Inactive Prothrombin Research Articles

Measuring serum prothrombin in patients treated with warfarin or direct oral anticoagulants

Warfarin therapy influences generation of γ-carboxyglutamyl (Gla) residues in prothrombin, causing reduced coagulation activity. It will leave such inactive prothrombin in serum after clot formation, resulting in serum prothrombin constituting total inactive prothrombin in these patients. An ELISA was developed to measure biologically inactive prothrombin in serum, and applied to serum from warfarin therapy causing a decrease in Gla residues or direct oral anticoagulant (DOAC) therapy as its contrast. The concentrations of serum prothrombin in both the warfarin and DOAC groups were higher than those in the healthy group (p<0.01 and p<0.001, respectively). When serum in the previous three groups was treated with barium carbonate to exclude prothrombin, which lost several Gla residues, the prothrombin concentration in the DOAC group decreased to the same level as that in the healthy group, indicating that prothrombin was obtained at a high level only in the warfarin group (p<0.01). Warfarin and DOAC led to increase in serum prothrombin concentration. The reason is that DOAC decreases prothrombin recruitment during fibrin clot formation, while warfarin leads to the accumulation of inactive prothrombin, which have a decreased number of Gla residues.

RELATIONSHIP BETWEEN PRE- AND POST-THROMBOSIS FACTORS IN PATIENTS WITH STAGE VD CKD TREATED BY LONG-TERM HEMODIALYSIS

The aim: To determine informative value of pre-thrombosis, post-thrombosis and anticoagulation factors as well as their correlations for assessment of hemostasis status in patients with stage VD CKD. Materials and methods: Potential predictors of thrombophilia development as well as their relationships depending on the level of molecular markers of hemostasis were studied in 88 patients with stage VD CKD undergoing long-term hemodialysis with the view to determine their informative value. Results: Accumulation of soluble fibrin (sF) was demonstrated to cause moderate reaction of D-dimer (D-d) being insufficient in the absence of reaction of anticoagulant component of hemostasis. Soluble fibrin levels were found to be associated with D-d concentration (r = 0.39) and functionally inactive prothrombin forms (FIPF) to some extent (r = -0.24). Accumulation of FIPF in individuals with high level of sF implies significant activation of blood coagulation system at the stage prior to thrombin formation. Absence of close relationship between pre- and post-thrombosis indices may be indicative of still preserved potential of anticoagulant component of hemostasis. Conclusions: Accumulation of FIPF is an early marker of activation of blood coagulation and possible thrombosis. Levels of sF correlate with pre-thrombosis (fibrinogen, FIPF) and post-thrombosis (D-d) factors being associated with inhibition of anticoagulation processes. Comprehensive study of basic components of hemostasis in patients with VD stage of chronic kidney disease offer broader opportunities in arranging prophylactic measures to prevent thrombophilia.

High Electrochemical Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction

Propagating cascade reactions based on two enzymes have been used to achieve high signal amplification in biosensors wherein one enzyme continually triggers the activation of the other enzyme that produces signaling species. However, in many cases, biosensors that use cascade reactions are not sensitive enough to allow low detection limits because of the inherent slowness of proteolytic reactions. This paper reports a sensitive electrochemical immunosensor using a high-signal-amplification method that combines a propagating cascade reaction and a redox cycling reaction. The cascade reaction uses ecarin and prothrombin: the ecarin label proteolytically converts inactive prothrombin into active thrombin, which then proteolytically liberates electroactive p-aminophenol (AP) from an AP-conjugated peptide. The liberated AP is electrochemically oxidized to p-benzoquinone imine (QI), regenerated by the reduction of QI by NADH, and then electrochemically reoxidized. This electrochemical−chemical (EC) redox cycling reaction significantly increases the electrochemical signal. The developed immunosensor is also compared with an immunosensor that uses only a propagating cascade reaction and an immunosensor that uses a single proteolytic reaction and an EC redox cycling reaction. The detection limits for thyroid-stimulating hormone (TSH) obtained using the three immunosensors are 3 pg/mL, 2 ng/mL, and 4 ng/mL, respectively, indicating that the newly developed immunosensor is more sensitive than the others. The measured concentrations of TSH in clinical serum were found to agree well with those determined using a commercial instrument, indicating that the immunosensor is highly promising for sensitive detection of biomolecules.Keyword: Electrochemical biosensor, Cascade reaction, Redox cycling, Proteolytic reaction

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 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 (r 2 = 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.

