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PubMed1987

Abnormal prothrombin in the plasma of rats carrying hepatic tumors.

Vitamin K is required for the posttranslational formation of gamma-carboxyglutamyl residues in a number of plasma clotting factors. Interference with vitamin K action results in the appearance of abnormal (des-gamma-carboxy) forms of prothrombin in human plasma. Vitamin K-sufficient patients with primary hepatocellular carcinoma also secrete significant quantities of abnormal prothrombin; this response has now been studied in a rat model. Normal Buffalo strain rats had 9 micrograms/mL of circulating plasma abnormal prothrombin, whereas Buffalo strain rats carrying the transplantable Morris hepatoma tumor no. 7777 had 33 micrograms/mL at 3 weeks after transplant. Vitamin K-dependent carboxylase activity was normal in the liver of these rats, but very low in the tumor tissue. Rats carrying Morris hepatoma tumors no. 9618A and 5123D did not secrete significant amounts of abnormal prothrombin. Carboxylase activity in these tumors was 15 times that of the 7777 tumor. The data suggest that the secretion of abnormal prothrombin by hepatocellular tumors is the result of normal expression of the prothrombin gene by those tumors and a failure of the tumor to express the carboxylase gene.

Adsorption↗
PubMed1986

Mechanism of the calcium-dependent self-association of bovine prothrombin. Use of a covalent cross-linking reagent to study the reaction.

The present study has made use of a covalent cross-linking agent, dithiobis(succinimidylpropionate), to study the self-association of prothrombin and has demonstrated that the covalent dimerization reaction involves the gamma-carboxyglutamic acid region of prothrombin (1-42 of 582). An essential role for the gamma-carboxyglutamic acid residues of prothrombin in the association reaction was demonstrated by experiments that converted gamma-carboxyglutamic acid residues to gamma-methylene glutamic acid or glutamic acid and resulted in a prothrombin species that was inactive in our cross-linking assay. Other experiments showed that very high concentrations of calcium ion inhibit the cross-linkage of prothrombin. This result is most consistent with an essential gamma-carboxyglutamic acid-calcium ion-gamma-carboxyglutamic acid bridge(s) in the calcium-dependent self-associated form of prothrombin.

1-Carboxyglutamic Acid↗
PubMed1979

Proteolysis of prothrombin by thrombin. Determination of kinetic parameters, and demonstration and characterization of an unusual inhibition by Ca2+ ions.

A discontinuous assay to measure the proteolysis of tritiated prothrombin to fragment 1 and prethrombin 1 by thrombin has been devised, based empirically on extracting the radiolabeled fragment 1 into 4% p-toluenesulfonic acid. The assay is shown to be valid for the measurement of the initial rate of reaction in simple mixtures and has been used to determine the Michaelis-Menten parameters. The Km, 127 microgram of prothrombin per ml, and the kcat, 6.9 s(-1), indicate that, in the absence of other controls, attack of thrombin on prothrombin would be overwhelming during coagulation in plasma and that the prothrombin concentration would be rate-limiting. However, Ca2+ ions at concentrations around 1 mM reduce the rate of proteolysis by at least 20-fold. This inhibition is rapidly reversible by the addition of chelating agents. Measurement of initial rates at Ca2+ concentrations up to 1.5 mM shows that the inhibition is multisite and perhaps involves cooperative binding of the metal ions. The effect of Ca2+ on the kinetic parameters has also been investigated. kcat could not be measured because thrombin could not be saturated with prothrombin under conditions amenable to assay, but the Km is increased, showing that the inhibition by Ca2+ is at least partially competitive. An independent qualitative demonstration that Ca2+ is inhibitory in the reaction of native prothrombin with thrombin has been obtained by measurement of intrinsic fluorescence.

Animals↗
PubMed1994

Activation of prothrombin by a novel membrane-associated protease. An alternative pathway for thrombin generation independent of the coagulation cascade.

