A new approach to treatment of bleeding episodes in young hemophilia patients: a single bolus megadose of recombinant activated factor VII (NovoSeven®)

Summary.  Recombinant activated factor VII (rFVIIa, NovoSeven^®) represents an effective treatment for hemophilia patients with inhibitors, but no consensus as to the best dosing regimen exists. We assessed the efficacy and safety of a rFVIIa 'megadose' (300 µg kg⁻¹ bolus) as treatment for bleeds in three young inhibitor patients. Of 114 bleeds, 95 responded to a single dose. Pain relief was faster and therapy duration significantly shorter than with continuous infusion (CI) regimens or standard boluses (90 µg kg⁻¹ every 3 h). Rebleeding occurred in 9.6% of cases and 19/114 episodes required a second bolus injection. Although rFVIIa consumption per bleed (median: 300 µg kg⁻¹) was higher than with standard boluses (180–270 µg kg⁻¹), patients found single bolus administration more convenient than recurrent injections or CI. With two exceptions, no complications occurred within 3 h of treatment, despite high FVII:C levels (median: 27.4 U mL⁻¹; range: 19.8–54 U mL⁻¹). Treatment of bleeds with a rFVIIa megadose in young inhibitor patients is effective and well tolerated.     

Effects of hemicolectomy on bile acid metabolism in relation to colon carcinogenesis in man

Bile acids are probably important in colon carcinogenesis. Regional differences in bile acid metabolism within the colon were studied to illuminate the preferential distal occurrence of colon cancer in Western countries. Faeces (24 h) were collected for bile acid measurement from 25 patients with hemicolectomy (nine left and 16 right) and 17 adenoma patients with an intact colon (control subjects). Duodenal bile and cytolytic and alkaline phosphatase activity of faecal water were also studied. The median percentage of deoxycholic acid (DCA) was lower in the hemicolectomy groups [left 48% (range 38–57%), right 45% (2–62%) vs. control subjects 59% (38–70%), P  < 0.05]. In duodenal bile, the proportion of DCA in left [4% (1–25%)] was lower than in the patients with right hemicolectomy [19% (0–69%)] and control subjects [24% (7–50%)], P  < 0.05. Faecal concentration of protonated DCA was higher in those with right hemicolectomy (0.101 μmol g⁻¹) than in those with left hemicolectomy (0.048 μmol g⁻¹), which coincided with a higher cytolytic [right 49% (3–93%), left 2% (1–37%)] and alkaline phosphatase activity [right 6.7 U mL⁻¹ (1.2–40.1 U mL⁻¹), left (2.0 U mL⁻¹ (1–25.7 U mL⁻¹), both P  < 0.02]. These findings suggest differences in bile acid metabolism between the proximal and distal colon that may contribute to the disparity in cancer risk.     

SSC/ISTH classification of hemophilia A: can hemophilia center laboratories achieve the new criteria?

Summary.  To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL⁻¹ were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1–29 U dL⁻¹ and 1–33 U dL⁻¹ respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1–5 U dL⁻¹. The median FVIII:C for the third sample was 5.8 U dL⁻¹ with a range of 1.5–36 U dL⁻¹. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL⁻¹; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.     

Specific detection of human coagulation factor IX in cynomolgus macaques

Summary.  After screening for species-specific antihuman factor (F)IX monoclonal antibodies, we found that antibody 3A6 did not bind to cynomolgus FIX. The 3A6 epitope was found to include Ala262 of human FIX. The 3A6 antibody was used as a catching antibody in an enzyme immunoassay (EIA) for specific detection of human FIX in cynomolgus macaque plasma. No significant increase of substrate hydrolysis was observed when EIA buffer containing cynomolgus macaque plasma was subjected to the 3A6-based EIA. Addition of up to 30% cynomolgus macaque plasma or canine plasma to the assay did not alter detection of human FIX. Three cynomolgus macaques were injected with human FIX (10 U kg⁻¹; i.v.) and the circulating human FIX was quantified in the macaque plasma. The FIX level in the circulation increased to 470 ± 37.6 ng mL⁻¹ at 1 h after the injection and gradually decreased to 1.79 ± 1.1 ng mL⁻¹ by day 5, which is approximately 0.06% of the normal human plasma FIX concentration. These data suggest that the cynomolgus macaque can be used as a primate model for studying hemophilia B gene therapy by transduction of macaque organs with vectors to express human FIX in vivo and detection of human FIX using the 3A6 monoclonal antibody.     

