Increased activity of vitamin K-dependent clotting factors in human hyperlipoproteinaemia - association with cholesterol and triglyceride levels

Levels of blood coagulation factors, cholesterol and triglyceride were measured in human plasma. Prothrombin was significantly elevated in type IIa hyperlipidaemia; prothrombin and factors VII, IX and X in type IIb; and prothrombin and factors VII and IX in type V. Multiple regression analysis showed significant correlation between the levels of these plasma lipids and the vitamin K-dependent clotting factors (prothrombin, factors VII, IX and X). Higher cholesterol levels were associated with higher levels of prothrombin and factor X while higher triglyceride levels were associated with higher levels of these as well as factors VII and IX. Prothrombin showed a significant cholesterol-triglyceride interaction in that higher cholesterol levels were associated with higher prothrombin levels at all levels of triglyceride, with the most marked effects in subjects with higher triglyceride levels. Higher prothrombin levels were noted in subjects with high or moderately elevated (but not low) cholesterol levels. Ultracentrifugation of plasma in a density of 1.21 showed activity for prothrombin and factors VII and X only in the lipoprotein-free subnatant fraction. Thus, a true increase in clotting factor protein was probably present. The significance of the correlation between levels of vitamin K-dependent clotting factors and plasma lipids remains to be determined.     

Critical role of factors II and X during coumarin anticoagulation and their combined measurement with a new Fiix-prothrombin time

Vitamin K antagonists (VKA) are monitored with prothrombin time (PT) based assays that are equally sensitive to reductions in factors II, VII or X. We compared the effect of vitamin K dependent (VKD) coagulation factors on PT and also on rotational thromboelastometric (ROTEM) parameters. The PT was equally sensitive to reductions in factors II, VII or X but ROTEM parameters correlated poorly with the PT in anticoagulated patients´ plasmas. ROTEM parameters were more affected by mild and moderate reductions in FII or FX than by FVII or FIX which had little influence except at very low coagulant activity. We developed a modified PT that was sensitive only to reductions in factors II and X. The Fiix-PT (Fiix-INR) correlated well with PT (INR) but the Fiix-INR fluctuated less than the INR in an anticoagulated patient reflecting its insensitivity to FVII. The ROTEM results suggest that mild to moderate reductions in factors II or X are more important during clot formation than factors VII or IX. Reductions in FII and X may better reflect anticoagulation with VKA than FVII or IX. The new Fiix-PT may more accurately reflect the degree of therapeutic anticoagulation in patients treated with VKA than the current PT which is subject to a confounding variation caused by FVII.     

Thrombin generation in newborn plasma is critically dependent on the concentration of prothrombin

The ability to generate thrombin is decreased and delayed in plasma from the healthy newborn infant compared to the adult. Only 30 to 50% of peak adult thrombin activity can be produced in neonatal plasma. To test whether this observation can be explained by the low neonatal levels of the contact or vitamin K dependent factors, we measured neonatal thrombin generation after raising the concentration of these factors to adult values. We also determined whether the addition of a variety of blood products to neonatal plasma improved thrombin generation. An amidolytic method was used to quantitate intrinsic (APTT) and extrinsic (PT) pathway thrombin generation in defibrinated pooled cord plasma from healthy term infants. Added individually, factors VII, IX, X or the contact factors (CF) failed to alter the rate or the total amount of thrombin generated in neonatal plasma. In contrast, the addition of prothrombin increased the total amount of thrombin generated to above adult values in both the APTT and the PT systems but did not alter the rate of thrombin generation. The rate of thrombin generation in cord plasma shortened after a combination of II, IX, X and CF was added to the APTT system or II, VII and X to the PT system. In both systems, the total amount of thrombin generated was linearly related to the initial prothrombin concentration. Each of fresh frozen plasma, cryoprecipitate, plasma from platelet concentrates, or factor IX concentrate (in amounts used therapeutically) caused an increase in the total amount of thrombin generated which was related to the increase in prothrombin concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relative importance of the factors II, VII, IX and X for the prothrombinase activity in plasma of orally anticoagulated patients

The individual importance of each of the four vitamin K-dependent clotting factors on the generation of prothrombinase activity in the plasma of orally anticoagulated patients has been investigated. Addition of purified factors VII, IX or X to plasma from deeply anticoagulated patients (International Normalized Ratio 2.8-4.8) did not influence the amount of prothrombinase activity or the amount of thrombin formed. Only the prothrombin level in the plasma determines the course of thrombin generation. Addition of increasing amounts of purified factor II, VII, IX or X to plasmas deficient in respectively factor II, VII, IX or X showed that the prothrombinase activity increases linearly with the concentration of factor II added and that the concentration below which the factors VII, IX and X start to have a measurable effect on prothrombinase activity are 5%, 20%, and 30%, respectively. Half maximal prothrombinase activity was found at about 1% factor VII, 5% factor IX and 8% factor X respectively. From these observations we conclude that primarily the variation in factor II level determines thrombin generation and hence presumably the antithrombotic effect of oral anticoagulant therapy. It therefore seems likely that, for the control of oral anticoagulant therapy, tests that reflect factor II activity would be suitable.     