We herein report that a novel membrane-associated protease capable of activating prothrombin is present in several mammalian cells. This protease can directly convert prothrombin to active thrombin and induces blood clotting both in vivo and in vitro but is apparently different from coagulation factor Xa, which has been thought to be the only physiological activator of prothrombin. This protease activity was initially found and was very high in 8C feline kidney fibroblast cells, and we characterized its enzymological features using this cell line. Activity was detected in neither the cytosolic fraction nor the culture medium but found in the membranes and identified on the surface of intact cells. The activation of prothrombin required Ca2+ ions, and the apparent Km value for prothrombin was 0.2 microM. The activity was irreversibly inhibited by exposure to EDTA, but various inhibitors for serine proteases including antithrombin III were without effect. Based on these results, we propose that this novel enzyme, membrane-associated prothrombin activator, catalyzes an alternative pathway for generation of thrombin, which is independent of the blood coagulation cascade, and that the thrombin generated is involved in certain pathological states and/or in activation of cells that are spatially separated from the bloodstream.

Amino Acid Sequence↗
PubMed1994

Phospholipid-specific conformational changes in human prothrombin upon binding to procoagulant acidic lipid membranes.

This paper provides evidence to demonstrate that human prothrombin undergoes conformational changes upon binding to procoagulant membranes specifically containing phosphatidylserine (PS). Fourier transform infrared spectroscopy was used to show a slight increase in ordered (alpha-helix, beta-sheet, beta-turns) secondary structure upon binding to PS-containing membranes. Thermograms representing prothrombin and prothrombin fragment 1 denaturation were obtained using differential scanning calorimetry. These were analyzed and interpreted in terms of changes in prothrombin domain organization associated with binding to PS-containing membranes. Changes in either secondary structure or domain organization upon binding to negatively-charged phosphatidylglycerol-containing membranes were, if they occurred at all, much less dramatic. The results paralleled results obtained previously with bovine prothrombin (1, 2). The implications of these results in terms of a possible molecular mechanism for the cofactor-like role of platelet membrane vesicles in prothrombin activation are discussed.

Acids↗
PubMedDate not supplied

Variations in prothrombin time and international normalized ratio over 24 hours in warfarin-treated patients.

STUDY OBJECTIVE: To determine the variation of prothrombin times and international normalized ratio (INR) over 24 hours in humans. DESIGN: Prospective, parallel study. SETTING: University-affiliated general clinical research center. PATIENTS: Six patients receiving long-term warfarin therapy and six sex-matched controls. INTERVENTIONS: Warfarin was administered to the patients at 6:00 P.M. MEASUREMENTS AND MAIN RESULTS: Prothrombin times and INR were determined every 2 hours over 24 hours. Time of study entry, meals, and sleep cycles were controlled. A significant cosinor rhythm for prothrombin times and INR (p < or = 0.03) occurred in warfarin-treated patients, suggesting that diurnal variation occurs. The mean difference between the peak and trough prothrombin times was 1.8 +/- 0.9 seconds (range 0.8-3 sec) with a mean change of 9.3% +/- 3.7%. The peak prothrombin time and INR values occurred between 4:00 A.M. and 8:00 A.M. in five patients, and trough values between 6:00 P.M. and midnight in five. No significant cosinor rhythm was noted for controls (p > 0.5). CONCLUSION: Significant variations in prothrombin time and INR occurred in patients receiving warfarin therapy, with the highest values occurring in the morning and the lowest in the evening. These results may have clinical implications for patients receiving either high- or low-intensity warfarin therapy.

Adult↗
PubMed1996

Characterization of an inducible endothelial cell prothrombin activator.

