Heparanase, galectin-3, and tissue factor mRNA are expressed in benign neoplasms of the thyroid

Objective: Heparanase, galectin-3, and tissue factor (TF) are overexpressed in solid malignant thyroid tumors. We studied their expression in multinodular goiters (MNGs). Design and Methods: Thyroid tissue specimens from 15 MNGs were obtained during surgery. mRNA expression for galectin-3, heparanase, and TF was assessed by RT-PCR. Results: Isolated expressions of heparanase and galectin-3 mRNA were expressed in 2 and 4 of the 15 MNGs, respectively; 8/15 MNGs were positive for both heparanase and galectin-3. TF mRNA was found in all MNG specimens. Conclusion: Galectin-3, heparanase, and TF RNA expression is prevalent in MNGs. Further studies will be needed to determine the prognostic significance of these findings.     

Activation of 125I-Factor IX and 125I-Factor X: Effect of Tissue Factor and Factor VII,Factor Xa and Thrombin

Activation of Factor IX and Factor X was studied by adding ¹²⁵I-Factor IX or ¹²⁵I-Factor X to reaction mixtures and quantitating cleavage products by reduced sodium dodecylsulfate gel electrophoresis. Thrombin failed to activate Factors IX or X; Factor X_a produced insignificant amounts of cleavage products of both factors. In contrast, the reaction product of tissue factor and Factor VII cleaved large amounts of both Factor IX and Factor X in purified systems and in plasma. In incubation mixtures of plasma containing added ¹²⁵I-Factor IX or ¹²⁵I-Factor X, tissue factor and Ca2+ions, the percentage of total radioactivity in the heavy chain peak of ¹²⁵I-IX_a and the heavy chain peak of ¹²⁵I-X_a increased at a similar rate. When the tissue factor was diluted, similar curves were obtained for percent cleavage of ¹²⁵I-Factor IX and percent cleavage of ¹²⁵I-Factor X plotted against tissue factor concentration. These findings support the hypothesis that activation of Facor IX by the tissue factor-Factor VII reaction product represents a physiologically significant step in normal haemostasis.     

Elevated circulating tissue factor procoagulant activity, factor VII, and plasminogen activator inhibitor-1 in childhood obesity: evidence of a procoagulant state

Childhood obesity is rapidly increasing in prevalence. We compared circulating membrane-bound tissue factor (FIII, F3) procoagulant activity (TF-PCA) and plasma markers of coagulation, fibrinolysis and endothelial dysfunction in 21 obese (10·1 ± 1·5 years, mean ± standard deviation) and 22 healthy weight children (9·9 ± 1·6 years), classified by Body Mass Index (BMI). TF-PCA and factor VII coagulant activity (FVII:C), plasminogen activator inhibitor (PAI-1, SERPINE1) and soluble vascular cell adhesion molecule 1 (sVCAM1) were higher in obese children. BMI correlated positively with TF-PCA, FVII:C, and PAI-1. Childhood obesity is associated with a procoagulant state and endothelial dysfunction. Studies are needed to assess whether weight reduction reverses these abnormalities.     

Activation of a recombinant human factor VII structural analogue alters its affinity of binding to tissue factor

A competitive enzyme-linked immunoadsorbent assay (ELISA) technique has been developed to facilitate quantitative analysis of the earliest step in the initiation of the extrinsic pathway of coagulation, i.e., complex formation of factor VII/VIIa with tissue factor. The ELISA measures the binding of biotinylated human plasma factor VII to relipidated recombinant human tissue factor. Quantitation of the relative affinity (expressed as IC₅₀) of any factor VII molecular population or structural analogue for tissue factor can be determined by competitive binding. Subnanomolar concentrations of both wild-type recombinant human factor VII (rFVII) and rFVII(R152Q), a mutation at the FVII activation site, competed effectively with biotinylated plasma-derived factor VII in binding to tissue factor. In contrast, the affinity of rFVII(R79Q), a mutation in the first epidermal growth factor-like domain, was 12-fold lower. Following activation of rFVII(R79Q), its affinity for tissue factor and enzymatic activity increased 4-fold and 6-fold, respectively. For wild-type rFVII, enzymatic activity rose significantly following activation. However, its affinity for tissue factor was unchanged. We conclude that both the activation state of factor VII and the mutation of amino-acid residues within the first epidermal growth factor-like domain may alter the affinity of factor VII for tissue factor. © 1996 Wiley-Liss, Inc.     