The activation of factor IX by tissue factor-factor VII in a bovine plasma system lacking factor X

The activation of Factor IX by tissue factor-Factor VII has been studied in a bovine plasma system under conditions that minimize the activation of Factor VII. The plasma was defibrinated, then passed twice through a column of anti-Factor X coupled to Sepharose in order to lower the Factor X level below its limit of assay ($\text{ca.}$ 5 ng/ml), and once through an anti-Factor IX column to remove Factor IX. Varying levels of tritium-labelled Factor IX were then added back to the plasma, permitting measurement of its activation upon the addition of tissue factor and Ca²⁺. Despite the absence of significant levels of Factor X in the system, the course of Factor IX activation was initially characterized by some upward curvature, which suggested activation of the plasma Factor VII during the incubation. In order to obtain linear activation of Factor IX three proteolytic inhibitors were added to the system: 1) a Factor X_a inhibitor, 1,2-bis-(5-amidinobenzimidazole)-ethane, 2) aprotinin, and 3) heparin. Under these conditions the apparent K_m of non-activated Factor VII (+ tissue factor) on Factor IX was 17.3 +/- 2.5 nM (SE), and the maximum velocity was 0.12 nM/min. In parallel experiments the plasma Factor VII was activated by first treating the plasma with Factor X_a for 30 seconds before the addition of inhibitors and the final addition of substrate. Under these conditions the maximum velocity rose to 4.2 nM/min, and the K_m increased to 53.3 +/- 6.0 nM (SE). This change in the K_m is highly significant (P < 0.002), and indicates that the activation of Factor IX by nonactivated plasma Factor VII cannot be due only to traces of Factor VII_a in the plasma. At least in part, activation of Factor IX in the presence of tissue factor is suggested to be a result of the action of Factor VII itself.     

Antiserum against factor IX shortens the bovine thromboplastin coagulation time of human plasma

The one-stage prothrombin time was determined with bovine thromboplastin after the addition of a high-titred sheep antiserum against human factor IX. In normal plasma and plasma from patients with hemophilia B and a normal amount of plasma factor IX antigen, the addition of antiserum resulted in a shortening of the prothrombin time. Serum from a sheep which had not been immunized had no such effect. The antiserum had no effect on the prothrombin time of plasma from patients with hemophilia B and no detectable plasma factor IX antigen (hemophilia B-). Incubation with antiserum also resulted in a shortening of the clotting time of normal plasma in the Thrombotest and had no effect on the clotting time of hemophilia B-plasma. Addition of purified factor IX to normal plasma increased the prothrombin time determined with bovine thromboplastin. The antiserum had no effect on the prothrombin time determined with human or rabbit thromboplastin. It is concluded that normal factor IX antigen and factor IX antigen from patients with hemophilia B+ probably act as inhibitors in the reaction between bovine thromboplastin, factor VII and factor X.     

The effect of intravenous vitamin E and menadiol sodium diphosphate on vitamin K dependent clotting factors

During a Phase I study of intravenous vitamin E-free alcohol (all-rac-a-tocopherol or Ephynal®) in patients with neuroblastoma, we noticed a bleeding diathesis in two patients receiving 2300 mg/m² daily for four or more days in succession. Both blood prothrombin time and acclerated partial thromboplastin times were prolonged. These spontaneously returned to normal levels three days after interrupting vitamin E infusions. It was also noted that factors VII, IX and X were decreased, which corresponded with the prolonged PT and APTT. It was found that by infusing menadiol sodium diphosphate just prior to the vitamin E, these inhibiting effects on procoagulant factors could be abrogated and high dosages of vitamin E-free alcohol safely given.     