In vivo prothrombin activation is thought to occur via a factor Xa/factor V-dependent mechanism. We investigated whether human venous endothelial cells (EC) could be induced to express a prothrombin activator. EC treated with lipopolysaccharide (LPS) or interleukin-1 activated prothrombin in the absence of exogenous factors Xa and V. This activity resided in the membrane fraction of EC and was not inhibited by an antibody to factor V. The apparent Km value was 3.3 +/- 0.3 mumol/L. Comparative studies of thrombin generation using a model system of phospholipid and factors Xa/V versus LPS-treated EC were performed to quantitate the effects of known inhibitors to factor Xa. The factor Xa inhibitor DEGR-chloromethyl ketone and an antibody to factor X inhibited prothrombin activation. However, the EC activator did not hydrolyze a factor Xa chromogenic substrate, and recombinant tick anticoagulant peptide did not suppress activity of the prothrombin activator. The apparent molecular weight of the EC activator was approximately 30 kD. Exogenous factor V enhanced the activity of the EC activator, such that in the presence of factor V, the apparent K(m) value was 1.28 +/- 0.10 mumol/L. Additionally, LPS-treated EC activated exogenous factor V. This activator has several characteristics of a previously described inducible murine monocyte prothrombin activator and may contribute to thrombin generation associated with pathologic stimuli.

Animals↗
PubMed1997

Co-inheritance of the 20210A allele of the prothrombin gene increases the risk of thrombosis in subjects with familial thrombophilia.

The presence of the 20210A allele of the prothrombin (PT) gene has recently been shown to be a risk factor for venous thromboembolism. This is probably mediated through increased plasma prothrombin levels. The aim of this study was to compare the prevalence of the prothrombin 20210A allele in control subjects and in subjects with recognised thrombophilia and to establish whether the additional inheritance of the PT 20210A allele is associated with an increased risk of venous thromboembolism. 101 subjects with a history of venous thromboembolism and diagnosed as having either factor V Leiden (R506Q) or heritable deficiencies of protein C, protein S or antithrombin were studied. The prevalence of the PT 20210A allele in this group was compared with the results obtained for 150 control subjects. In addition, the relationships were examined between genetic status and the number of documented thromboembolic episodes, and between plasma prothrombin levels and possession of the PT 20210A allele. 8 (7.9%) of the 101 patients were also heterozygous for the PT 20210A allele. This compares with 0.7% in the control subjects (p = 0.005). After exclusion of patients on warfarin, the mean plasma prothrombin of 113 subjects without 20210A was 1.09 U/ml, as compared with 1.32 U/ml in 8 with the allele (p = 0.0002). Among the 101 patients with either factor V Leiden, protein S deficiency, protein C deficiency or antithrombin deficiency, the age adjusted mean (SD) number of venous thromboembolic episodes at diagnosis was 3.7 (1.5) in those with the PT 20210A allele, as compared with 1.9 (1.1) in those without (p = 0.0001). We have demonstrated that the prevalence of the PT 20210A allele is significantly greater in subjects with venous thrombosis and characterised heritable thrombophilia than in normal control subjects and that the additional inheritance of PT 20210A is associated with an increased risk of venous thromboembolism. We have also confirmed that plasma prothrombin levels are significantly greater in subjects possessing the PT 20210A compared with those who do not.

Adolescent↗
PubMed1976

Role of gamma-carboxyglutamic acid. An unusual protein transition required for the calcium-dependent binding of prothrombin to phospholipid.