Racial differences in endotoxin-induced tissue factor-triggered coagulation

Summary.  Background: Racial differences in coagulation are poorly understood. While some studies suggest a 'prothrombotic' coagulation profile in blacks compared with whites, others report an increased bleeding risk for blacks in various clinical settings. Moreover, preclinical data suggest a link between the Duffy antigen (= DARC, Duffy antigen receptor of chemokines) and coagulation. Objectives: Based on our previous research in Duffy antigen negative Africans, we hypothesized that Africans have an attenuated procoagulant response compared with Caucasians in a model of lipopolysaccharide (LPS)-induced, tissue factor (TF)-triggered coagulation activation. Patients/methods: Healthy male volunteers (16 Duffy-negative Africans, 16 Duffy-positive Caucasians) received 2 ng kg⁻¹ LPS, and outcome parameters were measured using enzyme immunoassays and real-time polymerase chain reaction (RT-PCR, Taqman). Results: LPS increased microparticle (MP)-associated TF procoagulant activity (PCA) less in Africans than Caucasians. Africans had reduced in vivo thrombin formation compared with Caucasians: they generated less thrombin–antithrombin (TAT) complexes (10.4 pg mL⁻¹ vs. 23.0 pg mL⁻¹, P  < 0.0001) and less prothrombin fragments (F₁₊₂) (337 pmol mL⁻¹ vs. 819 pmol mL⁻¹, P  < 0.0001). Consistently, Africans also had decreased fibrin formation ( d -dimer: 0.3 pg mL⁻¹ vs. 0.5 pg mL⁻¹, P  = 0.02). Conclusion: Duffy-negative subjects of African descent have a markedly reduced procoagulant response in a model of LPS-induced, TF-triggered coagulation activation compared with Duffy-positive healthy Caucasians.     

High titers of autoantibodies to tissue factor pathway inhibitor are associated with the antiphospholipid syndrome

Summary.  As the activity of the tissue factor pathway inhibitor (TFPI) may be impaired in patients with antiphospholipid antibodies (aPL), 162 aPL patients were evaluated for autoantibodies to recombinant TFPI (anti-TFPI) using an optimized ELISA. Anti-TFPI (>18 U mL⁻¹ for IgG and/or > 15 U mL⁻¹ for IgM) were detected in 54 patients with aPL (33.3%) and in three out of 79 normal controls (3.8%, P  < 0.0001). Among aPL patients, the prevalence of positive anti-TFPI was 38.3 and 28.4% in those with or without diagnosis of definite antiphospholipid syndrome (APS). Patients with definite APS had a significantly greater frequency of high titer (>50 U mL⁻¹) anti-TFPI than aPL patients from the no definite APS group (18.5% vs. 6.2%, OR 3.7, P = 0.017). Most aPL recognized full-length TFPI, but not a truncated form of TFPI lacking the C-terminus of the molecule. Isolated IgGs from subjects with anti-TFPI impaired the dose-dependent inhibitory effect of TFPI on factor Xa activity in the presence, but not in the absence of phospholipid vesicles. Thus, aPL with high titer anti-TFPI limit TFPI action and are associated with the APS.     

Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor

Summary.  Background:  Oral contraceptives (OC) containing different types of progestogens induce different sensitivities to activated protein C (APC) measured with the thrombin generation-based APC-resistance test. These differences in APC resistance may be the biological explanation for the differences in thrombotic risk of the various pills. The mechanistic basis of APC resistance observed in OC users is unknown. Our objective was to study the effect of OC on the two main determinants of the APC-resistance test, free protein S and free tissue factor pathway inhibitor (TFPI). Patients/methods:  We measured free protein S and free TFPI in 156 users of various types of OC. Results:  Users of desogestrel-containing OC, known to double the risk of thrombosis compared with levonorgestrel-containing OC, had lower free protein S (24 vs. 33 U dL⁻¹) and TFPI free antigen (2.9 vs. 3.6 ng mL⁻¹) levels than users of OC containing levonorgestrel. Women using cyproterone acetate-containing OC, known to confer a high thrombotic risk, had the lowest free protein S (19 U dL⁻¹) and free TPFI antigen (2.5 ng mL⁻¹) levels. Users of OC containing drospirenone had lower free protein S (23 U dL⁻¹) and TFPI antigen levels (3.2 ng mL⁻¹) than users of levonorgestrel-containing OC. Low free protein S and low free TFPI antigen levels were associated with an increased resistance to APC, an established risk factor for thrombosis. Conclusions:  This study observed that the differences in APC resistance induced by OC containing different progestogens can at least in part be explained by different effects of OC on free protein S and TFPI.     

Sulfatides inhibit platelet adhesion to von Willebrand factor in flowing blood

Summary.  Sulfatides are sulfated glycosphingolipids present on cell surfaces that bind to adhesive proteins such as von Willebrand factor (VWF), P-selectin, laminin and thrombospondin. Previous studies have localized the sulfatide-binding site of VWF to amino acid residues Gln626–Val646 in the A1 domain. The A1 domain also contains the binding site for platelet glycoprotein Ib (GP Ib), a site that has been reported to be distinct from the sulfatide-binding site. In this study, we analyzed the interaction of sulfatides with VWF and its effect on GP Ib-mediated platelet adhesion under flow conditions. Recombinant VWF A1 domain (rVWF-A1) bound specifically and saturably to sulfatides (half-maximal concentration of ∼12.5 µg mL⁻¹), binding that was blocked by dextran sulfate (IC₅₀≈100 µg mL⁻¹) but not by heparin at concentrations up to 100 U mL⁻¹. Furthermore, sulfatides (125 µg mL⁻¹) prevented the adhesion of platelets or glycocalicin-coupled polystyrene beads to a rVWF-A1-coated surface under high shear stress. In addition, plasma VWF prebound to a sulfatide-coated surface failed to support subsequent platelet adhesion. These results provide firm evidence that sulfatides bind the VWF A1 domain at a site overlapping the GP Ib-binding site.     

Coagulation factor XII (FXII) activity, activated FXII, distribution of FXII C46T gene polymorphism and coronary risk

Summary.  Background:  Whether factor XII (FXII) activity, its 46C>T polymorphism and activated FXII (FXIIa) are associated with coronary heart disease (CHD) remains to be determined. Methods:  FXII, FXIIa and the FXII 46C>T polymorphism were determined in a hospital-based cohort of 2615 patients undergoing coronary angiography. Results:  Fifty-seven per cent of the participants were identified as wild-type (46CC), 38% as heterozygous (46CT) and 5% as homozygous (46TT) for FXII 46C>T. FXII and FXIIa levels were significantly lower in carriers of the T-allele: 132 (97–151) U dL⁻¹ FXII in 46CC, 87 (77–99) U dL⁻¹ FXII in 46CT and 53 (42–67) U dL⁻¹ FXII in 46TT carriers ( P  <   0.001), and 2.8 (2.3–3.5) μg L⁻¹ FXIIa in CC, 2.1 (1.6–2.6) μg L⁻¹ FXIIa in CT and 1.2 (0.9–1.5) μg L⁻¹ FXIIa in TT carriers ( P  <   0.001; medians, lower and upper quartiles). Patients with stable CHD ( n  =   935), a history of myocardial infarction ( n  =   785) or who were suffering from acute coronary syndromes (ACS; n  =   323) had significantly lower FXII levels than controls ( n  =   572). The differences remained statistically significant after adjustments for age, sex, diabetes mellitus, smoking, hypercholesterolemia and hypertension. Significantly reduced FXIIa levels in ACS patients lost significance once adjusted for covariates. FXII genotype was not associated with any clinical phenotype. Conclusion:  Lower FXII activity represents an independent risk for CHD and ACS. This is not the case for FXIIa levels or the FXII 46C>T variation.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.     