In vitro characterization of various heat-treated prothrombin complex concentrates (PCC)

All of 6 heat-treated prothrombin complex concentrates (PCC) tested contained adequate levels of factor IX but factor VII content was low. Levels of factors II, X, protein C and protein S were variable and antigen levels were always greater than those of functional activities. On crossed-immunoelectrophoresis factor IX showed variable anodal shift in all concentrates tested and in some activated factor IX was demonstrated by immunoblotting technique. These findings suggested some activation and/or denaturation during production and/or heating. Modest amount of factor VIII clotting activity by solid-phase amidolytic method and of factor VIII antigen was demonstrated in some concentrates but none contained more than a trace factor VIII inhibitor bypassing activity. The results suggested that heat-treated PCC should provide safe therapeutic products for hemophilia B.     

Localization of blood coagulation factors in situ in pancreatic carcinoma.

Blood coagulation is activated commonly in pancreatic carcinoma but the role of the tumor cell in this activation is undefined. Immunohistochemical procedures were applied to fixed sections of 22 cases of resected adenocarcinoma of the pancreas to determine the presence of components of coagulation and fibrinolysis pathways in situ. Tumor cell bodies stained for tissue factor: prothrombin: and factors VII, VIIIc, IX, X, XII, and subunit "a" of factor XIII. Fibrinogen existed throughout the tumor stroma, and tumor cells were surrounded by fibrin. Staining for tissue factor pathway inhibitor, and plasminogen activators was minimal and inconsistent. Plasminogen activator inhibitors -1, -2, and -3 were present in the tumor stroma, and on tumor cells and vascular endothelium. Extravascular coagulation activation exists associated with pancreatic carcinoma cells in situ that is apparently unopposed by naturally occurring inhibitors or the plasminogen activator-plasmin system. We postulate that such local coagulation activation may regulate growth of this malignancy. These findings provide a rationale for testing agents that modulate the blood coagulation/fibrinolytic system (that inhibit tumor growth in other settings) in pancreatic carcinoma.     

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.     

Metabolism of the coagulation factors of the prothrombin complex in hypothyroidism in man.

The metabolic rate of prothrombin, factors VII, IX and X, was studied in nine hypothyroid patients. Disappearance rates of the four vitamin-K-dependent factors, called the prothrombin complex, were measured after assumedly complete blocking of their synthesis with adequate doses of a coumarin congener (acenocoumarol). Reappearance rates were assessed by induction of synthesis with high doses of vitamin K1 (phytomenadion) when stable hypocoagulability had been achieved. Normal values for these rates were derived from earlier studies in our laboratory. In hypothyroid patients the rates of both disappearance and reappearance were significantly slower for all factors tested. Practical consequences of these observations are discussed. The initial level of factor-IX activity in all nine patients was substantially lower than in normal individuals. Therapy by thyroxine-substitution led to normal levels of factor-IX. This implies a divergency in the retardation of the breakdown and production rates in hypothyroidism. The reappearance rate was indeed found to be more retarded than the disappearance rate.     

Manufacturing of a prothrombin complex concentrate aiming at low thrombogenicity

The paper describes the production of a prothrombin complex concentrate (PCC) with high virus safety and a well-balanced content of vitamin K-dependent clotting factors and inhibitors. Solid-phase extraction is followed in a second step by optimized anion exchange chromatography using a radial column. A step for virus removal by nanofiltration is introduced in addition to the solvent/detergent step. By speeding up the chromatographic step, the period of time required for production is reduced considerably. The activities of the four vitamin K-dependent clotting factors II, VII, IX and X are in ratios of about 1:1:1:1. Protein C, Protein S, and Protein Z are also present in therapeutically effective concentrations. The product shows no thrombogenicity, in either in vivo nor in vitro models. Clinical investigations show that the PCC is a safe and efficient preparation for the substitutive treatment of FIX or FVII in patients suffering from the respective deficiencies. All bleeding episodes have been efficiently controlled with relatively low doses of the concentrate. The surgical procedures have been conducted without any problems in severely FIX and FVIII deficient patients.     

Consumption of abnormal prothrombin in the presence of different types of thromboplastin

An abnormal form of prothrombin is invariably found in the plasma of patients on oral anticoagulants. When plasma from these patients is clotted with the addition of calcium the abnormal prothrombin remains in serum (Ganrot et Nilehn, 1968). However, Josso et al (1968) reported that both normal and abnormal prothrombin were consumed in the presence of thromboplastin. The behaviour of abnormal prothrombin in the presence of different types of thromboplastin was studied using Laurell crossed immunoelectrophoresis. Three types of human thromboplastin and one bovine reagent caused complete consumption of abnormal prothrombin. Of the eight rabbit thromboplastins studied, only one, of high sensitivity to coumarin induced defect, caused the consumption of abnormal prothrombin. With other rabbit thromboplastins the abnormal prothrombin always remained in serum. The addition of factors V, VII, IX and X did not cause the consumption in the presence of rabbit reagents. The consumption of abnormal prothrombin correlated with the thromboplastin sensitivity to the coumarin induced defect expressed as sensitivity ratio and can be used as an additional indicator of thromboplastin sensitivity to this defect, in particular to the changes on prothrombin itself.     

