Vitamin K-dependent Factors Research Articles

Biological Rhythms in the Physiology and Pharmacology of Blood Coagulation

This article reviews the current knowledge on time-dependent variations in the physiology of blood coagulation and in the anticoagulant effect of heparin and warfarin. Animal data indicated that the shortest blood clotting time and the highest levels of coagulation factors II, VII, and IX were recorded during the resting period of the animal. These circadian rhythms were not altered by modifications of the lighting regimens. In healthy volunteers, the prothrombin time was longer at the end of the afternoon than early in the morning; the acrophases of activated partial thromboplastin time and thrombin time occurred in the evening or during the night. The acrophases of fibrinogen, factors II, VII, VIII, and a-1-antitrypsin were obtained in the morning. There is no agreement on the chronobiology of platelet aggregation, and differences can be found in the time of maximal aggregability. The chronopharmacological studies of heparin infused at a constant rate to patients with thromboembolic diseases suggested that maximal effectiveness occurred at 04:00, while it was minimal at 08:00. Animal data indicated that oral administration of warfarin at the end of the activity period of rats produced maximal inhibition of vitamin K-dependent factors. This was the time of day when warfarin interference with the vitamin K cycle of the liver was highest. Further studies are needed to determine the clinical significance of biological rhythms in the physiology and pharmacology of blood coagulation.

Thrombin Generation Is Not Increased in the Blood of Hemophilia B Patients After the Infusion of a Purified Factor IX Concentrate

Prothrombin complex concentrates (PCC), licensed for the treatment of hemophilia B, are known to carry a significant risk of thromboembolic complications. Although the reasons for thrombogenicity are not completely understood, several manufacturers have developed purified factor IX concentrates that contain negligible amounts of the other vitamin K-dependent factors. To evaluate whether or not the infusion of such a factor IX concentrate is followed by lesser activation of the hemostatic system than by the infusion of a PCC, we performed a series of coagulation assays on 11 hemophilia B patients before and after the administration of these two types of concentrate using a randomized cross-over design. The levels of prothrombin fragment F1 + 2, a sensitive measure of the in vivo cleavage of prothrombin by factor Xa, was significantly increased in plasma after PCC, but not after factor IX concentrate. Plasma fibrinopeptide A, a sensitive index of the enzymatic activity of thrombin on fibrinogen, also increased significantly after PCC but not after factor IX concentrate. The fragment B beta 15-42, a sensitive index of the enzymatic action of plasmin on fibrin II, did not change after either concentrate. There were also no differences in less sensitive coagulation measurements, such as plasma fibrinogen, antithrombin III, and fibrin monomers, nor in indices of platelet activation, such as beta-thromboglobulin and platelet factor 4. These findings show that the infusion of a purified factor IX concentrate can result in substantially less activation of the coagulation cascade than may be seen with PCC.

Thrombin generation is not increased in the blood of hemophilia B patients after the infusion of a purified factor IX concentrate

Prothrombin complex concentrates (PCC), licensed for the treatment of hemophilia B, are known to carry a significant risk of thromboembolic complications. Although the reasons for thrombogenicity are not completely understood, several manufacturers have developed purified factor IX concentrates that contain negligible amounts of the other vitamin K-dependent factors. To evaluate whether or not the infusion of such a factor IX concentrate is followed by lesser activation of the hemostatic system than by the infusion of a PCC, we performed a series of coagulation assays on 11 hemophilia B patients before and after the administration of these two types of concentrate using a randomized cross-over design. The levels of prothrombin fragment F1 + 2, a sensitive measure of the in vivo cleavage of prothrombin by factor Xa, was significantly increased in plasma after PCC, but not after factor IX concentrate. Plasma fibrinopeptide A, a sensitive index of the enzymatic activity of thrombin on fibrinogen, also increased significantly after PCC but not after factor IX concentrate. The fragment B beta 15–42, a sensitive index of the enzymatic action of plasmin on fibrin II, did not change after either concentrate. There were also no differences in less sensitive coagulation measurements, such as plasma fibrinogen, antithrombin III, and fibrin monomers, nor in indices of platelet activation, such as beta-thromboglobulin and platelet factor 4. These findings show that the infusion of a purified factor IX concentrate can result in substantially less activation of the coagulation cascade than may be seen with PCC.

