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Actinomycin D inhibits vitamin K induced formation of prothrombin in chicks deficient in vitamin K. The administration of actinomycin in doses which inhibit prothrombin formation also inhibits synthesis in the liver of RNA from adenosine triphosphate as detected with adenine-8-C1(4). The results are consistent with a genetic action of vitamin K in inducing RNA formation for the synthesis of clotting proteins
Infusion of prothrombin complex concentrates into pigs resulted in evidence of disseminated intravascular coagulation manifested by positive fibrin monomer tests, depletion of coagulation factors and platelets, and the presence of fibrin in small blood vessels at autopsy. All of the nine prothrombin complex concentrates were found to be thrombogenic. The response appeared to be dose-related, and the two activated materials were more thrombogenic than the non-activated products. In contrast, a purified factor IX concentrate resulted in minimal transient changes in only 2 of 5 animals tested, and autopsy findings were negative for fibrin deposition in all. Four of these animals received 200 factor IX units/kg, which was twice the dose used for any of the other products. Control animals received human plasma or albumin with no evidence of coagulation changes or fibrin deposition at autopsy. The porcine model is more sensitive than other animal models for detection of the thrombogenic effects of prothrombin complex concentrates and may be useful for testing new products found to be non-thrombogenic in other test procedures.
The crude venom of Oxyuranus scutellatus (Taipan snake) was found to cleave single-chain human factor VII to yield a two-chain molecule indistinguishable from authentic factor VIIa by SDS-polyacrylamide gel electrophoresis. A protease that activates factor VII was purified from this venom by a combination of gel permeation and ion-exchange chromatography. Characterization of the venom factor VII activator revealed its apparent identity with the Oxyuranus scutellatus prothrombin activator. The purified venom prothrombin activator was observed to activate factor VII by limited proteolysis in a reaction that was greatly potentiated by calcium and phospholipids (75% phosphatidyl choline/25% phosphatidylserine). Treatment of the venom protease with 0.8 M NaSCN weakly inhibited its ability to activate factor VII indicating that, in contrast to prothrombin activation, the factor Va-like component of this oligomeric enzyme complex was not essential for the activation of factor VII.
Autoantibodies to prothrombin, first described almost 50 years ago, are paradoxically associated with thrombosis. Described is an unusual case of fatal hypercoagulability in a patient with multiple arterial and venous thromboembolic complications despite intense anticoagulation while being bridged to transplantation with a left ventricular assist device. Serum analysis revealed the presence of prothrombin autoantibodies and high titers of anti-nuclear antibodies, and autopsy revealed pulmonary arteriolar vasculitis. These findings suggest an autoimmune basis for the presence of anti-prothrombin antibodies and the hypercoagulable state observed in the present case.
Hereditary deficiency of prothrombin is a rare autosomal recessive bleeding disorder, with severe bleeding diathesis in homozygotes, but rarely resulting in intracranial haematoma. We describe two infants of consanguineous parents, presenting with acute subdural haematoma. Because such haematomas in infancy are highly indicative of trauma caused by child battering and because the socio-economic status of the family was unstable, there was a suspicion of child battering. However, further investigations revealed a bleeding diathesis due to a prothrombin deficiency. DNA analysis of the prothrombin gene showed homozygosity for a novel mutation, substituting Lys for Glu at codon 7 and resulting in decreased specific clotting activity. We discuss the probability of bleeding diathesis versus child battering in the aetiology of intracranial haematoma.
OBJECTIVE: To evaluate the efficacy and safety of intravenous infusions of an improved prothrombin complex concentrate (PCC) formulation. PATIENTS AND METHODS: Twenty-two adults with haemostatic defects due to severe liver disease (Quick's test 50%), which required rapid haemostasis because of bleeding or before urgent surgery or invasive intervention. Laboratory follow-up, including the response and in-vivo recovery of the substituted coagulation factors II, VII, IX and X and protein C took place before, then 10 min, 30 min and 60 min after PCC substitution. Clinical efficacy (avoidance or cessation of bleeding) was assessed using a scale ranging from 'very good' to 'none'. RESULTS: Patients received a median PCC dose of 25.7 IU/kg. The response of factor IX and protein C was 1.2-1.4 (IU/dl)/(IU/kg), the in-vivo recovery was 49.7-57.4%, and the Quick's test increased from 39% to a maximum of 65%. Levels of activation markers of the coagulation system factor VIIa, prothrombin fragment 1 + 2 and thrombin antithrombin complex (TAT) increased, but without evidence of any thromboembolic events. Clinical efficacy was judged as 'very good' in 76% of patients after the first (n = 21) treatment. There were no changes in serological status regarding transmission of HIV, hepatitis A virus, hepatitis B virus and hepatitis C virus. No PCC-related adverse reactions occurred. CONCLUSIONS: The infusion of pasteurized, nanometre-filtered PCC is an effective, well-tolerated method of correcting prothrombin complex deficiency in patients with severe liver disease with haemorrhage, or before an urgent surgical or invasive diagnostic intervention.
The origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa-factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P. textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at approximately 280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.
The cardiovascular and haematological effects of purified prothrombin activator derived from the venom of the Australian Common Brown Snake (Pseudonaja textilis) were studied in anaesthetised, mechanically ventilated dogs. Severe depression of systemic blood pressure and cardiac output and a rise in central venous pressure were observed. Thrombocytopenia, prolongation of both prothrombin time and activated partial thromboplastin time and a reduction in serum fibrinogen were also observed. All of these observed effects were prevented by the prior administration of heparin--a naturally occurring anticoagulant. We conclude that the prothrombin activator in Pseudonaja textilis venom may cause cardiovascular depression due to myocardial dysfunction secondary to disseminated intravascular coagulation.
The availability of the relevant mutant mouse lines provided an opportunity to test the doctrine that platelet activation and fibrin formation account for the importance of thrombin for hemostasis. Prothrombin-deficient mice that survive to birth exsanguinate in the perinatal period. By contrast, protease-activated receptor 4 (PAR4)-deficient mice, which have platelets that fail to respond to thrombin, survive to adulthood with only a mild bleeding diathesis, and fibrinogen-deficient mice show perinatal bleeding but those that survive this period can have a relatively normal life expectancy. We now report that mice that lacked both PAR4 and fibrinogen exsanguinated at birth like prothrombin-deficient mice. However, while approximately half of prothrombin-deficient embryos die during midgestation, mice lacking both PAR4 and fibrinogen developed normally. At face value, these results suggest that platelet activation and fibrin formation are together sufficient to account for the importance of thrombin for hemostasis but not for its importance for embryonic development.
In this paper we report a method via which enzymatically active products formed during prothrombin activation can be detected by simple photographic means after SDS-gel electrophoresis, blotting onto a nitrocellulose membrane and visualization with the chromogenic substrate, S2238. After amidolytic detection the same nitrocellulose membrane can also be used for immunologic detection of prothrombin activation products, thus allowing a complete description of product formation during prothrombin activation. The detection limit of the so-called "amidoblot" is approximately 3 ng thrombin per gel sample which is comparable to the sensitivity of immunoblotting. It is further shown that the amidoblot technique can also be applied to other coagulation factors for which a suitable chromogenic substrate is available (factor XIIa, kallikrein, factor XIa, factor Xa, plasmin and activated protein C).
Thrombogenicity of prothrombin complex concentrates can be diminished by adding heparin to human citrate plasma, preferably before or during the adsorption of the prothrombin complex. Time, extent, and procedure of the heparin addition are dependent on the conditions of the initial material, the realization of the procedure and the provided application of the plasma supernatant after isolating the prothrombin complex. The procedure involves the use of buffer solutions containing heparin and antithrombin III. An activity of a procoagulatory effect cannot be identified in these preparations.
A fatal myocardial infarction in a 22-year-old man with hemophilia A and a factor VIII inhibitor is described. The catastrophic event occurred while the patient was receiving high doses of unactivated prothrombin complex concentrates. Autopsy examination revealed myocardial hemorrhage with no evidence of coronary artery disease or thrombosis. There also was postmortem evidence of previous myocardial infarctions. This is the fourth documented case of myocardial infarction occurring in a young hemophiliac patient using unactivated prothrombin complex concentrates. It is concluded that utilization of prothrombin complex concentrates in hemophiliac patients must be limited and closely monitored. Therapeutic guidelines are recommended.
PURPOSE: We present the association of a lupus anticoagulant with hypoprothrombinemia in a 5-year-old girl, who presented with ecchymoses and a hematoma. This coagulopathy should be included in the differential of bleeding in the previously healthy children. PATIENTS AND METHODS: Coagulation and immunology laboratory evaluation was performed at the time of presentation with bleeding and 2 months later, after complete clinical recovery. RESULTS: A 5-year-old girl presented with ecchymoses and a hematoma after after an upper respiratory illness. Laboratory evaluation showed prolongation of both the prothrombin time (PT) and activated partial thromboplastin time (aPTT) due to the presence of a strong lupus anticoagulant associated with a decreased level of prothrombin (15 U/dl). Hypocomplementemia was also detected. Bruising resolved spontaneously, and the PT and aPTT gradually normalized. Reevaluation 2 months later showed that the lupus anticoagulant had disappeared and the prothrombin deficiency was markedly improved. CONCLUSIONS: This case demonstrates that transient lupus anticoagulants must be included in the differential for bleeding in young children. Also, in children with lupus anticoagulants, neither the association of hypoprothrombinemia nor the presence of evidence of activation of the immune system appears to predict whether a patient will have or develop systemic lupus erythematosus.