Hemostatic derangement in dengue hemorrhagic fever

Dengue hemorrhagic fever (DHF) is a more severe manifestation of dengue virus infection. Patients with DHF exhibit abnormal hematological indices, including high hematocrit, low white blood cells, low neutrophils, high lymphocytes, increased atypical lymphocytes, low platelets, slightly prolonged activated partial thromboplastin time, prothrombin time, and thrombin time. Abnormal platelet functions manifest as impaired platelet aggregation to ADP, and concurrent increases in plasma thromboglobulin and platelet factor 4 levels are also seen. Variable reductions in the activities of coagulation factors including prothrombin, V, VII, VIII, IX, and X may be present. The plasma level of antithrombin is typically normal, but protein C and protein S are modestly reduced. Within the fibrinolytic system, slightly increased levels of tissue-plasminogen activator accompanied by slightly increased plasminogen activator inhibitor-1 and decreased thrombin activatable fibrinolysis inhibitor have been demonstrated. These derangements are prominent in patients with DHF grades III and IV, collectively known as dengue shock syndrome. Moreover, patients with excessive depletion of intravascular volume from plasma leakage and/or massive bleeding from endothelial dysfunction, thrombocytopenia, platelet dysfunction, and coagulopathy may exhibit shock, prolonged shock and repeated shock. DIC is also commonly found in these complicated patients. However, most patients recover spontaneously with normalization of abnormal laboratory profiles during the convalescent stage or within one to two weeks after defervescence.     

Effect of factor XII deficiency on pregnancy and parturition.

The clotting parameter of a primigravida with factor XII deficiency was studied during her third trimester of pregnancy, labor and post-partum; and compared with those of her newborn male infant. Sharp increases in factors VII, VIII, IX, X and moderate increases in factors II and XI were documented during pregnancy and at labor. All factors had returned to normal or near normal levels 24 hours after delivery. Factor XII remained at 0.0 level throughout. In the infant the clotting factor levels reflected depression of vitamins K-dependent factors II, IX, and X and a factor XII level of 40.0%. No undue bleeding was noted in the mother at delivery or placental separation, and no bleeding manifestation was apparent in the infant. These findings suggest that factor XII does not play a major role in triggering or modulating the course of normal labor, nor is its absence necessarily associated with bleeding complications during parturition or placental separation.     

The activation of human factor X in sodium citrate: the role of factor VII.

Human factor X was purified by several different procedures yielding products which had varying amounts of factor VII and factor IX. Treatment with CHCl3 during the fractionation of the factor X removed 95% of the factor VII and factor IX activity and the resulting factor X activated more slowly when incubated in 25% sodium citrate. Removal of residual factor VII by DEAE cellulose chromatography yielded a factor X which activated still more slowly and less completely. When the factor VII, removed by chromatography, was added to the chromatographed factor X, the ability to be activated in 25% sodium citrate was restored. Confirmatory evidence for the role of factor VII in this reaction was the inhibition of the conversion of the factor X by both DFP and SBTI.     

The effect of suramin on laboratory tests of coagulation.

The antitrypanosomal drug suramin, which has recently been under investigation as a cancer chemotherapeutic agent, has previously been found to induce heparin-like anticoagulants in treated patients. In the currently reported work suramin is shown to have an additional anticoagulant activity that is due to direct effects of the drug on procoagulant proteins. The studies were conducted with pooled normal plasma treated in vitro with suramin and with plasma samples obtained from patients who had received the drug intravenously for 2 weeks. It is demonstrated that in plasma suramin inhibits factors V, VIII, IX, X, XI, and XII, while thrombin, prothrombin, and factor VII are unaffected. The inhibition of factor V is virtually irreversible, although the effect of suramin on the other factors is readily reversed by dilution.     

Extrinsic activation of human coagulation factors IX and X on the endothelial surface.

