pH increased from 6.8 to 7.0, and then decreased when pH increased to 7.2. The influence of pH on the yield of FIX and X was completely different from FII and VII, and was just opposite to FVII. In Figure 3, the influence of different levels on four factors purity emerges. Obviously, the influence of levels of NaCl on the factors purity was consistent with that of sodium citrate, in which the purity increased with their concentrations increasing. However, the influence of pH on purity was not wholly uniform. For purity of FII, VII, and X, the influence was consistent, in which the purity increased for pH 6.8–7.0, and then
FIGURE 3 Influence of different levels of sodium citrate, NaCl, and pH on the purity of FII (A), FVII (B), FIX (C), and FX (D). These data are mean values and the error bars represent SD.
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decreased for pH 7.0–7.2 (Figure 3, A, B, and D). As seen from Figure 3C, the effect of pH on FIX purity was not the same as on the other three factors, and FIX purity was a little lower at pH 7.0.
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Influence of Washing Conditions on Four Coagulation Inhibitors The orthogonal test results for four inhibitors of PCCs were analyzed and are listed in Table 3. Based on R value, the influence orders of the three variables on yield of the four inhibitors were not exactly the same. The order for PC yield was B > A > C, as well as for PS, but for PZ, B > C > A, and for AT, A > B > C. The influence orders on purity of PC, PS, PZ, and AT were C > B > A, A > B > C, B > A > C, and A > B > C, respectively. From the preceding, it was demonstrated that NaCl had a stronger effect on the yield of four inhibitors, but for purity, three variables showed different effects. The influence of the levels on yield of four inhibitors is presented in Figure 4. The influence of NaCl on the yield of PC, PS, and PZ exhibited the same tendency, and in the range of 0.1–0.2 mol=L NaCl, the higher the concentrations, the lower is the yield of PC, PS, and PZ. The influence of NaCl on AT yield was different from this. When NaCl was 0.15 mol=L, AT TABLE 3
Results Analysis of L9 (3)3 Orthogonal Test for Four Coagulation Inhibitors Yield
Coagulation Inhibitor PC
PS
PZ
AT
a A
k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R k1 k2 k3 R
A
B
24.59 0.63a 21.80 2.38 13.95 2.12 10.64b 20.43 1.26 21.19 1.89 16.72 1.57 4.47 48.92 0.23 48.15 1.83 44.90 0.45 4.02 0.03 0.02 3.48 0.08 3.13 0.02 3.45
25.00 1.11 22.43 0.86 12.92 2.71 12.08 26.28 0.60 20.81 0.56 11.25 0.68 15.03 50.20 0.80 48.00 0.81 43.82 1.25 6.38 2.27 0.40 2.13 0.38 2.24 0.38 0.14
ki calculated according to Table 2. R calculated according to Table 2. c C ki calculated according to Table 2. d R calculated according to Table 2. b
Purity C
A
16.00 1.34 0.62 0.05 19.30 3.10 0.61 0.03 25.05 0.58 0.47 0.06 9.05 0.15 19.00 1.65 0.46 0.01 18.81 2.11 0.56 0.02 20.58 1.04 0.59 0.02 1.77 0.13 43.46 1.09 2.17 0.15 48.68 1.16 2.29 0.09 49.83 0.46 2.92 0.20 6.37 0.75 2.23 0.40 0.001 0.001 2.24 0.40 0.09 0.007 2.17 0.37 0.10 0.006 0.07 0.10
B
C
0.54 0.03 0.68 0.02 0.47 0.07 0.21 0.57 0.01 0.57 0.03 0.47 0.003 0.10 1.79 0.06 2.37 0.06 3.21 0.14 1.42 0.04 0.008 0.07 0.01 0.09 0.01 0.05
0.51 0.03c 0.46 0.06 0.72 0.30 0.26d 0.54 0.02 0.50 0.01 0.57 0.02 0.07 2.29 0.15 2.63 0.24 2.45 0.09 0.34 0.08 0.01 0.06 0.01 0.05 0.008 0.03
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FIGURE 4 Influence of different levels of sodium citrate, NaCl, and pH on the yield of PC (a), PS (b), PZ (c), and AT (d). These data are mean values and the error bars represent SD.
