A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes
Warfarin is a narrow therapeutic index anticoagulant drug and its use is associated with infrequent but significant adverse bleeding events. The international normalized ratio (INR) is the most commonly used biomarker to monitor and titrate warfarin therapy. However, INR is derived from a functional...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2020-07-01
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| Series: | Clinical and Translational Science |
| Online Access: | https://doi.org/10.1111/cts.12757 |
| _version_ | 1818526443753177088 |
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| author | Abdul Basit Bhagwat Prasad Joanne K. Estergreen Daniel E. Sabath Nathan Alade David L. Veenstra Allan E. Rettie Kenneth E. Thummel |
| author_facet | Abdul Basit Bhagwat Prasad Joanne K. Estergreen Daniel E. Sabath Nathan Alade David L. Veenstra Allan E. Rettie Kenneth E. Thummel |
| author_sort | Abdul Basit |
| collection | DOAJ |
| description | Warfarin is a narrow therapeutic index anticoagulant drug and its use is associated with infrequent but significant adverse bleeding events. The international normalized ratio (INR) is the most commonly used biomarker to monitor and titrate warfarin therapy. However, INR is derived from a functional assay, which determines clotting efficiency at the time of measurement and is susceptible to technical variability. Protein induced by vitamin K antagonist‐II (PIVKA‐II) has been suggested as a biomarker of long‐term vitamin K status, providing mechanistic insights about variation in the functional assay. However, the currently available antibody‐based PIVKA‐II assay does not inform on the position and number of des‐carboxylation sites in prothrombin. The assay presented in this paper provides simultaneous quantification of carboxy and des‐carboxy prothrombin that are essential for monitoring early changes in INR and, thus, serves as the superior tool for managing warfarin therapy. Additionally, this assay permits the quantification of total prothrombin level, which is affected by warfarin treatment. Prothrombin recovery from plasma was 95% and the liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) assay was linear (r2 = 0.98) with a dynamic range of 1–100 µg/mL. The assay interday precision was within 20%. A des‐carboxy peptide of prothrombin (GNLER) was negatively correlated with active prothrombin (Pearson r = 0.99, P < 0.0001), whereas its association was positively linked with INR values (Pearson r = 0.75, P < 0.015). This novel LC‐MS/MS assay for active and inactive prothrombin quantification can be applied to titrate anticoagulant therapy and to monitor the impact of diseases, such as hepatocellular carcinoma on clotting physiology. |
| first_indexed | 2024-12-11T06:23:01Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1752-8054 1752-8062 |
| language | English |
| last_indexed | 2024-12-11T06:23:01Z |
| publishDate | 2020-07-01 |
| publisher | Wiley |
| record_format | Article |
| series | Clinical and Translational Science |
| spelling | doaj.art-606c3ebd3fce494c85b4dd491ba170ea2022-12-22T01:17:46ZengWileyClinical and Translational Science1752-80541752-80622020-07-0113471872610.1111/cts.12757A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced ChangesAbdul Basit0Bhagwat Prasad1Joanne K. Estergreen2Daniel E. Sabath3Nathan Alade4David L. Veenstra5Allan E. Rettie6Kenneth E. Thummel7Department of Pharmaceutics University of Washington Seattle Washington USADepartment of Pharmaceutics University of Washington Seattle Washington USADepartments of Laboratory Medicine and Medicine University of Washington Seattle Washington USADepartments of Laboratory Medicine and Medicine University of Washington Seattle Washington USADepartment of Pharmaceutics University of Washington Seattle Washington USADepartment of Pharmacy University of Washington Seattle Washington USADepartment of Medicinal Chemistry University of Washington Seattle Washington USADepartment of Pharmaceutics University of Washington Seattle Washington USAWarfarin is a narrow therapeutic index anticoagulant drug and its use is associated with infrequent but significant adverse bleeding events. The international normalized ratio (INR) is the most commonly used biomarker to monitor and titrate warfarin therapy. However, INR is derived from a functional assay, which determines clotting efficiency at the time of measurement and is susceptible to technical variability. Protein induced by vitamin K antagonist‐II (PIVKA‐II) has been suggested as a biomarker of long‐term vitamin K status, providing mechanistic insights about variation in the functional assay. However, the currently available antibody‐based PIVKA‐II assay does not inform on the position and number of des‐carboxylation sites in prothrombin. The assay presented in this paper provides simultaneous quantification of carboxy and des‐carboxy prothrombin that are essential for monitoring early changes in INR and, thus, serves as the superior tool for managing warfarin therapy. Additionally, this assay permits the quantification of total prothrombin level, which is affected by warfarin treatment. Prothrombin recovery from plasma was 95% and the liquid chromatography‐tandem mass spectrometry (LC‐MS/MS) assay was linear (r2 = 0.98) with a dynamic range of 1–100 µg/mL. The assay interday precision was within 20%. A des‐carboxy peptide of prothrombin (GNLER) was negatively correlated with active prothrombin (Pearson r = 0.99, P < 0.0001), whereas its association was positively linked with INR values (Pearson r = 0.75, P < 0.015). This novel LC‐MS/MS assay for active and inactive prothrombin quantification can be applied to titrate anticoagulant therapy and to monitor the impact of diseases, such as hepatocellular carcinoma on clotting physiology.https://doi.org/10.1111/cts.12757 |
| spellingShingle | Abdul Basit Bhagwat Prasad Joanne K. Estergreen Daniel E. Sabath Nathan Alade David L. Veenstra Allan E. Rettie Kenneth E. Thummel A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes Clinical and Translational Science |
| title | A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes |
| title_full | A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes |
| title_fullStr | A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes |
| title_full_unstemmed | A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes |
| title_short | A Novel LC‐MS/MS Assay for Quantification of Des‐carboxy Prothrombin and Characterization of Warfarin‐Induced Changes |
| title_sort | novel lc ms ms assay for quantification of des carboxy prothrombin and characterization of warfarin induced changes |
| url | https://doi.org/10.1111/cts.12757 |
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