Simvastatin increases osteoblasts and osteogenic proteins in ovariectomized rats

Background  Previous reports have indicated that statins could prevent bone loss in ovariectomized (OVX) rats and increase the expressions of osteogenic genes in cultured osteoblasts. In this study, we hypothesized that simvastatin might increase osteoblast number and protein expressions of osteogenic markers localized in bones in concomitance with the prevention of bone loss in OVX rats.
Materials and methods  Fifty-four 3-month-old OVX and sham-operated (SHAM) female Sprague–Dawley rats were used. Simvastatin (10–20 mg kg⁻¹ day⁻¹) was administrated orally for 6 weeks. Trabecular volume, osteoblast number and osteogenic proteins including BMP2, collagen type I and osteocalcin on bone sections obtained from lumbar vertebral body, distal femur and proximal tibia were measured.
Results  The results showed that SHAM rats had significantly less trabecular bone volume and osteoblast number than that of OVX rats 6 weeks after operation. Oral simvastatin treatment (10–20 mg kg⁻¹ day⁻¹) increased bone volume and osteoblast number in the distal femurs, proximal tibiae and vertebrae of OVX rats. Furthermore, the osteoblastic cells with immuno-stained BMP2, collagen type I and osteocalcin in vertebral bones were significantly increased by simvastatin treatment (20 mg kg⁻¹ day⁻¹) in OVX rats.
Conclusions  This study demonstrates that simvastatin enhances the production of osteogenic proteins in bone and this effect may contribute to the prevention of bone loss in OVX rats.     

The role of recombinant factor VIIa (FVIIa) in fibrin structure in the absence of FVIII/FIX

Summary.  Patients with hemophilia have an impaired thrombin generation and therefore form loose fibrin hemostatic plugs that are easily dissolved by fibrinolysis. This prevents maintained hemostasis in these patients, resulting in a severe bleeding disorder. Recombinant (F)VIIa has been shown to enhance thrombin generation on already thrombin-activated platelets in the absence of FVIII and FIX. An efficacy rate of 80–90% has been found in hemophilia patients with inhibitors against FVIII or FIX both in association with major surgery and in the treatment of serious bleedings. In a model measuring fibrin clot permeability in a platelet-containing system described by Blombäck et al . (1994) this was demonstrated to be dependent on the concentration of FVIII and FIX. The addition of rFVIIa in concentrations of 1.9, 4.8 and 9.6 µg mL⁻¹ normalized fibrin clot permeability. The concentration of 1.9 µg mL⁻¹ of rFVIIa normalized clot permeability in this system and the higher concentrations of rFVIIa added only slightly to the effect. No further decrease in clot permeability was found when rFVIIa in a concentration of 1.9 µg mL⁻¹ was added to a sample with a normal concentration (100%) of FVIII or FIX. Higher concentrations of rFVIIa added to the plasma containing 100% of FVIII or FIX induced only a slight further decrease of fibrin permeability constant, arguing against any unwanted effect of extra rFVIIa on clot permeability in the case of a normal hemostasis. Furthermore, the fibrin network was studied with 3D microscopy and the loose network found in the absence of FVIII or FIX increased in density with increasing FVIII or FIX concentrations. The addition of rFVIIa to FVIII- or FIX-deficient systems altered the network structure, making the fibers thinner and more tightly packed.     