A first order calcium-dependent transition can be monitored by a decrease in the intrinsic fluorescence of the isolated "pro" (Fragment1) region of prothrombin. The maximum fluorescence change is -40% for Fragment 1, and only about -6% for whole prothrombin. The most remarkable features of this transition are its rate and activation energy. The half-life for the transition at 0 degrees is about 100 min, and the temperature dependence shows an activation energy of 21 kcal/mol. The rate constant for the forward reaction is zero order in calcium and is not affected by the presence of phospholipid membranes. The equilibrium for the transition, however, is affected by phospholipid. At 30 degrees, [Ca]eq (the calcium concentration where half of the protein has undergone the transition) is 0.4 mM and the Hill coefficient is 2.6. Under the same conditions but in the presence of phospholipid [Ca]eq is 0.24 mM and the Hill coefficient is about 4.5. The transition is triggered by binding 3 or 4 calcium ions. The rate of Fragment 1 binding to phospholipid vesicles was tested using gel filtration techniques at 0 degrees. The rate constants, activation energy, and [Ca]eq values for this process were shown to correspond to the properties of the fluorescence change. The rate constants, activation energy, and Hill coefficients for binding of whole prothrombin to phospholipid correspond to the same parameters for Fragment 1 but the [Ca]eq values are lower. At 0 degrees, the [Ca]eq is 0.19 mM for the prothrombin transition and 0.1 mM for the transition in the presence of phospholipid. These results demonstrate that Fragment 1 and prothrombin undergo a transition when exposed to calcium ions which necessarily precedes protein-phospholipid interactions. In addition to its role in determining the correct protein structure, calcium plays a second role in prothrombin-phos-pholipid interaction which is in the actual formation of the protein-phospholipid bond. The [Ca]eq for binding protein (after its transition) to phospholipid is about 0.06 mM.

Binding Sites↗
PubMed1998

Prothrombin time in retinitis pigmentosa.

The prothrombin time was recorded for 87 primary retinitis pigmentosa (RP) patients belonging to three different clinical categories. All categories showed prothrombin time higher than normal. There was no correlation between the age of onset and the prothrombin time, nor between duration of disease and the prothrombin time. The high prothrombin time in patients with RP suggests that further study of prothrombin time and related factors may help in better understanding of the pathogenesis of RP.

Adolescent↗
PubMed1976

Role of gamma-carboxyglutamic acid. Cation specificity of prothrombin and factor X-phospholipid binding.

Divalent cations are required for two roles in prothrombin-phospholipid interaction. The first role, catalysis of a prothrombin protein transition has a reaction half-life of 100 min at 0 degrees and is a prerequisite to phospholipid binding. The binding sites required for the transition have a very low cation specificity. All di- and trivalent cations tested were effective in this role with the exception of beryllium. Barium catalyzed the transition but only at high concentrations (6.6 mM was required for half-reaction). Blood-clotting Factor X, another gamma-carboxyglutamic acid-containing protein, also undergoes a cation-catalyzed protein transition which is a prerequisite to Factor X-phospholipid binding. In both proteins, the transition can be monitored by a decrease in the protein's intrinsic fluorescence. Compared to prothrombin, the Factor X transition occurs much more rapidly, has a somewhat greater specificity for cations, and requires higher concentrations of cations. This indicates that the cation binding sites provided by gamma-carboxyglutamic acid are not completely uniform in all proteins. The second role of divalent cations in prothrombin-phospholipid interaction is in the actual protein-phospholipid binding. This interaction was studied by protein fluorescence quenching resulting from excitation energy transfer to a chromophore attached to the phospholipid membrane. Only strontium and barium satisfactorily replaced calcium in this role. A number of other cations form protein-phospholipid complexes but of the wrong structure. These cations inhibit the prothrombinase complex (Factor Xa, calcium, phospholipid, Factor V). The cation specificity for Factor X-phospholipid binding is the same as for prothrombin except that higher concentrations of cations are required. Factor Xa (generated by action of Russell's viper venom on Factor X) displayed the same calcium requirements for the protein transition and phospholipid interaction as Factor X. The cation requirements of the prothrombinase complex correlate with the cation requirements of prothrombin and Factor X-phospholipid binding. Strontium is the only cation that will singly replace calcium. Barium is ineffective alone because the concentrations required to catalyze the protein transitions cause precipitation of the phospholipid. Combination of certain other cations with barium will, however, substitute for calcium. The other cations (specifically magnesium or manganous ion) catalyze the protein transitions and barium forms the correct protein-phospholipid complexes.