Incomplete gamma carboxylation of human coagulation factor VII: differential effects on tissue factor binding and enzymatic activity

The integrity of the γ-carboxylic glutamic acid (GLA) residues of coagulation factor VII are thought to be essential for both the interaction of factor VII with its cell-surface lipoprotein receptor tissue factor and for the activated protein to manifest its serine protease activity. During the course of transiently expressing recombinant human factor VII in monkey COS cells it was noted that the factor VII synthesized in the absence of added vitamin K had < 20% of expected procoagulant activity yet retained 65% of its binding activity to recombinant human tissue factor. Similar results were obtained when vitamin K was omitted from human 293 cell cultures permanently expressing recombinant factor VII. In contrast, both transient and permanent expression of factor VII in human 293 cell cultures containing physiological concentrations of vitamin K resulted in the synthesis of fully functional factor VII. Furthermore, factor VII in plasma samples from 24 patients undergoing warfarin therapy bound quantitatively to tissue factor whereas factor VII procoagulant activity averaged 65% of normal. Thus, data from both in vitro and in vivo situations indicated that factor VII molecules with suboptimal GLA content retained most of their ability to bind tissue factor but exhibited reduced procoagulant activity.     

Comparison of the behavior of normal factor IX and the factor IX BM variant hilo in the prothrombin time test using tissue factors from bovine,human, and rabbit sources

A subset of hemophilia B patients have a prolonged bovine-brain prothrombin time. These CRM⁺ patients are classified as having hemophilia Bm. The prolongation of the prothrombin time has been reported only with bovine brain (referred to as ox brain in some literature) as the source of thromboplastin; prothrombin times determined with thromboplastin from rabbit brain or human brain are not reported to be prolonged. Factor IX from a hemophilia Bm patient (factor IX Hilo) was isolated. The activity of factor IX Hilo was compared to that of normal factor IX in prothrombin time assays when the thromboplastin source was of bovine, rabbit, or human origin. Factor IX, either normal or Hilo, prolonged a prothrombin time regardless of the tissue factor source. However, unless thromboplastin was from a bovine source, this prolongation required high concentrations of factor IX. Further, factor IX normal was as effective as factor IX Hilo in prolonging the prothrombin time when rabbit or human thromboplastin was used. With bovine thromboplastin, factor IX Hilo was significantly better than factor IX normal at prolonging the prothrombin time. The amount of prolongation was dependent on the amount of factor IX Hilo added. In addition, the prolongation was dependent on the concentration of factor × present in the sample. The prothrombin time changed as much as 20 seconds when the factor × concentration was varied from 50% to 150% to normal (fixed concentration of factor IX Hilo). These results demonstrate the difficulty of classifying the severity of a hemophilia Bm patient based on the bovine brain prothrombin time unless both the factor IX and factor × concentrations are known.     

Successful treatment of severe intra-abdominal bleeding associated with disseminated intravascular coagulation using recombinant activated factor VII

Recombinant activated factor VII (rFVIIa) is indicated mainly for the treatment of patients with haemophilia and inhibitors. However, little information is available on the use of rFVIIa in the treatment of the severe bleeding associated with disseminated intravascular coagulation (DIC). We report a pregnant woman with DIC, who developed severe intra-abdominal bleeding after caesarean section. Despite treatment with fresh-frozen plasma, fibrinogen, platelet transfusions and surgery, the abdominal bleeding persisted and intravenous treatment with rFVIIa was initiated. The response to treatment was rapid, with control of the bleeding and resolution of the coagulopathy. No side-effects related to rFVIIa were noted. This case suggests a potential role for rFVIIa in the treatment of severe and refractory bleeding associated with DIC.     

Pancreatic cancer complicated by disseminated intravascular coagulation associated with production of tissue factor

Received: April 21, 2000 / Accepted: December 22, 2000     

Tissue factor pathway inhibitor is the main determinant of thrombin generation in haemophilic patients

The thrombin generation (TG) assay evaluates haemostatic balance, which is influenced by the levels of many coagulation factors and inhibitors. Our objective was to identify the determinant factors of TG in haemophilia A (HA) and haemophilia B (HB) patients and to compare them to those in healthy controls. Coagulation factor and inhibitor levels, and TG, were measured in platelet‐poor plasma from 40 patients with HA, 32 patients with HB and 40 healthy subjects. Data were analysed using multiple regression models. In HA patients, factor VIII was a positive determinant of endogenous thrombin potential (ETP) and peak, whereas tissue factor pathway inhibitor (TFPI) and factor V were negative determinants of ETP and peak. In HB patients, FIX was a positive determinant of ETP and peak, FVII being a positive determinant of peak. Antithrombin and protein S (PS) were negative determinants of ETP while FX was a negative determinant of peak. Above all, in HB patients, TFPI was a negative determinant of ETP and peak. In healthy subjects, FVIII was a positive determinant of ETP and peak, whereas FX and protein S were negative determinants of these parameters. TFPI was not a negative determinant of either peak or ETP. In haemophilic patients, the determinant factors of TG are all implicated in FXa generation and inhibition, the crucial determinant factor being TFPI whatever the type of haemophilia, A or B. These findings contribute to the rationale that recently place TFPI as a target for innovative therapies of haemophilia.     