A standardized chromogenic assay of prothrombin (factor II, FII) was compared with a conventional coagulation assay and global tests (Quick prothrombin time, Hepatoquick) in patients on oral anticoagulant therapy. Twenty patients with chronic liver disease were also investigated. Studies of precision showed a coefficient of variation ranging between 2.9 and 4.1% in both normal and anticoagulant samples. Accuracy and variability studies yielded satisfactory results. There was a highly significant positive correlation between chromogenic and coagulation assays, whereas correlation with global coagulation tests was much lower, particularly when normal plasmas were tested. In patients with liver disease, FII assay gave reduced values and appeared at least as sensitive as global coagulation tests in showing the synthetic defect occurring in liver disease. These findings suggest that the chromogenic assay can be employed in the clinical laboratory, even though prospective studies are warranted to establish its predictive value.
Two groups of 15 patients each with disseminated intravascular coagulation in association with septic disease were treated with low-molecular-weight heparin (lmw-heparin) in different dosages (group I: 1.5-5 IE/kg body weight (BW) per hour; group II: 8-15 IE/kg BW). We studied the levels of D-dimer, thrombin-antithrombin III complex (TAT), prothrombin fragments 1 and 2 (PTF), and global tests of coagulation like prothrombin time (PT), activated partial thromboplastin time (PTT), thrombin time (TT) and platelet count, plasminogen activation (PA) and fibrinogen concentration to estimate the success of heparin therapy in the two groups. TT and fibrinogen concentration were not suitable to follow the course of the coagulation disorder, PT, PTT, platelet count progressively PA, D-dimer, TAT, and PTF normalised progressively after heparinisation. However, only the last three parameters were sensitive enough to show different effects of variable dosages of lmw-heparin. D-dimer, TAT, and PTF levels declined in proportion with heparin concentrations, and thus appear to be the most useful parameters for monitoring the therapeutic effect of heparin in septic coagulopathies.
An acute coagulopathy developed in a 49 year old woman with severe liver disease after she received an infusion of prothrombin complex concentrate. The concentrate used in the infusion was subsequently studied by observing the effect of the concentrate on the partial thromboplastin times of various plasmas. The evidence suggests that activated coagulation factors, including activated factor X, were present in the concentrate, and probably played a role in initiating the acute change in the patient's coagulation status. Mechanisms whereby liver disease predisposes toward the development of such a coagulopathy are discussed. It would appear that prothrombin complex concentrates should be used in patients with liver disease only with utmost caution.
This study was undertaken to establish a correlation between prothrombin activation fragment F 1 + 2 and one-stage prothrombin time (PT) ratios in patients receiving oral anticoagulant therapy. One hundred consecutive patients on warfarin treatment were utilized for this study. The patients had received warfarin for not less than four days prior to entry into the study. F 1 + 2 levels and PT ratios were found to be 0.28 +/- 0.24 nM/L (mean +/- SD) ranging from 0.01 to 1.5 nM/L and 1.62 +/- 0.46 (mean +/- SD) ranging from 0.97 to 3.11, respectively. Most patients on oral anticoagulants with PT ratios between 1.2 - 1.7 exhibited decreased concentrations of F 1 + 2. Normal control values of F 1 + 2 were established for this study in 40 healthy individuals; they were 0.40 +/- 0.23 nM/L (median +/- SD) ranging from 0.11 to 1.19 nM/L. Mean plasma levels of F 1 + 2 were significantly lower in the anticoagulated patients as compared to the healthy controls (t = 2.377, p < 0.05). The relationship between F 1 + 2 levels and PT ratios in the 100 anticoagulated patients was analyzed by linear regression. No significant correlation (r = -0.208) was found between F 1 + 2 levels and PT ratios. It is concluded that the degree of reduction in F 1 + 2 levels is not proportional to the intensity of therapy reflected by the PT ratios in anticoagulated patients.
In the present study, human factor X and factor IX were each digested with chymotrypsin, and the Gla-peptide from each protein was purified by QAE-Sephadex chromatography. The effect of each Gla-peptide on the activation of human prothrombin by a complex of factor Xa, phospholipid, and calcium was studied using an amidolytic assay for generated thrombin. Prothrombin activation was half-maximally inhibited by factor X Gla-peptide at a concentration of 0.7 microM. Factor IX Gla-peptide was markedly less inhibitory and inhibited this reaction half-maximally at a concentration of 3.7 microM. Kinetic analyses revealed that the factor X Gla-peptide inhibited this reaction in an apparent competitive manner, whereas the factor IX Gla-peptide yielded an exponential Dixon plot. Heat decarboxylation experiments revealed that 3-4 gamma-carboxyglutamic acid residues are critical for the expression of inhibitory activity in each peptide. These studies indicate that, in spite of their structural homology, the ability of each of these Gla-peptides to act as a prothrombinase inhibitor is markedly different.