Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties
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2023
Authors
Dunjić Manevski, SofijaCumbo, Marija
Pruner, Iva
Gvozdenov, Maja
Tomić, Branko
Taxiarchis, Apostolos
Antović, Jovan
Đordjević, Valentina
Article (Published version)
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Introduction Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. Methods Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) t...o determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. Results No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. Conclusions Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.
Keywords:
fibrin / microscopy / mutation / prothrombin / thrombosisSource:
International Journal of Laboratory Hematology, 2023, n/aFunding / projects:
- Ministry of Science, Technological Development and Innovation of the Republic of Serbia, institutional funding - 200042 (University of Belgrade, Institute of Molecular Genetics and Genetic Engineering) (RS-MESTD-inst-2020-200042)
- addition, this study was a part of ERASMUS+ KA1 exchange
URI
https://onlinelibrary.wiley.com/doi/abs/10.1111/ijlh.14195https://imagine.imgge.bg.ac.rs/handle/123456789/2199
Institution/Community
Institut za molekularnu genetiku i genetičko inženjerstvoTY - JOUR AU - Dunjić Manevski, Sofija AU - Cumbo, Marija AU - Pruner, Iva AU - Gvozdenov, Maja AU - Tomić, Branko AU - Taxiarchis, Apostolos AU - Antović, Jovan AU - Đordjević, Valentina PY - 2023 UR - https://onlinelibrary.wiley.com/doi/abs/10.1111/ijlh.14195 UR - https://imagine.imgge.bg.ac.rs/handle/123456789/2199 AB - Introduction Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. Methods Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. Results No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. Conclusions Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture. T2 - International Journal of Laboratory Hematology T1 - Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties VL - n/a DO - 10.1111/ijlh.14195 ER -
@article{ author = "Dunjić Manevski, Sofija and Cumbo, Marija and Pruner, Iva and Gvozdenov, Maja and Tomić, Branko and Taxiarchis, Apostolos and Antović, Jovan and Đordjević, Valentina", year = "2023", abstract = "Introduction Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. Methods Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. Results No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. Conclusions Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.", journal = "International Journal of Laboratory Hematology", title = "Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties", volume = "n/a", doi = "10.1111/ijlh.14195" }
Dunjić Manevski, S., Cumbo, M., Pruner, I., Gvozdenov, M., Tomić, B., Taxiarchis, A., Antović, J.,& Đordjević, V.. (2023). Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties. in International Journal of Laboratory Hematology, n/a. https://doi.org/10.1111/ijlh.14195
Dunjić Manevski S, Cumbo M, Pruner I, Gvozdenov M, Tomić B, Taxiarchis A, Antović J, Đordjević V. Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties. in International Journal of Laboratory Hematology. 2023;n/a. doi:10.1111/ijlh.14195 .
Dunjić Manevski, Sofija, Cumbo, Marija, Pruner, Iva, Gvozdenov, Maja, Tomić, Branko, Taxiarchis, Apostolos, Antović, Jovan, Đordjević, Valentina, "Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties" in International Journal of Laboratory Hematology, n/a (2023), https://doi.org/10.1111/ijlh.14195 . .