Covert self poisoning with brodifacoum, a ‘superwarfarin’

The clinical course of a patient poisoned with the 'superwarfarin' brodifacoum and a method for estimation of plasma levels is described. It was characterised by prolonged depression of Vitamin K-dependent clotting factors poorly responsive to Vitamin K administration.

Hereditary deficiency of all vitamin K‐dependent procoagulants and anticoagulants

Hereditary combined deficiency of vitamin K-dependent factors is a rare entity. We report a 7-year-old girl of Arab origin with hereditary deficiency of the procoagulants factors II, VII, IX and X and the natural anticoagulants proteins C and S. The patient is the tenth offspring of a consanguinous marriage and presented at 6 weeks with spontaneous intracerebral haemorrhage. Symptoms improved following plasma infusion. A sibling died at 5 d from uncontrollable umbilical bleeding. Blood coagulation work-up at 6 years showed: factor II:C (activity) 12 U/dl, factor II:Ag (antigen) 40 U/dl; factor VII:C 12 U/dl; factor IX:C 36 U/dl, factor IX:Ag 57 U/dl; factor X:C 17 U/dl, factor X:Ag 54 U/dl; protein C activity 43 U/dl; protein C:Ag 45 U/dl; protein S:Ag 34 U/dl; levels of factors V:C and VIII:C were normal. Assays of coagulation factors in the parents and five of the siblings were within the normal range. Following acute infection and dilantin therapy procoagulant activity levels were reduced further and were partially increased after vitamin K infusion. Crossed immunoelectrophoresis of prothrombin in the presence of calcium lactate revealed a population of des-carboxyprothrombin. Serum vitamin K epoxide levels were undetectable. The data suggest that the defect in our patient stems from abnormal carboxylation of the vitamin K-dependent proteins and that the mode of inheritance is autosomal recessive.

Hepatic Impairment in Fetuses of Preeclamptic Mothers

Cord blood from preeclamptic and normal gestations were analyzed for the vitamin K-dependent proteins, factors II, VII, IX, X, and protein C, and for fibrinogen and albumin. Factor II, factor IX, protein C, and albumin protein levels were reduced in the preeclamptic group, whereas there was no significant change in the fibrinogen or factor X protein levels. The data suggest that these findings are probably due to decreased synthesis and are not indicative of vitamin K deficiency.

Clinical Pharmacokinetic Considerations in the Control of Oral Anticoagulant Therapy

Aspects of the pharmacokinetics of warfarin that are clinically relevant are reviewed here. Since warfarin is normally completely absorbed, resistance to treatment due to impaired absorption is unusual, even in severe short bowel syndrome. Warfarin is highly albumin-bound; thus, hypoalbuminaemic states result in an increased free fraction of the drug and a decreased half-life but, as might be expected, there is no evidence of altered response at steady-state. Warfarin is completely metabolised by the liver to hydroxy-warfarins and warfarin alcohols, and although the latter have some biological activity they do not contribute significantly to the drug effect. No information is available concerning the metabolism of warfarin in chronic liver disease, but there is evidence of increased sensitivity due to impaired vitamin K-dependent clotting factor synthesis. Impaired renal function does not appear to alter the effect of warfarin. Lowered response to the drug may be secondary to poor compliance, kinetic resistance or pharmacodynamic resistance. These factors can be identified using algorithms based on population values for plasma warfarin concentrations and clearances at steady-state. The pharmacokinetics and pharmacodynamics of warfarin indicate that several days' overlap with heparin on initiation of warfarin, and gradual (rather than sudden) discontinuation of warfarin, might theoretically be necessary. However, those studies which have been performed have indicated that a long overlap and gradual discontinuation are not associated with greater safety or efficacy of the drug. Because of the long elimination half-life of warfarin and the short elimination half-life of vitamin K, many days' treatment with phytomenadione may be required after warfarin overdose. The elimination half-life and therefore the duration of therapy may be reduced by regular oral cholestyramine, although the means by which the latter enhances warfarin elimination is still unknown.

Methods for Memorizing Coagulation Interactions

A method for memorizing coagulation enzymatic sequences is presented. The diagrams stress symmetry to simplify memorization. Methods for evaluating coagulation tests, vitamin K-dependentfactors, contact factors, and thrombin-sensitive factors are incorporated into the diagrams.