The presence of antiphospholipid antibodies is not related to increased levels of annexin A5 in plasma

Summary.  Annexin A5 has been proposed to be important for shielding of negatively charged phospholipids from blood, thereby preventing the binding of clotting factors. It has been suggested that antiphospholipid antibodies can disrupt the binding of annexin A5 from negatively phospholipid-containing surfaces, resulting in uncontrolled coagulation. If this hypothesis is correct, than the plasma levels of annexin A5 will be increased in patients with antiphospholipid antibodies. Therefore, we have measured plasma levels of annexin A5 of 175 patients with systemic lupus erythematosus (SLE), of which 104 had antiphospholipid antibodies and 23 patients had primary antiphospholipid syndrome. The annexin A5 levels were compared with the annexin A5 plasma levels measured in 23 patients with diabetes mellitus type 2 and 35 healthy volunteers. We found a significant increase of annexin A5 plasma levels in patients with SLE (median 6.7 ng mL⁻¹) and primary antiphospholipid syndrome (median 7.1 ng mL⁻¹) as compared to patients with diabetes mellitus type 2 (median 3.3 ng mL⁻¹) and healthy volunteers (median 3.9 ng mL⁻¹). However, no correlation was found with the presence of antiphospholipid antibodies or with a history of thromboembolic complications. Based on these observations, we conclude that displacement of annexin A5 from cellular surfaces by antiphospholipid antibodies is not a common mechanism in patients with antiphospholipid antibodies.     

Helper-dependent adenoviral gene therapy mediates long-term correction of the clotting defect in the canine hemophilia A model

Summary.  Background:  Adenoviral vector-mediated gene therapy might have potential for long-term correction of the monogenic disease hemophilia A. Objective:  In this study, we tested the efficacy of administering a helper-dependent adenoviral vector (HDV) designed for maximal liver-restricted canine factor VIII (cFVIII) expression on three out-bred hemophilia A dogs. Methods:  Three FVIII-deficient animals from the University of North Carolina colony were injected with 1 × 10¹² (Dog A), and 3 × 10¹² (Dog B and C) vp kg⁻¹ helper-dependent adenoviral vector, and we performed systematic analysis of toxicity, persistence of therapeutic gene expression, and molecular analysis of gene transfer. Results:  We observed acute dose-dependent elevation in liver enzymes and thrombocytopenia after injection, although both were transient and resolved within 2 weeks. The whole blood clotting time (WBCT), plasma FVIII concentration, FVIII activity, and activated partial thromboplastin time in all animals improved significantly after treatment, and two animals receiving a higher dose reached near normal WBCT with low-level FVIII activity until terminal sacrifice at 3 months, and 2 years. Importantly, the treated dogs suffered no bleeding events after injection. Moreover, we observed persistent vector-specific DNA and RNA in liver tissue collected from one high-dose animal at days 18 and 79, and could not detect the formation of inhibitory antibodies. Conclusion:  Although vector-associated toxicity remains an obstacle, a single injection of HDV led to long-term transgene expression and vector persistence in two FVIII-deficient animals with conversion of their severe phenotype to a moderate one.     

Break-through bleeding in relation to predicted factor VIII levels in patients receiving prophylactic treatment for severe hemophilia A

Summary.  Background: The role of prophylactic factor VIII (FVIII) to decrease hemophilic bleeding and arthropathy is well established. The rationale for this strategy is to convert patients with severe hemophilia A to a moderate clinical phenotype by reducing time spent with a FVIII level <1 IU dL⁻¹. Studies to date, however, have not demonstrated a strong link between FVIII level and the bleeding rate. Objectives : To assess the effect of FVIII level on break-through bleeding in patients with severe hemophilia A on prophylaxis. Patients/methods: This study analysed data from 44 patients aged 1–6 and 99 patients aged 10–65 years with severe hemophilia A (FVIII <1 IU dL⁻¹) who were treated with prophylactic FVIII as part of clinical studies assessing pharmacokinetics, safety and efficacy of a recombinant FVIII (Advate^®). Each patient had pharmacokinetic measurements and FVIII infusions recorded, and these were used to calculate time spent with a FVIII below 1, 2 and 5 IU dL⁻¹. Results: The data demonstrate that increasing time with a FVIII below 1 IU dL⁻¹ is associated with increased total bleeds and hemarthroses. Lack of adherence to the intended frequency of FVIII infusion was the most important determinant of low FVIII and increased bleeding. In children aged 1–6 years, the rate of bleeding was also influenced by FVIII half-life and clearance. Conclusions: These data have important implications for the management of patients with severe hemophilia.     