Combined Signal Amplification Using a Propagating Cascade Reaction and a Redox Cycling Reaction for Sensitive Thyroid-Stimulating Hormone Detection

Propagating cascade reactions based on two proteases are promising for obtaining high signal amplification. However, in many cases, biosensors that use cascade reactions do not have low detection limits because of the inherent slowness of proteolytic reactions. Here, we report a sensitive electrochemical immunosensor using a high-signal-amplification method that combines a propagating cascade reaction and a redox cycling reaction. The cascade reaction uses ecarin and prothrombin: the ecarin label proteolytically converts inactive prothrombin into active thrombin, which then proteolytically liberates electroactive p-aminophenol (AP) from an AP-conjugated peptide. The liberated AP is electrochemically oxidized to p-benzoquinone imine (QI), regenerated by the reduction of QI by NADH, and then electrochemically reoxidized. This electrochemical-chemical (EC) redox cycling reaction significantly increases the electrochemical signal. The developed immunosensor is also compared with an immunosensor that uses only a propagating cascade reaction and an immunosensor that uses a single proteolytic reaction and an EC redox cycling reaction. The detection limits for thyroid-stimulating hormone (TSH) obtained using the three immunosensors are 3 pg/mL, 2 ng/mL, and 4 ng/mL, respectively, indicating that the newly developed immunosensor is more sensitive than the other two. The measured concentrations of TSH in clinical serum are found to agree well with those determined using a commercial instrument.

Outcomes Following Three-Factor Inactive Prothrombin Complex Concentrate Versus Recombinant Activated Factor VII Administration During Cardiac Surgery

To compare outcomes following inactive prothrombin complex concentrate (PCC) or recombinant activated factor VII (rFVIIa) administration during cardiac surgery. Retrospective propensity-matched analysis. Academic tertiary-care center. Patients undergoing cardiac surgery requiring cardiopulmonary bypass who received either rFVIIa or the inactive 3-factor PCC. Outcomes following intraoperative administration of rFVIIa (263) or factor IX complex (72) as rescue therapy to treat bleeding. In the 24 hours after surgery, propensity-matched patients receiving PCC versus rFVIIa had significantly less chest tube outputs (median difference -464 mL, 95% confidence interval [CI] -819 mL to -110 mL), fresh frozen plasma transfusion rates (17% v 38%, p = 0.028), and platelet transfusion rates (26% v 49%, p = 0.027). There were no significant differences between propensity-matched groups in postoperative stroke, deep venous thrombosis, pulmonary embolism, myocardial infarction, or intracardiac thrombus. Postoperative dialysis was significantly less likely in patients administered PCC versus rFVIIa following propensity matching (odds ratio = 0.3, 95% CI 0.1-0.7). No significant difference in 30-day mortality in patients receiving PCC versus rFVIIa was present following propensity matching. Use of rFVIIa versus inactive PCCs was significantly associated with renal failure requiring dialysis and increased postoperative bleeding and transfusions.

Detection of Prethrombin 1 in Human Blood Plasma

Background: Prethrombin 1 is one of prothrombin derivatives which appears in the presence of thrombin in vitro. However, existence of prethrombin 1 in vivo remained questionable. The aim of present work was to detect of prethrombin 1 in vivo at abdominal aortic aneurysm, hip replacement after fracture and stroke. Methodology: Blood plasma samples of patients with abdominal aortic aneurysm, hip replacement after fracture, stroke, and patients which were treated with warfarine were collected. Detection of prethrombin 1 was completed using three independent approaches: combination of prothrombin index and ecamulin (ecarin) index; the APTT-test modified by addition of exogenous prothrombin; Western-blotting of blood plasma using polyclonal antibody to prothrombin. Results: Prethrombin 1 presence in patients’ blood plasma at studied pathologies was proven. Inactive prothrombin (descarboxy-prothrombin), but not prethrombin 1 was found in blood plasma of patients which were treated with warfarine. Developed approach can be used for testing the prethrombin 1 in clinical diagnostics. Conclusion: Prethrombin 1 at different pathologies, accompanied by coagulation disorders was detected in patients’ blood plasma directly.

Differential activation and inhibition of human platelet thrombin receptors by structurally distinct α-, β- and γ-thrombin

The development of drugs to neutralize the action of thrombin has to date focused on the α form of the protease. It is generally agreed that inactive prothrombin is proteolytically converted to active α-thrombin which may be further hydrolyzed to β- and γ-thrombin. While all three forms of the enzyme retain catalytic activities, only α-thrombin is presumed to be physiologically important. The β- and γ-thrombin are presumed to be degradation products of no physiological significance. Our demonstration that β- and γ-thrombin selectively activate PAR-4 in this and a previous report (J. Biol. Chem. 276, 21173–21183, 2001) necessitates a reevaluation of how we view their physiological roles and how we approach the pharmacological regulation of their actions. β-Thrombin, like γ-thrombin, at nM levels selectively activates PAR-4. This was demonstrated by full retention of aggregatory activity with platelets whose PAR-1 and GP Ib receptors were inactivated. Furthermore, the β-thrombin response was abrogated by desensitizing platelets with suboptimal levels of the thrombin receptor activating peptide for PAR-4 (TRAP-4). For β-thrombin and γ-thrombin to have a physiological role, it is necessary to show they can be generated under physiological conditions. We demonstrate, for the first time, that α-thrombin is hydrolyzed in less than 1 min by activated factor X at physiological pH, in vitro. This implies that α-thrombin may be rapidly converted to β-thrombin and/or γ-thrombin in vivo in the proper microenvironment. The differential activation of the three platelet thrombin receptors by α-, β- and γ-thrombin implies selective structural variations between these thrombin species. Structural differences are likely to account for the marked differential responses observed with the antithrombotic, hirudin, which inhibits α-thrombin, is a slightly weaker inhibitor of β- thrombin and a very weak inhibitor of γ-thrombin-induced platelet aggregations. The converse order of inhibition is observed with the physiological protease inhibitor, α1-antitrypsin. Finally, a non-traditional inhibitor, histone-1, selectively inhibits only β- and γ-thrombin, primarily at the receptor level of PAR-4 rather than on the thrombin molecule. Trypsin, like β- and γ-thrombin, activates PAR-4 and is also inactive with TRAP-4 desensitized platelets. Therefore, it was reasoned that trypsin would be more structurally similar to γ-thrombin than to α-thrombin. The analysis of the crystalline structures of α-, γ-thrombin and trypsin from the databases confirm that this is the case. These findings should help to elucidate structure-function relationships of the different thrombins and may aid in the development of new anti-thrombotic drugs.