In previous kinetic studies, the catalytic efficiency of the activation of human coagulation factors IX and X by factor VIIa in the presence of purified tissue factor apoprotein was found to be essentially equal. These activation reactions were now studied on the surface of human umbilical vein endothelial cells. The cells were stimulated with endotoxin to express tissue factor. This tissue factor activity was saturable with factor VIIa and could be inhibited by rabbit antibodies against human tissue factor apoprotein. Only stimulated cells supported factor VIIa activity. No difference in the reactivity of factor VII and VIIa was observed in the presence of factor X, due to rapid feedback activation of factor VII by factor Xa. However, the activation of factor IX by factor VII shows a 10 min lag-phase, which reflects that the activation of factor VII by factor IXa is a less efficient process. The kinetic parameters for the factor VIIa dependent activation of factor IX and factor X on the endothelial surface were: Km 0.09 microM, Vmax 0.13 pmol/min, and Km 0.071 microM, Vmax 0.41 pmol/min, respectively. The same ratio between the Vmax for factor X and factor IX activation was observed as in a cell free system. However, the Km of factor IX was 4-fold higher on the endothelial surface than in the cell free system. Together, these kinetic parameters will favour factor X activation 5-fold over factor IX activation at physiological concentrations of these proteins. The activation of factor X by factor VIIa on the endothelial surface was characterized by a short lag-phase, which was absent in factor IX activation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunological aspects of some vitamin K-dependent factors and preparation of depleted plasmas

The specificity of the immune response elicited in rabbits to purified bovine prothrombin, autoprothrombin III (Factor X), Factor IX, and Protein C was studied. The immunizing proteins were homogeneous on polyacrylamide gel electrophoresis. By immunodiffusion analysis antigenic similarities were not detected between them in spite of known stretches of amino acid sequence homology. Immunoelectrophoresis of bovine plasma and each purified protein gave identical single precipitin lines with the homologous antisera. This experiment established that the antibodies prepared against each one of the purified vitamin K-dependent factors studied reacted with a single component in bovine plasma. Immunologic crossreactions were found between human, bovine, dog, rat and chicken prothrombin, Protein C, Factor X, and Factor IX. They were not species specific. Purified bovine prothrombin has at least four antigenic determinant sites. Incubation of the purified proteins with suitable proteolytic enzymes yielded fragments and intermediate products which were purified and tested for their potential capacity to react with antibodies in the native antisera. In immunoelectrophoresis tests both prothrombin fragments 1 and 2 and the intermediate product prethrombin 1 gave immunoprecipitates which were separately identified by their differing mobilities, while the enzyme portion, thrombin, did not react with antithrombin serum. In addition to prothrombin, bovine plasma contained prethrombin 1, prothrombin fragments 1 and 2, as well as prethrombin 2 and/or thrombin. Autoprothrombin III$\text{m}$ (Factor Xβ) and autoprothrombin C (Factor Xa) crossreacted with anti-autoprothrombin III and the active form of Protein C. Gamma globulins of antisera to prothrombin, to autoprothrombin III, to Factor IX, and to Protein C were fractionated by using saturation ammonium sulfate solution and coupled to activated Agarose A-15m beads. The insolubilized antibodies were used to absorb the antigen from the plasma. Each depleted plasma served as test material for a specific clotting factor. The prolonged prothrombin time of the prothrombin depleted plasma was normalized by additions of purified prothrombin, but not by purified prethrombin 1. The prothrombin time of autoprothrombin III depleted plasma was shortened by fresh plasma, purified autoprothrombin III, and by purified autoprothrombin III$\text{m}$ (Factor Xβ). In the case of Factor IX depleted plasma, the partial thromboplastin time was used. The depleted bovine plasmas are suitable for assay purposes. They serve the same role as the corresponding deficient plasma in bioassay coagulation tests.     

Studies on the anticoagulant action of Aspilia africana

An anticoagulant activity was identified and isolated from the leaves of a West African plant, Aspilia africana by gel filtration on Sephadex G-100. The anticoagulant factor had an apparent molecular weight of approximately 60,000 d. Upon incubation with plasma, it prolonged the partial thromboplastin time, prothrombin time, thrombin and reptilase time. The factor decreased the fibrinogen content of plasma as well as the activity of coagulation factors V, VIII and IX but not factor VII, X or XI activities. After incubation with fibrinogen, the thrombin clotting time was prolonged and the quantity of clottable fibrinogen reduced. The action on fibrinogen was characterized by sequential lytic breakdown of the A-alpha-chain and B-beta-chain, the gamma-chain being lysed last, after prolonged incubation. Benzamidine, Epsilon aminocaproic acid or soybean trypsin inhibitor did not impede lysis.