yield was the minimum. The influence of sodium citrate on PC and PZ did not accord with PS and AT. Within 0.01–0.02 mol=L of sodium citrate, the higher the concentrations, the lower is the yield of PC and PZ; however the yield of PS and AT increased when sodium citrate increased from 0.01 mol=L to 0.015 mol=L, and then decreased when sodium citrate increased to 0.02 mol=L. The influence of pH on yield was not still accordant. In the range of pH 6.8–7.2, the higher pH, the higher was the yield of PC and PZ, but PS yield decreased when pH increased from 6.8 to 7.0, and then increased when pH increased to 7.2. The variation tendency of AT yield with levels of pH was exactly contrary to that of PS. The influence of different levels of three variables on four inhibitors purity is shown in Figure 5. The results display that the NaCl influence on purity of PC and PS was distinctly different from that on purity of PZ and AT. For PC and PS, the purity increased when NaCl increased from 0.1 mol=L to 0.15 mol=L, and then decreased when NaCl increased to 0.2 mol=L; nevertheless, PZ and AT purity constantly increased with NaCl.
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FIGURE 5 Influence of different levels of sodium citrate, NaCl, and pH on the purity of PC (a), PS (b), PZ (c), and AT (d). These data are mean values and the error bars represent SD.
The influence of sodium citrate on PC was different from that on PS, PZ, and AT. For 0.01–0.02 mol=L of sodium citrate, PS, PZ, and AT purity gradually increased with sodium citrate, but PC was just contrary. The influence of pH on purity was as follows: When pH increased from 6.8 to 7.0, PC and PS purity decreased but PZ purity increased, and when pH increased to 7.2, PC and PS purity increased but PZ purity decreased. At pH 6.8–7.2, AT purity decreased with increase of pH.
SDS-PAGE The purity of PCCs in the nine test groups was reflected by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) (Figure 6). The molecular masses of coagulation factors and inhibitors were about 50 and 70 kD, respectively. As shown in Figure 6, there was a regular variation in PCCs from group 1 to group 9. PCCs from groups 3 (lane 4), 6 (lane 7), and 9 (lane 10) were purer than those from their
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FIGURE 6 SDS-PAGE analysis for PCCs in the orthogonal test. Lanes: 1, Protein weight marker; 2, PCCs from group 1; 3, PCCs from group 2; 4, PCCs from group 3; 5, PCCs from group 4; 6, PCCs from group 5; 7, PCCs from group 6; 8, PCCs from group 7; 9, PCCs from group 8; 10, PCCs from group 9. PCCs were separated under reduced conditions (color figure available online).
preceding two groups, respectively, and PCCs from group 9 (lane 10) were the purest. These results displayed by SDS-PAGE were consistent with that in Figure 2B.
CONCLUSION The present research first in the field demonstrated the influence regularity of washing conditions on the yield, purity, and balance of coagulation factors and inhibitors in PCCs. It was found that the different washing conditions produced a significant discrepancy in their yield, purity, and balance. In three variables of sodium citrate, NaCl, and pH, for most coagulation factors and inhibitors, NaCl was the first and main determinant for the yield and purity and for the balance, respectively, and sodium citrate was the first determinant for both the yield of AT and the purity of FVII, PS, and AT, and pH was the first determinant for only the PC purity. In different levels of three variables, the influence of NaCl on the yield and purity was generally consistent with that of sodium citrate, while with the increase of NaCl and sodium citrate, the yield of coagulation factors and inhibitors except FII, PS, and AT decreased, and the purity except PC and PS increased. However, the influence of pH showed an inconsistent tendency. According to the regularity already described, the yield, purity, and balance of coagulation factors and inhibitors can be improved by adjusting washing conditions in different batch procedures. Furthermore, for certain factors or inhibitors, there were some inconsistencies of influence of sodium citrate, NaCl, and pH on the yield or purity, and those can be used to further improve the yield or purity of specific factor or inhibitor.
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ACKNOWLEDGMENTS
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The authors gratefully acknowledge financial support from the Public Welfare Industry of Health of China (200902008), the National High Technology Research and Development Program of China (863 Program; 2012AA021903), the Open Fund (PLN1111) of State Key Laboratory of Oil and Gas Reservoir Geology and Exploitation of Southwest Petroleum University, and the Specialized Research Fund for the Doctoral Program of Higher Education of China (20115121120005).
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