Acenocoumarol therapy in pediatric patients

Summary.  To determine guidelines for administering and monitoring acenocoumarol therapy in children, 93 patients (median 5.1 years, range: 0.2–18 years) were prospectively evaluated over a 33-month period. The loading doses used were: <1 year, 0.20 mg kg⁻¹; >1–5 years, 0.09 mg kg⁻¹; 6–10 years, 0.07 mg kg⁻¹; 11–18 years, 0.06 mg kg⁻¹. In this study, the loading dose and the dose to achieve and maintain target therapeutic range (TTR) for acenocoumarol are age-dependent, with infants having the highest and teenagers having the lowest requirements. The use of a different loading dose according to age has allowed most of the children (80%) in all the age groups to achieve TTR in less than 1 week. No patients had serious bleeding or thrombotic complications. We conclude that there is an age-dependent response to acenocoumarol in pediatric patients. The implementation of an age-adjusted loading dose regimen reduces the length of hospitalization required to achieve effective anticoagulant therapy.     

Stimulation of arginine vasopressin secretion by a small increase in blood ionized calcium in normal men

Evidence has been provided for an increase in baseline serum corticotrophin (ACTH) levels in response to a rise in circulating ionized calcium (Ca_i) levels within the physiological range. In order to establish whether small Ca_i increments are also able to modify the basal secretion of arginine vasopressin (AVP), we infused calcium gluconate through an intravenous infusion pump in eight healthy male subjects (25–31 years old). Serum Ca_i, ACTH and AVP concentrations were measured every 10 min over an infusion period lasting 90 min. A significant progressive rise in serum Ca_i (baseline: 42 ± 0.9 mg dL⁻¹; 90 min: 47.2 ± 0.9 mg dL⁻¹, P  < 0.001), ACTH (baseline: 30.7 ± 1.3 pg mL⁻¹; mean peak at 80 min: 37.4 ± 2.4 pg mL⁻¹, P  < 0.01) and AVP levels (baseline: 2.1 ± 0.6 pg mL⁻¹; mean peak at 80 min: 3.2 ± 0.5 pg mL⁻¹, P  < 0.01) was observed during calcium infusion. Furthermore, a significant positive correlation ( r  = 0.71; P  < 0.001) was observed between ACTH and AVP responses to calcium infusion at 60, 70, 80 and 90 min. These data demonstrate that AVP secretion is stimulated by a slight rapid increase in serum Ca_i levels even though absolute serum Ca_i levels remain within the normal range. In addition, the positive correlation between Ca_i-induced ACTH and AVP increments suggests that AVP plays a releasing role on ACTH secretion during calcium infusion.     

The promoting effect of epinephrine on lipopolysaccharide-induced interleukin-8 production in whole blood may be mediated by thromboxane A2