Animals↗
PubMed1985

The effect of low-dose estroprogestinic preparations on prothrombin complex factors: no significant increase after an 8-month trial.

The behavior of the prothrombin complex factors in 16 healthy women during low-dose estroprogestinic treatment (laevonorgestrel 0.15 mg and ethynilestradiol 0.30 mg) at basal conditions and during 8 months of therapy has been investigated. We found a statistically significant decrease of the PTT (Partial Thromboplastin Time). The prothrombin time, on the other hand, became slightly decreased, but not to a statistically significant extent. Among the prothrombin time derived tests for evaluating the prothrombin complex only the PP test (Prothrombin Proconvertin test) was significantly shortened. Of the coagulation factors (factors II, VII and X) only a modest, but not statistically significant, increase in Factor VII and Factor X was noted. We conclude that, during the 8 month observation period, prothrombin complex factors are not altered substantially.

Adult↗
PubMed1991

Modulation of Human Neurite Outgrowth by Serine Proteases: A Comparison of the Interaction of Thrombin and Prothrombin with Glia-Derived Nexin.

Neurite outgrowth from cells of neuroepithelial origin is under the reciprocal control of thrombin and the thrombin inhibitor-glia-derived nexin (GDN). The neurite retraction activity of thrombin is blocked when GDN complexes with the enzyme and inhibits its proteolytic activity. However, we have previously shown that enzymically inactive proenzyme is also capable of inducing neurite retraction. We present evidence here to show that GDN does not bind to prothrombin in solution. When a mixture of prothrombin and GDN is subjected to either polyacrylamide gel electrophoresis or immunoprecipitation, a stable complex cannot be detected. This is in direct contrast to thrombin, which exhibits stable complexes with GDN under both conditions. At the cell surface, however, GDN is able to inhibit the biological activity of prothrombin. When a mixture of proenzyme and inhibitor is applied to previously differentiated transformed retinoblasts (Ad12 HER10), the ability of prothrombin to induce neurite retraction is blocked. Furthermore, following 1 h exposure to Ad12 HER10 cells, a solution of prothrombin was found to contain half the potential enzyme activity as detected by chromogenic assay. These results have been interpreted as evidence for the ability of neuronal cells to cleave prothrombin and subsequently release activated enzyme.

Journal Article↗
PubMed1975

Prothrombin complex concentrate: use in controlling the hemorrhagic diathesis of chronic liver disease.

A prothrombin complex concentrate was used in attempts to control life-threatening hemorrhage in 4 patients with chronic liver disease. The population manifested profuse bleeding from varices and/or hemorrhagic gastritis; 3 had Laennec's cirrhosis and 1 had postnecrotic cirrhosis from childhood hepatitis. In all patients the complex was given in amounts needed to raise the prothrombin (factor II) level to approximately 100% of normal. In all 4 cases the prothrombin time and prothrombin complex factors approached normal within 1-2 hr after beginning the infusion. In all patients bleeding ceased with correction of the clotting status. One patient rebled several hours after completing the infusion. In several patients, increases in factors V and VIII were noted following infusion of the concentrate. A further unexpected finding was a spontaneous increase in factors II and IX at 3 days postinfusion. Prothrombin complex concentrate appears to be useful in controlling the hemorrhage of chronic liver disease when used alone or in combination with other modalities to correct specific hemostatic defects; however, patients may be expected to rebleed when the effect of the concentrate wears off. Its use, therefore, should probably be restricted to those patients who are to undergo corrective surgery of the bleeding point once hemostasis is achieved.

Adult↗
PubMed1971

Stability of prothrombin and factor VII in freeze-dried plasma.