Plasma tissue factor plus activated peripheral mononuclear cells activate factors VII and X in cardiac surgical wounds

OBJECTIVES: The purpose of this study was to test the hypothesis that activated monocytes with soluble plasma tissue factor (pTF) activate factors VII and X to generate thrombin. BACKGROUND: Despite heparin, thrombin is progressively generated during cardiac surgery with cardiopulmonary bypass (CPB), produces intravascular fibrin and fibrinolysis, and causes serious thromboembolic and nonsurgical bleeding complications. Thrombin is primarily produced in the surgical wound, but mechanisms are unclear. METHODS: In 13 patients, interactions of mononuclear cells, platelets, pTF, and pTF fractions to activate factors VII and X were evaluated in pre-bypass, perfusate, and pericardial wound blood before and during CPB. RESULTS: Monocytes are activated in wound, but not in pre-bypass or perfusate plasma (monocyte chemotactic protein-1 = 29.5 +/- 2.1 pmoles/l vs. 2.8 +/- 1.2 pmoles/l and 3.3 +/-1.4 pmoles/l, respectively). Wound pTF is substantially elevated compared to other locations (3.64 +/- 0.45 pmoles/l vs. 0.71 +/- 0.65 pmoles/l and 1.31 +/- 1.4 pmoles/l). Supernatant wound pTF contains 81.7% of TF antigen; wound microparticle pTF contains 18.3%. Wound monocytes and all C5a-stimulated monocytes (but not activated platelets) completely convert factor VII to factor VIIa with wound pTF. Activated monocytes more efficiently activate factor X with wound supernatant TF/factor VII(VIIa) complex than with wound microparticle TF/factor VII(fVIIa). The correlation coefficient (r) between wound thrombin generation (F1.2) and wound pTF concentration is 0.944 (p = 0.0004). CONCLUSIONS: During cardiac surgery with CPB, wound monocytes plus wound pTF or wound microparticle-free supernatant pTF preferentially accelerate activation of factor VII and factor X. This system represents a novel mechanism for thrombin generation via the TF coagulation pathway.     

Activation of human factor VII by activated factors IX and X

Factor VII clotting activity increases about five-fold when blood is clotted in glass. Prior studies suggested that this results from activation induced by activated factor IX (IXa). However, in purified systems containing phospholipid and calcium, activated factor X (Xa) is known to activate factor VII rapidly. Therefore, we studied activation of factor VII by IXa and X, in systems using purified human factors. Concentrations of IXa and Xa were calculated from total activated protein concentrations rather than from active site concentrations. In the presence of phospolipid and calcium, both IXa and Xa activated factor VII 25-fold; however, Xa was roughly 800 times more efficient than IXa. Without added phospholipid, activation of factor VII by both Xa and IXa was markedly slowed, and Xa was roughly 20 times more efficient than IXa. When both phospholipid and calcium were omitted, activation of factor VII by either enzyme was negligible. Adding normal prothrombin, but not decarboxylated prothrombin, substantially slowed activation of factor VII by both Xa and IXa. Adding thrombin-activated factor VIII and antithrombin-III did not change rates of factor VII activation by either enzyme. These results from purified systems do not provide an explanation for the prior data from plasma systems.     

Newer concepts of blood coagulation

Summary. In this report we describe an in vitro model of blood coagulation reactions that mimics as closely as possible the in vivo condition. Our model indicates that the tissue factor—factor VIIa complex initiates coagulation by activating small amounts of both factor IX and factor X in the environment of the tissue factor bearing cell. Factor Xa and factor IXa formed in the initial reaction then play very distinct roles in the subsequent interactions of the clotting mechanism leading to a burst of thrombin generation on the platelet surface. Our results also indicate that factor XI can be activated by thrombin in the absence of factor XII and that the function of factor XI is simply to enhance conversion of factor IX to factor IXa resulting in enhanced thrombin generation on the platelet surface.     