Studies on anemia of fish. XII. Hemostatic disorder in common carp induced by exposure to the herbicide molinate.

Hemostatic disorders in common carp induced by exposure to the herbicide molinate were investigated. Fish were exposed to molinate at 0.50ppm for 9 days in Experiment 1, and at 0.32ppm for 13 days in Experiment 2. Blood was collected from all specimens at various intervals, and activated partial thromboplastin times (APTT) and prothrombin times (PT) were determined. A prothrombin correction test was applied to the fish exposed to molinate, and the effect of menadione dimethylpyrimidinol bisulfite (MPB) in preventing the prolongation of clotting time was examined. The hemostatic disorders induced by exposing fish to molinate are summarized as follows: 1) Prolongations were observed in both APTT and PT after 6-7 days. Thereafter, the prolongation of PT became more pronounced than that of APTT. 2) The prolongation of PT was corrected by the addition of stored serum, suggesting that depleted coagulation factors exist in the serum. 3) Menadione was highly effective in preventing the prolongation of clotting times. These facts suggest that the hemostatic disorders resulted from a depletion of vitamin K-dependent factors.

Severe Acquired Deficiency of Vitamin K-Dependent Factors and Resistance to Multiple Vitamin K Injections

Severe Acquired Deficiency of Vitamin K-Dependent Factors and Resistance to Multiple Vitamin K Injections

Extrinsic-pathway enzyme-linked coagulation assay (EP-ELCA). A clot-based alternative to prothrombin time for measurement of extrinsic pathway factors in plasma.

This solid-phase colorimetric microtiter-plate clotting assay is much more sensitive than standard clotting tests. In enzyme-linked coagulation assay (ELCA), enzyme-labeled fibrinogen and solid-phase fibrinogen are the substrate for thrombin generated in the clotting cascade. We used this assay to measure the factors of the extrinsic pathway by an extrinsic pathway-specific assay (EP-ELCA) and to determine the individual factors of the extrinsic pathway (VII, X, V, II) in plasmas of coumadin-treated and heparin-treated patients, with prothrombin time (PT) values used as a reference. In the ELCA method, samples and controls are incubated on the same plate, eliminating the requirement for pre-standardization of the substrate "plasma" before the factor assay is done. Concentrations of factors are determined by serially diluting sample and control plasmas to yield equivalent activity at given dilutions, a more direct approach for measuring specific factors than determining log concentrations vs log clotting time. Changes in the concentrations of clotting factors are seen before changes are apparent by PT. For coumadin-treated patients, all vitamin K-dependent factors were significantly (P less than or equal to 0.001) less than in normal controls, whereas factor V concentrations were normal, as expected. For patients treated with heparin, concentrations of factors X and VII were less than in normal controls (P less than 0.01) and results for EP-ELCA, II, and V assays were normal. This methodology can readily be automated.

The Frequency of Type I Heterozygous Protein S and Protein C Deficiency in 141 Unrelated Young Patients with Venous Thrombosis

The frequency of heterozygous protein C and protein S deficiency, detected by measuring total plasma antigen, in a group (n = 141) of young unrelated patients (less than 45 years old) with venous thrombotic disease was studied and compared to that of antithrombin III, fibrinogen, and plasminogen deficiencies. Among 91 patients not receiving oral anticoagulants, six had low protein S antigen levels and one had a low protein C antigen level. Among 50 patients receiving oral anticoagulant therapy, abnormally low ratios of protein S or C to other vitamin K-dependent factors were presented by one patient for protein S and five for protein C. Thus, heterozygous Type I protein S deficiency appeared in seven of 141 patients (5%) and heterozygous Type I protein C deficiency in six of 141 patients (4%). Eleven of thirteen deficient patients had recurrent venous thrombosis. In this group of 141 patients, 1% had an identifiable fibrinogen abnormality, 2% a plasminogen abnormality, and 3% an antithrombin III deficiency. Thus, among the known plasma protein deficiencies associated with venous thrombosis, protein S and protein C deficiencies (9%) emerge as the leading identifiable associated abnormalities.

Effect of Oral Aspirin on Plasma Levels of Vitamin K-Dependent Clotting Factors – Studies in Healthy Volunteers

Effect of Oral Aspirin on Plasma Levels of Vitamin K-Dependent Clotting Factors – Studies in Healthy Volunteers

Abnormalities of blood coagulation tests in patients with cancer.