The stability of prothrombin and factor VII was studied using accelerated degradation tests in three preparations of freeze-dried pooled normal plasmas. In a previous report (Brozović, Gurd, Robertson, and Bangham, 1971) factor X was shown to be relatively unstable in these preparations of freeze-dried plasma: it was calculated that up to 8% of the original factor X activity would be lost after 10 years at -20 degrees C, up to 54% at 4 degrees C, and up to 90% at room temperature. The losses of factor VII activity were estimated to be negligible at -20 degrees C, between 2 and 18% at 4 degrees C, and between 20 and 70% of the original activity at 20 degrees C, after 10 years of storage. Prothrombin was found to be less stable than factor VII: the expected loss in 10 years at -20 degrees C may be up to 4%, at 4 degrees C up to 30%, and at 20 degrees C up to 83% of the initial activity. These findings indicate that in freeze-dried plasma prothrombin as well as factor X may be insufficiently stable for plasma to serve as long-term reference material for the standardization of the one-stage prothrombin time. Moreover, the loss of prothrombin and factor X in freeze-dried plasma stored at 4 degrees C may be so high that when it is required to preserve these factors it may be necessary to store freeze-dried plasma at lower temperatures.

Cold Temperature↗
PubMed1983

Determination of prothrombin by a micro-coagulation method.

In contrast to earlier assays, the prothrombin determination described here uses a two-step reaction: first, prothrombin is activated with human factor Xa, phospholipids, Ca++ and factor V; second, the amount of activated prothrombin is assayed by the proteolysis of added fibrinogen. The assay is dependent only on prothrombin, and independent of other components of thrombin generation. Carboxy- and decarboxyprothrombin can be differentiated by means of using Echis carinatus venom instead of factor Xa as activator. The micro-coagulation assay is compared with a conventional one-stage coagulation test, with a prothrombin determination using chromogenic substrate and immunologically by Laurell electrophoresis.

Calcium↗
PubMed1963

COUMARIN THERAPY. PROTHROMBIN ACTIVITY AFTER TERMINATION OF TREATMENT.

Twelve patients receiving coumarin type hypoprothrombinemic agents were studied before, during and after termination of therapy, the prothrombin proconvertin method having been used to assay the prothrombin activity complex. In no instance was post treatment "rebound" demonstrated. Prothrombin activity levels returned to pretreatment values only after ten days following termination of coumarin or Dicumarol administration. If a reactivation of thrombotic tendency occurs following discontinuance of anticoagulant therapy, it would not appear to be related to a "rebound" of prothrombin activity above that which is "normal" for the individual patient. Patients tend to return to the same level of prothrombin activity present before initiation of coumarin therapy.

Anticoagulants↗
PubMed2001

Acquired bleeding disorder in a patient with malignant lymphoma: antibody-mediated prothrombin deficiency.

BACKGROUND: Bleeding manifestations secondary to acquired hemostatic abnormalities in cancer patients have been well described. Bleeding due to the development of hemostatic inhibitors is observed less frequently. In this report, the authors describe a patient with a low grade lymphoma who presented with an acquired bleeding disorder and abnormal hemostatic screening tests. METHODS: Patient plasma samples were collected initially and during the course of treatment. Mixing studies and specific coagulation factor assays were performed to detect and confirm any deficiencies. Patient immunoglobulin G was isolated from plasma, and binding to prothrombin was demonstrated by immunoblot method and enzyme-linked immunosorbent assay (ELISA) techniques. RESULTS: Initial prolongations in the prothombin time and the activated partial thromboplastin time suggested a factor deficiency in the common pathway of coagulation. Factor assays confirmed that the coagulation abnormality in this patient was the result of an acquired prothrombin (factor II) deficiency. This was confirmed by an immunoassay for prothrombin antigen. Further studies demonstrated the presence of a noninhibitory antibody to prothrombin that interacted with a calcium dependent epitope. CONCLUSIONS: Successful treatment of the lymphoma resulted in clearance of the antibody and complete correction of all hemostatic abnormalities and manifestations. An acquired prothrombin deficiency has not been reported previously in association with a malignancy, and this patient represents the first such documented case.

Antineoplastic Combined Chemotherapy Protocols↗