Characterization of a tissue factor/factor VIIa-dependent model of thrombosis in hypercholesterolemic rabbits

Summary.  Tissue factor (TF) expressed in arterial atherosclerotic plaque plays a key role in activating the extrinsic coagulation pathway and triggering acute coronary syndromes. In this study, we developed and characterized a TF–factor (F)VIIa-mediated thrombosis model in rabbits. Balloon catheter-induced endothelial denudation in the femoral artery and a 4-week high cholesterol diet produced a localized atherosclerotic plaque at the injured site. High levels of TF mRNA and TF protein antigen (152 ± 25 vs. 49 ± 12 pg mg⁻¹ protein in normal vessels) were detected in these atherosclerotic plaques. Plasma FVII coagulant activity (FVII:C) was significantly increased in the hypercholesterolemic rabbits (36 ± 1 s) compared with the normal rabbits (44 ± 1 s, P  < 0.0001). Plaque rupture was induced by balloon angioplasty, which resulted in thrombus formation in the injured vessel segment after a brief period of stasis. FVIIai, a specific TF–FVIIa inhibitor, was administered intravenously to rabbits before plaque rupture at 0.3 and 1.0 mg kg⁻¹. FVIIai dose-dependently reduced thrombus mass (14.7 ± 2.5 and 5.9 ± 2.2 mg, respectively, vs. 21.6 ± 1.9 mg in the control group). PD198961, a novel factor Xa inhibitor, and argatroban, a thrombin inhibitor, also dose-dependently inhibited thrombosis. These results indicate that thrombus formation in this model is initiated by the activation of TF–FVIIa pathway, which is attributed to TF expression in the atherosclerotic plaque and enhanced plasma FVII coagulant activity. This model may be useful for evaluating in vivo efficacy of new antithrombotic drugs, particularly TF–FVIIa inhibitors.     

Effect of vascular injury on inhibition of venous thrombosis with ZK-807834, a direct inhibitor of factor Xa

Summary.  Inhibition of factor Xa with the small molecule inhibitor ZK-807834 (Mr 527 Da, K_i 0.11 nM) attenuates progression of thrombosis, but the ED₅₀ is substantially lower for venous compared with arterial thrombosis in experimental animals. To determine whether this reflects differences in the extent of vascular injury, we compared the dose–response of ZK-807834 for inhibition of venous thrombosis induced with a cotton thread and copper wire device in the presence and absence of balloon catheter-induced injury to the vena cava in rabbits. ZK-807834 administration over 2 h (total dosages of 0.0023–2.3 µmol kg⁻¹, n  = 6/group) resulted in dose-dependent reductions in clot weight compared with vehicle controls, but the ED₅₀ was 0.03 µmol kg⁻¹ for non-injured veins and 0.42 µmol kg⁻¹ for injured veins. We conclude that vascular injury invokes a tissue factor-mediated response that increases the dose requirements for inhibition of venous thrombosis with ZK-807834.     

Combination of aspirin and metoclopramide produces a synergistic antithrombotic effect in a canine model of coronary artery thrombosis

Summary— We compared the antithrombotic properties of low doses of aspirin (0.03, 0.1 mg kg⁻¹ intravenously [iv]) and metoclopramide (0.1, 0.3 mg kg⁻¹ iv) alone or in combination. The animal model chosen for this study involved the generation of cyclic flow variations (CFV) in the circumflex coronary artery of anaesthetized dogs as a result of a critical coronary stenosis associated with a controlled arterial lesion at the site of stenosis. Subsequent regular CFV represent sequential thrombus formation and embolization in the damaged vessel. Neither aspirin nor metoclopramide alone demonstrated antithrombotic properties at the doses tested. However, the combination of aspirin 0.1 mg kg⁻¹ iv and metoclopramide 0.3 mg kg⁻¹ iv produced a significant antithrombotic effect, reducing the frequency of large CFV from 6.7 ± 0.5 to 0.8 ± 0.4 cycles h⁻¹ ( P < 0.01) and increasing minimum mean coronary blood flow from 5.0 ± 1.1 to 23.7 ± 2.6 mL min⁻¹ ( P < 0.01). This result apparently reflects an antithrombotic synergism between aspirin and metoclopramide since the effects of the combination were greater than the combined effects of the individual treatments. The antithrombotic influence of metoclopramide could be due to its 5HT₂-antagonist or α₂-antagonist properties, both of which would inhibit platelet aggregation. This demonstration of a synergistic antithrombotic action of the combination of aspirin and metoclopramide is of interest since these two agents are often combined in clinical use. Its therapeutic relevance, however, remains to be established.     

Insulin inhibits tissue factor expression in monocytes

Summary.  Objectives:  Platelets from healthy subjects are inhibited by insulin but type 2 diabetes mellitus (T2DM) platelets have become insulin-resistant, which might explain their hyperactivity. In the present study we investigated whether monocytes are responsive to insulin. Methods and results:  LPS-induced tissue factor (TF) upregulation was measured in human monocytes and monocytic THP-1 cells in a factor Xa generation assay. Insulin (0.1–100 nmol L⁻¹) induced a dose-dependent inhibition in both cell types and in monocytes 100 nmol L⁻¹ insulin inhibited cytosolic, membrane-bound and microparticle TF by 32 ± 2, 27 ± 3 and 52 ± 4% ( n  = 3). Insulin induced Tyr phosphorylation of the insulin receptor (INS-R) and formation of an INS-R – G_iα₂ complex, suggesting interference with LPS-induced cAMP control. Indeed, insulin interfered with LPS-induced cAMP decrease and TF upregulation in a manner similar to an inhibitor of G_i (pertussis toxin) and agents that raise cAMP (iloprost, forskolin, IBMX) reduced TF upregulation. Although LPS failed to raise cytosolic Ca²⁺, quenching of Ca²⁺ increases (BAPTA-AM) reduced and induction of Ca²⁺ entry (ionophore, P2X7 activation) enhanced upregulation of TF mRNA and procoagulant activity. Insulin interfered with MCP-1-induced Ca²⁺ mobilization but not with ATP-induced Ca²⁺ rises. Conclusions:  Insulin inhibits TF expression in monocytes and monocyte-derived microparticles through interference with G_iα₂-mediated cAMP suppression, which attenuates Ca²⁺-mediated TF synthesis.     