Routine blood coagulation tests were performed on 431 consecutive patients enrolled in a study of the role of anticoagulation in cancer treatment (VA Cooperative Study #75). Two hundred sixteen control patients were treated with standard therapy, and 215 patients were treated with standard therapy plus sodium warfarin. At the time of entry into the study, the most common abnormalities were elevated fibrinogen levels, platelet counts, and fibrinopeptide A levels. Serial studies demonstrated a steady increase in platelet count and fibrinogen levels before death. Anticoagulation lowered FPA levels but had no significant effect on fibrinogen levels, platelet counts, or euglobulin clot lysis times. An unexpected finding was a dramatic increase in fibrin split product levels after institution of anticoagulation (means +/- SEM = 42.6 +/- 116.4 vs. 2.9 +/- 7.0 mg/L in control subjects; P less than 0.02). This study supports the presence of subclinical activation of blood coagulation in most patients with cancer. Moreover, the preferential activation of fibrinolysis in anticoagulated patients suggests a role for a vitamin K-dependent factor(s) in the regulation of fibrinolysis in patients with cancer.

Decrease in blood coagulation factors II (prothrombin), VII, IX and X in the rat after a single oral dose of butylated hydroxytoluene

Male Sprague-Dawley rats were given butylated hydroxytoluene (BHT) in a dose of 800 mg/kg body weight orally, and 0.5–72 hr later plasma concentrations of factors II, VII, IX and X and hepatic levels of BHT and BHT quinone methide (2,6-di- tert-butyl-4-methylene-2,5-cyclohexadienone) were determined. Levels of factors II, VII, X and IX were reduced 36–60 hr after BHT treatment, but by 72 hr, those most affected (VII and IX) showed some recovery and X had returned to normal. Hepatic levels of BHT reached a maximum 3 hr (a major peak) and 24 hr after BHT dosing and BHT quinone methide reached a maximum at 6 and 24 hr (a major peak). In rats given BHT orally in doses of 200, 400 and 800 mg/kg, factors II, VII and X decreased after 48 hr only in rats given the highest dosage, but factor IX was more susceptible to BHT and showed a dose-dependent decrease. Phylloquinone (1 mg/rat) injected ip 24 hr after the administration of 800 mg BHT/kg maintained normal levels of factors VII and X and an almost normal level of factor IX, but had little effect on the level of factor II. In studies of the effects of drug-metabolizing-enzyme modifiers, neither ip pretreatment with 75 mg phenobarbital sodium/kg for 3 days nor the feeding of 1% cysteine in the diet throughout the experiment prevented the decrease in vitamin-K-dependent factors by 800 mg BHT/kg, but 2-day ip pretreatment with 60 mg cobaltous chloride/kg/day maintained normal levels of factors II and VII and reduced the BHT effect on factors IX and X. SKF 525A (50 mg/kg) injected ip either 30 min before or 12 hr after BHT treatment partially prevented the decrease in factors II, VII and X, or in all four factors, respectively. Thus the decrease in vitamin K-dependent factors may be the same with a single oral dose of BHT as with dietary BHT, and the anticoagulant effect may require the metabolic activation of BHT.

The biochemical basis of warfarin therapy.

Vitamin K is required for a liver microsomal enzyme that catalyzes the posttranslational conversion of specific glutamyl residues in precursors of the vitamin K-dependent clotting factors to gamma-carboxyglutamyl residues in the plasma form of these proteins. A second product of this carboxylation reaction is the 2,3-epoxide of the vitamin. The anticoagulant warfarin blocks the enzyme which reduces this epoxide to vitamin K quinone, and also blocks one of the microsomal pathways which converts the quinone to the active coenzyme form of the vitamin, the hydroquinone. Warfarin anticoagulation therefore reduces the activity of the vitamin K-dependent carboxylase and results in the secretion of vitamin K-dependent clotting factors that are under carboxylated. The presence of gamma-carboxyglutamyl residues is essential for the normal Ca++/phospholipid-mediated activation of prothrombin, and the des-gamma-carboxy forms of prothrombin secreted by patients receiving warfarin therapy lack biological activity and have a reduced thrombotic potential.