Summary.  Epinephrine is known to enhance lipopolysaccharide (LPS)-induced interleukin (IL)-8 secretion in a platelet dependent manner. To determine whether thromboxane A₂ (TxA₂; a product from activated platelets) is involved in this process, blood samples drawn either before or 2 h after oral administration of 440 mg acetylsalicylic acid (ASA) were stimulated with LPS (5 ng mL⁻¹) and different concentrations of epinephrine were added (0.1–100.0 µmol L⁻¹). ASA ingestion significantly (global P  < 0.05) reduced the enhancing effect of epinephrine on LPS-induced IL-8 release by 15–28%. To further explore whether TxA₂ may be involved in this process, a TxA₂ agonist (U46619) was added to whole blood together with LPS instead of epinephrine. U46619 mimicked the epinephrine effect: 20 ng mL⁻¹ U46619 enhanced LPS-induced IL-8 release by 39% ( P  < 0.05). Furthermore, preincubation of whole blood with 75 µmol L⁻¹ or 150 µmol L⁻¹ SQ29548, a TxA₂ receptor antagonist, completely blocked epinephrine's promoting effect on LPS-induced IL-8 release. Since thrombin-activated platelets have been reported to be important in the production of IL-8 in monocytes through the activation of monocytes by exposed RANTES in a P-selectin-dependent reaction, we suggest that the epinephrine effect is mediated by enhanced TxA₂ production and subsequent rise in the exposure of RANTES and P-selectin on the platelets of whole blood.     

A human monoclonal antibody inhibiting partially factor VIII activity reduces thrombus growth in baboons

Summary.  Background: The inhibitory activity of an anti-factor VIII (FVIII) antibody can be modulated through glycosylation of the antigen binding site, as has recently been described. This offers the opportunity to develop an optimized anticoagulant agent targeting partial FVIII inhibition. Objectives: We investigated in non-human primates the antithrombotic activity, pharmacokinetics,and pharmacodynamics of a human monoclonal antibody, Mab-LE2E9Q, inhibiting FVIII activity partially. Methods: The ability of Mab-LE2E9Q to prevent thrombosis was evaluated in baboons after administration of 1.25 and 5 mg kg⁻¹ antibody or saline as a single intravenous (i.v.) bolus. Thrombus development was recorded in expansion ('venous') and in Dacron^® ('arterial') thrombosis chambers incorporated in an extracorporeal arteriovenous shunt implanted between the femoral vessels 1 h, 24 h and 7 days after the administration of Mab-LE2E9Q. Results: Mab-LE2E9Q reduced thrombus growth to a similar extend 1 h, 1 day and 1 week after administration of the antibody. Ex vivo pharmacodynamic analysis indicated that the evaluation of the residual FVIII activity was strongly dependent on the type of FVIII assay and on the phospholipid concentration in the assay. No significant difference in bleedings was observed between animals treated with Mab-LE2E9Q or with saline. Conclusions: Understanding the role of glycosylation in FVIII inhibition by a human monoclonal antibody allowed selection of an antibody inhibiting only moderately FVIII activity while significantly reducing thrombus development in a baboon extracorporeal model. As that antibody did not increase the bleeding tendency, it may represent a novel type of a long-acting antithrombotic agent with an optimal safety/efficacy profile.     

Effect of colchiceine and ursodeoxycholic acid on hepatocyte and erythrocyte membranes and liver histology in experimentally induced carbon tetrachloride cirrhosis in rats

Colchiceine and ursodeoxycholic acid (UDCA) are drugs currently in use as therapy for different types of liver damage. We evaluated their ability to reverse the damage induced by carbon tetrachloride (CCl₄) in rats. Six groups were analysed: (1) CCl₄ (0.4 g kg⁻¹, i.p., three times a week) for 13 weeks; (2) CCl₄ for 8 weeks followed by colchiceine (60 μg kg⁻¹) + CCl₄ for 5 weeks; (3) CCl₄ for 8 weeks and thereafter UDCA (25 mg kg⁻¹) + CCl₄ for 5 weeks. Groups 4, 5 and 6 were appropriate controls of colchiceine, UDCA and vehicles respectively. Na⁺,K⁺- and Ca²⁺-ATPase activities and the cholesterol–phospholipid (CH/PL) ratio from erythrocyte and hepatocyte membranes were quantified. Membrane enzymatic activities and CH/PL ratios were affected more in group 1 than groups 2 and 3. We concluded that colchiceine and UDCA were effective drugs in this model of liver damage.