EFFECT OF THIOCYANATE ON THE ACTIVITY OF BLOOD CLO?TING FACTOR VIII
(Abstract release date: 05/19/16)
EHA Library. Shurko N. 06/09/16; 135124; PB2224
Dr. Nataliya Shurko
Contributions
Contributions
Abstract
Abstract: PB2224
Type: Publication Only
Background
The transmissions of viral agents have been associated with blood transfusion, particularly with the factor coagulation concentrates (for example, factor VIII, vWF, factor IX and prothrombin complex). The blood clotting concentrates should have not only a high specific activity, but to be viral safety. The safety of the final plasma protein сoncentrates has a paramount importance. The specific steps for the inactivation of the virus have been included in each process to enhance the safety of the products by reducing the virus load during manufacturing. Currently, there are several technologies to help ensure the safety of plasma products: solvent-detergent method, heat treatment, sodium caprylate and low pH, chromatography, nanofiltration and chemical reagents (for example, sodium hydroxide, guanidine chloride, sodium or ammonium thiocyanate) for purification of proteins and sanitizing chromatography systems.
Aims
studies of the effect of ammonium thiocyanate (NH4SCN) on the activity of blood clotting factor VIII (FVIII).
Methods
The commercial concentrate of FVIII («Immunate») was initial raw material for the work (working activity 1.6 IU/ml). Determination of activity of FVIII conducted the one-stage clotting method which is based upon the activated partial thromboplastin time (APTT) and using of congenital severe hemophilia A plasma or artificially prepared of FVIII deficiency plasma as a substrate. To investigate the effect of NH4SCN on the activity of FVIII, to enzyme working solution were added a buffered solutions of different concentrations of reagent. Removing of NH4SCN was performed in stages by ultrafiltration (used the filters Amicon Ultra-0.5 10k; Centrifuge Eppendorf 5702-R).
Results
Virus inactivation by NH4SCN is shown earlier in studies of various scientists. NH4SCN was used as an antiviral agent in the manufacture of factors coagulation from plasma of blood. We conducted the study of this effect on the activity of clotting FVIII, to determine the possibility of its use in the manufacture of investigational factor.Experimental studies were performed in stages: in the reaction mixture was added buffered solutions of various concentrations of NH4SCN and was measured activity of FVIII.As a result of the research we showed that thiocyanate reversibly inhibits the activity of the investigated FVIII (Tabl.1). When it had removed from the reaction mixture, factor activity recovered. Table 1. – Comparative characteristics of FVIII activity depending on the concentration of thiocyanate.
Conclusion
Thiocyanate may be used for antiviral treatment and as the eluting agent of coagulation FVIII in chromatographic process purification.
Session topic: E-poster
Keyword(s): Factor VIII, Hemophilia A, Transfusion
Type: Publication Only
Background
The transmissions of viral agents have been associated with blood transfusion, particularly with the factor coagulation concentrates (for example, factor VIII, vWF, factor IX and prothrombin complex). The blood clotting concentrates should have not only a high specific activity, but to be viral safety. The safety of the final plasma protein сoncentrates has a paramount importance. The specific steps for the inactivation of the virus have been included in each process to enhance the safety of the products by reducing the virus load during manufacturing. Currently, there are several technologies to help ensure the safety of plasma products: solvent-detergent method, heat treatment, sodium caprylate and low pH, chromatography, nanofiltration and chemical reagents (for example, sodium hydroxide, guanidine chloride, sodium or ammonium thiocyanate) for purification of proteins and sanitizing chromatography systems.
Aims
studies of the effect of ammonium thiocyanate (NH4SCN) on the activity of blood clotting factor VIII (FVIII).
Methods
The commercial concentrate of FVIII («Immunate») was initial raw material for the work (working activity 1.6 IU/ml). Determination of activity of FVIII conducted the one-stage clotting method which is based upon the activated partial thromboplastin time (APTT) and using of congenital severe hemophilia A plasma or artificially prepared of FVIII deficiency plasma as a substrate. To investigate the effect of NH4SCN on the activity of FVIII, to enzyme working solution were added a buffered solutions of different concentrations of reagent. Removing of NH4SCN was performed in stages by ultrafiltration (used the filters Amicon Ultra-0.5 10k; Centrifuge Eppendorf 5702-R).
Results
Virus inactivation by NH4SCN is shown earlier in studies of various scientists. NH4SCN was used as an antiviral agent in the manufacture of factors coagulation from plasma of blood. We conducted the study of this effect on the activity of clotting FVIII, to determine the possibility of its use in the manufacture of investigational factor.Experimental studies were performed in stages: in the reaction mixture was added buffered solutions of various concentrations of NH4SCN and was measured activity of FVIII.As a result of the research we showed that thiocyanate reversibly inhibits the activity of the investigated FVIII (Tabl.1). When it had removed from the reaction mixture, factor activity recovered. Table 1. – Comparative characteristics of FVIII activity depending on the concentration of thiocyanate.
| № | Concentration,NH4SCN, М | Time, s | Activity of factor VIII, IU/ml |
| 1 | 0.1230 | > 180 | not determined |
| 2 | 0.0610 | >180 | not determined |
| 3 | 0.0240 | 90 | 0.16 |
| 4 | 0.0122 | 69 | 0.74 |
| 5 | 0.0024 | 63 | 1.24 |
| 6 (control) | 0 | 60 | 1.6 |
Conclusion
Thiocyanate may be used for antiviral treatment and as the eluting agent of coagulation FVIII in chromatographic process purification.