Low levels of activated factor VII in systemic sclerosis

Introduction. Recent investigations show that activated factor VII, the primary enzyme in the extrinsic pathway of blood coagulation, exerts additional extra-coagulant functions, such as apoptosis and angiogenesis. On the basis of these recent acquisitions, the present study was aimed to evaluate activated factor VII in patients with systemic sclerosis and to establish a potential association with pathogenesis and complications of this severe autoimmune disorder. Materials and methods. Activated factor VII level was measured in twenty-eight consecutive scleroderma patients (2 men and 26 women, mean age 49.7 ± 14.8 years). The main clinical correlates of disease, such as disease activity, renal function, skin, vascular and lung involvement, were evaluated by clinical and instrumental investigations. Activated factor VII level was also evaluated in 28 sex and age matched controls. Results. Systemic sclerosis patients exhibited plasma activated factor VII activities significantly lower than those of healthy matched controls (15.2 versus 37.7 U/l, respectively; p < 0.001). No correlation was observed between plasma activated factor VII concentration and age, disease duration, disease subset, disease activity, renal, lung, skin and microvascular involvement. Conclusions. Results of our investigation provide first evidence of low activated factor VII activity in patients with systemic sclerosis. Reduced activated factor VII activity might be involved in the pathogenesis of the ischemic complications, by modulating apoptotic and angiogenetic processes. Contributed equally to this work.     

Recombinant, activated factor VII for surgery in factor VII deficiency: a prospective evaluation - the surgical STER

Excessive bleeding represents a major complication of surgical interventions and its control is especially relevant in patients with Congenital Bleeding Disorders (CBD). In factor VII (FVII) deficiency, scanty data on surgery is available to guide treatment strategies. The STER (Seven Treatment Evaluation Registry) is a multi-centre, prospective, observational, web-based study protocol providing the frame for a structured and detailed data collection. Inhibitor occurrence was checked in a centralized fashion. Forty-one surgical operations (24 'major' and 17 'minor') were performed in 34 subjects with a carefully characterized FVII deficiency under the coverage of recombinant activated Factor VII (rFVIIa). Bleeding occurred during three major interventions of orthopaedic surgery, but rFVIIa was given at very low dose in each case. An antibody to FVII was observed in one patient who underwent a multiple dental extraction. No thromboses were reported during the 30-d follow up period. Replacement therapy with rFVIIa proved effective when suitable doses were used, which, during the period of maximum bleeding risk (the day of operation), were calculated (Receiver Operated Characteristic analysis) to be of at least 13 μg/kg/body weight per single dose and no less than three administrations. This indication is important especially in the case of major surgery.     

Continuous infusion of recombinant activated factor VII during caesarean section delivery in a patient with congenital factor VII deficiency

Recombinant activated factor VII (rFVIIa) can be used as an alternative therapy in patients with FVII deficiency. However, as the drug has a very short half-life, continuous infusion could be a meaningful administration modality. We report the case of a 30-year-old woman with moderate FVII deficiency and human immunodeficiency virus infection who underwent a caesarean section delivery. She was treated with a continuous infusion of rFVIIa and did not suffer any bleeding complication. The continuous infusion of rFVIIa was a safe and effective therapeutic approach for our patient, maintaining her levels of FVII:C and avoiding bleeding during caesarean section and afterwards.     

Variation in the use of recombinant activated factor VII in critical bleeding

Background: Recombinant activated factor VII (rFVIIa) is being increasingly used as a treatment option in settings of uncontrolled bleeding. Despite this, national practice guidelines are lacking, resulting in widespread practice variation between providers. This investigation aimed to describe the differences in use of rFVIIa across Australian and New Zealand hospitals. Methods: Data were extracted from the Haemostasis Registry that collects both contemporaneous and retrospective cases of off‐licence (i.e. in non‐haemophilia patients) rFVIIa use in participating institutions. Hospitals were classified according to geographical location and service provision. Results: 2075 cases from 87 hospitals were recorded on the Haemostasis Registry. Across all hospital categories, over 41% of cases received rFVIIa in relation to cardiac surgery. Case complexity varied between providers, with large urban centres treating more severely ill patients. This was reflected in significant differences in the use of blood components and products before rFVIIa administration. Despite differences in patient complexity and use of blood products between hospital categories, response to treatment and patient outcomes remained similar across providers, with survival rates ranging from 68.29% to 70.41%. Conclusion: This is the largest study of off‐licence use of rFVIIa. There is significant regional variation in the administration of rFVIIa in Australian and New Zealand hospitals, with little documentation of adherence to guidelines. National consensus guidelines based on available evidence should be developed and promulgated to ensure optimal outcomes.