Recurrent Coumarin-Induced Skin Necrosis in a Patient With an Acquired Functional Protein C Deficiency

An elderly woman who had been receiving long-term oral anticoagulant therapy developed skin and subcutaneous fat necrosis on five repeated occasions of extreme hypocoagulability, associated with coinciding periods of congestive cardiac failure. In each episode, the skin necrosis developed within days after the prothrombin time (as determined with Thrombotest) exceeded 200 s (International Normalized Ratio greater than 5.4). Widespread thrombosis in the subcutaneous vasculature and interstitial bleeding, as observed in a skin biopsy specimen, were consistent with a diagnosis of coumarin necrosis. On two occasions, an acquired functional protein C deficiency was present. It is hypothesized that an imbalance between anticoagulant and procoagulant vitamin K-dependent factors contributed to the pathogenesis of coumarin-induced skin necrosis. This imbalance was related to repeated periods of congestive heart failure.

The Effect of Calcium Ions on the Activated Partial Thromboplastin Time of Heparinized Plasma

The effect of calcium chloride (CaCl2) on the activated partial thromboplastin time (aPTT) of heparinized plasma was studied. The aPTT ratio (heparinized plasma:control plasma) increased as the CaCl2 concentration to recalcify the plasma was increased from 15 to 35 mmol/L CaCl2. Platelet-poor plasma from patients receiving intravenous heparin, and in vitro heparinized plasmas from either coumarinized patients or plasma depleted of the vitamin K-dependent factors, displayed the calcium-dependent increase in the aPTT ratio. The magnitude of the calcium-dependent change in the aPTT ratio was similar for the three partial thromboplastins studied. Heparinized blood collected in 3.2% and 3.8% citrate demonstrated the calcium-dependent increase in the aPTT ratio. The authors have also studied the effect of the divalent cations (Ca+2, Mg+2, Zn+2, and Sr+2) on the anti-Factor Xa activity of heparin to determine whether the calcium-dependent increase in the aPTT was due to an increase in the anti-Factor Xa activity. The anti-Factor Xa activity of heparin was measured using chromogenic substrate S-2251, purified Factor Xa, and excess antithrombin III. The anti-Factor Xa activity of heparinized plasma increased 2.4-2.8-fold as the divalent cation concentration was increased from 0-5 mmol/L. Similar results were obtained using purified bovine Factor Xa, antithrombin III, and heparin in the absence of plasma. These results suggest that divalent cations play an important role in modulating heparin's anticoagulant activity in vitro. In addition, the CaCl2 concentration used to recalcify plasma is an important variable that modifies heparin sensitivity of the aPTT. Furthermore, divalent cations play an important role in regulating the anti-Factor Xa activity of heparin in vitro.

Haemophilia B Leyden in Greece

In this paper, a five generation Greek family is described with haemophilia B. The disease is characterized by a normal ox-brain prothrombin time, normal levels of the vitamin-K dependent clotting factors VII and X and a proportional reduction of factor IX activity and antigen levels all of which is consistent with the cross-reacting material negative form of haemophilia B. However, in this family the factor IX levels in the three patients of generation V are around 1 U/dl while the three older patients in generation III have factor IX levels ranging from 28 to 44 U/dl. In the oldest patient of generation V we observed a rise of the factor IX level from 1 U/dl up to the age of 13 to 10 U/dl at age 14. In addition, the older patients have very mild bleeding symptoms or none at all, while the young ones have occasional spontaneous haemorrhages in muscles and joints, compatible with severe or moderately severe haemophilia. The disease appears to be similar to haemophilia B Leyden which has been described in a Dutch family.

Immunoradiometric Assays for Human Coagulation Factor VII Using Polyclonal Antibodies Against the Ca(II)-Dependent and Ca(II)-Independent Conformation

Human coagulation factor VII is a trace plasma protein belonging to the vitamin K-dependent factors. Two specific and sensitive immunoradiometric assays for factor VII were developed using immunopurified rabbit antibodies against the Ca(II)-independent and Ca(II)-dependent conformation of factor VII. Both assays were insensitive to the activation state of factor VII. The distribution of factor VII antigen was studied in 40 healthy individuals and the antigen level in normal plasma was calculated to be 0.52-0.62 micrograms/ml. The two assays were used in a comparative study of factor VII procoagulant activity and factor VII antigen in patients treated with oral anticoagulants.