Session topic: E-poster
Keyword(s): Factor VIII, Hemophilia A, Transfusion
Abstract: PB2224
Type: Publication Only
Background
The transmissions of viral agents have been associated with blood transfusion, particularly with the factor coagulation concentrates (for example, factor VIII, vWF, factor IX and prothrombin complex). The blood clotting concentrates should have not only a high specific activity, but to be viral safety. The safety of the final plasma protein сoncentrates has a paramount importance. The specific steps for the inactivation of the virus have been included in each process to enhance the safety of the products by reducing the virus load during manufacturing. Currently, there are several technologies to help ensure the safety of plasma products: solvent-detergent method, heat treatment, sodium caprylate and low pH, chromatography, nanofiltration and chemical reagents (for example, sodium hydroxide, guanidine chloride, sodium or ammonium thiocyanate) for purification of proteins and sanitizing chromatography systems.
Aims
studies of the effect of ammonium thiocyanate (NH4SCN) on the activity of blood clotting factor VIII (FVIII).
Methods
The commercial concentrate of FVIII («Immunate») was initial raw material for the work (working activity 1.6 IU/ml). Determination of activity of FVIII conducted the one-stage clotting method which is based upon the activated partial thromboplastin time (APTT) and using of congenital severe hemophilia A plasma or artificially prepared of FVIII deficiency plasma as a substrate. To investigate the effect of NH4SCN on the activity of FVIII, to enzyme working solution were added a buffered solutions of different concentrations of reagent. Removing of NH4SCN was performed in stages by ultrafiltration (used the filters Amicon Ultra-0.5 10k; Centrifuge Eppendorf 5702-R).
Results
Virus inactivation by NH4SCN is shown earlier in studies of various scientists. NH4SCN was used as an antiviral agent in the manufacture of factors coagulation from plasma of blood. We conducted the study of this effect on the activity of clotting FVIII, to determine the possibility of its use in the manufacture of investigational factor.Experimental studies were performed in stages: in the reaction mixture was added buffered solutions of various concentrations of NH4SCN and was measured activity of FVIII.As a result of the research we showed that thiocyanate reversibly inhibits the activity of the investigated FVIII (Tabl.1). When it had removed from the reaction mixture, factor activity recovered. Table 1. – Comparative characteristics of FVIII activity depending on the concentration of thiocyanate.
Conclusion
Thiocyanate may be used for antiviral treatment and as the eluting agent of coagulation FVIII in chromatographic process purification.
Session topic: E-poster
Keyword(s): Factor VIII, Hemophilia A, Transfusion
Type: Publication Only
Background
The transmissions of viral agents have been associated with blood transfusion, particularly with the factor coagulation concentrates (for example, factor VIII, vWF, factor IX and prothrombin complex). The blood clotting concentrates should have not only a high specific activity, but to be viral safety. The safety of the final plasma protein сoncentrates has a paramount importance. The specific steps for the inactivation of the virus have been included in each process to enhance the safety of the products by reducing the virus load during manufacturing. Currently, there are several technologies to help ensure the safety of plasma products: solvent-detergent method, heat treatment, sodium caprylate and low pH, chromatography, nanofiltration and chemical reagents (for example, sodium hydroxide, guanidine chloride, sodium or ammonium thiocyanate) for purification of proteins and sanitizing chromatography systems.
Aims
studies of the effect of ammonium thiocyanate (NH4SCN) on the activity of blood clotting factor VIII (FVIII).
Methods
The commercial concentrate of FVIII («Immunate») was initial raw material for the work (working activity 1.6 IU/ml). Determination of activity of FVIII conducted the one-stage clotting method which is based upon the activated partial thromboplastin time (APTT) and using of congenital severe hemophilia A plasma or artificially prepared of FVIII deficiency plasma as a substrate. To investigate the effect of NH4SCN on the activity of FVIII, to enzyme working solution were added a buffered solutions of different concentrations of reagent. Removing of NH4SCN was performed in stages by ultrafiltration (used the filters Amicon Ultra-0.5 10k; Centrifuge Eppendorf 5702-R).
Results
Virus inactivation by NH4SCN is shown earlier in studies of various scientists. NH4SCN was used as an antiviral agent in the manufacture of factors coagulation from plasma of blood. We conducted the study of this effect on the activity of clotting FVIII, to determine the possibility of its use in the manufacture of investigational factor.Experimental studies were performed in stages: in the reaction mixture was added buffered solutions of various concentrations of NH4SCN and was measured activity of FVIII.As a result of the research we showed that thiocyanate reversibly inhibits the activity of the investigated FVIII (Tabl.1). When it had removed from the reaction mixture, factor activity recovered. Table 1. – Comparative characteristics of FVIII activity depending on the concentration of thiocyanate.
| № | Concentration,NH4SCN, М | Time, s | Activity of factor VIII, IU/ml |
| 1 | 0.1230 | > 180 | not determined |
| 2 | 0.0610 | >180 | not determined |
| 3 | 0.0240 | 90 | 0.16 |
| 4 | 0.0122 | 69 | 0.74 |
| 5 | 0.0024 | 63 | 1.24 |
| 6 (control) | 0 | 60 | 1.6 |
Conclusion
Thiocyanate may be used for antiviral treatment and as the eluting agent of coagulation FVIII in chromatographic process purification.
Session topic: E-poster
Keyword(s): Factor VIII, Hemophilia A, Transfusion
{{ help_message }}
{{filter}}