Intracellular precursor forms of plasma proteins: their functions and possible occurrence in plasma.

An attempt is made to review the steps in the biosynthesis of plasma proteins by the liver and to point out intermediate precursor forms that might escape to the plasma. With the use of immunochemical techniques such as immunofixation electrophoresis it is possible to identify these precursors when they present an unusual bands on protein electrophoresis. The discovery of the role of vitamin K in the formation of the blood-clotting proteins, for example, followed the immunochemical detection of an abnormal form of inactive prothrombin in plasma. The chief types of precursors likely to occur in blood are forms that are incompletely glycosylated, phosphorylated, or sulfated, or which are single peptide chains of multichain proteins. Precursors containing basic propeptides such as proinsulin or proalbumin may also appear. By being alert to the possible appearance of intracellular forms in plasma, the clinical chemist may be able to relate their presence to the nature of the disease process responsible.

The Purification and Properties of an Abnormal Prothrombin Protein Produced by Dicumarol-treated Cows: A COMPARISON TO NORMAL PROTHROMBIN

A physiologically inactive prothrombin protein has been purified from the plasma of dicumarol-treated cows to a high level of purity. This protein gave 80% as much precipitation in an assay against bovine prothrombin antibody as did normal bovine prothrombin. This could be due to a difference in antigenicity, or it could be a reflection of nonhomogeneity of the preparation. It was very similar to normal prothrombin in its carbohydrate and amino acid composition, its ability to be activated by several nonphysiological prothrombin activators, and its molecular weight as judged by gel electrophoresis. Its disc gel electrophoretic pattern also showed little difference from that of normal prothrombin unless calcium was included in the gel buffers. Under these conditions it was apparent that the inactive prothrombin did not bind calcium to the same degree as the normal prothrombin. The other significant differences between these two proteins are their physiological activity and their adsorption onto barium citrate precipitates. In every case the abnormal prothrombin failed to have these properties. It appears that these three differences are due to the same property of prothrombin and that this is a result of vitamin K action. That is, vitamin K action is required in the metabolic step which is needed to produce the calcium-binding sites, and these are necessary for physiological activity and are responsible for barium citrate adsorption. These data suggest that vitamin K acts by chemically altering prothrombin in some unknown fashion, or in the attachment of a previously unrecognized prosthetic group.

Vitamin K and prothrombin formation

Abstract The site of action of vitamin K in the pathway of the formation of active prothrombin has been re-examined. The controversy as to whether the vitamin functions at a transcription, a translation or a later level has been resolved by finding that labeling of prothrombin by labeled amino acids given to vitamin K deficient rats along with a curative dose of vitamin K is (1) no different than the labeling of prothrombin in vitamin K normal untreated rats and (2) no different than the labeling of a protein which appears to be inactive prothrombin present in the plasma of vitamin K deficient rats. These data indicate that the site action of vitamin K is in the formation of active prothrombin from some “unfinished” inactive precursor and not at a level of de novo synthesis.

The Determinant of the Prothrombin Time in Normal Human Plasma

SummaryNormal adult human plasma has a constant prothrombin time when determined under standardized conditions and with carefully prepared reagents. An explanation for this constancy is offered; namely, that a plasma factor, designated as “prothrombin time fixing agent” (PTFA), is responsible for maintaining a constant fraction of the total prothrombin in the active state. This fraction is measured quantitatively by the prothrombin time, while the inactive prothrombin or prothrombinogen, has no influence on the test. The concentration of PTFA is fixed by heredity and appears to be a dominant. The results of studies on two subjects, one with true hypoprothrombinemia, the other with a prolonged prothrombin time but a normal total prothrombin and no deficiency of accessory prothrombin factors are reported and interpreted according to this hypothesis. A deficiency of PTFA can be differentiated from all other known hypoprothrombinemic states by observing a prolonged prothrombin time when mixing the plasma with an equal volume of fresh normal plasma.