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.     

Prolonged procoagulant activity on overstretch-injured coronary arteries in pigs

Summary.  This study was designed to assess the time course and nature of the vascular procoagulant response after 1.5-fold balloon overstretch injury of the coronary arteries in pigs. Arteries were excised for chromogenic assay of bound factor (F)Xa and thrombin at 24 h, 3 days, 1 week, or 2 weeks after injury. FXa at the site of injury remained elevated for 1 week (4.9 ± 5.9 µg cm⁻², n  = 10), compared with non-injured control arteries (0.4 ± 0.2 µg cm⁻², n  = 18, P  = 0.00025), while thrombin was increased only at 24 h. Tissue factor protein was abundant in non-injured coronaries (10 ± 6 ng µg⁻¹ total protein, n  = 9) and levels were unchanged by injury (13 ± 11 ng µg⁻¹, n  = 6) or 24-h administration of tissue factor pathway inhibitor (16 ± 6 ng µg⁻¹, n  = 6). Persistent tissue factor-mediated procoagulant activity may explain the need for prolonged anticoagulation to attenuate neointimal formation after balloon-induced coronary injury.     

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.     

Influence of food and body weight on the pharmacokinetics of penticainide

Summary— The pharmacokinetics of penticainide, a class Ic antiarrhythmic drug, was studied in 16 healthy adults (eight males and eight females) after a single 300-mg oral dose in fasting conditions and with a standard meal. Penticainide concentrations in plasma and urine were measured by hplc. The pharmacokinetic parameters of penticainide including C_max, t_max, AUC and t_1/2 were not significantly altered in the presence of food. AUC values (mean ± sd) were 50.68 ± 10.8 mg·h·l⁻¹ and 49.52 ± 9.87 mg·h·l⁻¹ in the absence and presence of food, respectively. However, a significant difference was observed between males and females in both fasting and fed conditions with a higher value of the apparent oral clearance in the second group. The values of apparent oral clearance, expressed in weight-normalized units were 1.33 ± 0.35 ml·mn⁻¹·kg⁻¹ (male) and 1.93 ± 0.34 ml·mn⁻¹·kg⁻¹ (female) in fast conditions ( P < 0.01) and 1.38 ± 0.28 ml·mn⁻¹·kg⁻¹ (male) and 1.93 ± 0.49 ml·mn⁻¹·kg⁻¹ (female) in fed conditions ( P < 0.02), respectively. The pharmacokinetics of penticainide is not modified by the presence of food, but an influence of body weight may be considered.     

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.     

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.     

Ex vivo inhibition of thrombus formation by an anti-glycoprotein VI Fab fragment in non-human primates without modification of glycoprotein VI expression

Summary.  Objectives:  Glycoprotein (GP)VI is an attractive target for the development of new antithrombotic drugs. Its deficiency protects animals in several models of thrombosis, arterial stenosis and ischemia-–reperfusion while inducing no major bleeding tendency. The Fab fragment of one anti-GPVI monoclonal antibody (9O12.2) inhibits all GPVI functions in vitro . The aim of this study was to determine the ex vivo effects of 9O12.2 Fab on hemostasis, coagulation and thrombosis in non-human primates. Methods and results:  Blood samples were collected from cynomolgus monkeys before and after (30, 90 and 150 min, 1 and 7 days) a bolus injection of 9O12.2 Fab (4 mg kg⁻¹) or vehicle. Platelet counts and coagulation tests (prothrombin time, activated partial thromboplastin time) were not modified following Fab injection. The PFA-100 closure time increased during the first hours and returned to initial values on day + 1. Platelet-bound Fab was detected from 30 min to 24 h after Fab injection without GPVI depletion at any time. Collagen-induced platelet aggregation was selectively and fully inhibited at 30 min. Thrombus formation on collagen in flowing whole blood (1500 s⁻¹) was delayed and decreased, and collagen-induced or tissue factor-induced thrombin generation in platelet-rich plasma was profoundly inhibited. Conclusion:  The anti-GPVI 9O12.2 Fab inhibits thrombus formation ex vivo in non-human primates with a composite effect on platelet activation and thrombin generation in the absence of GPVI depletion.     

Recombinant human factor VIIa and a factor VIIa-analogue reduces heparin and low molecular weight heparin (LMWH)-induced bleeding in rats

Summary.  Background:  Heparin and low molecular weight heparin (LMWH) are widely used for prevention and treatment of thromboemobolic events, but may occasionally cause uncontrollable bleeding. Heparin can readily be antagonized with protamine, but this is less effective against LMWH. Objectives:  To test the effects of rFVIIa or an analogue of rFVIIa, NN1731, on heparin- and LMWH-induced bleeding in rats. Methods:  Initially the doses of heparin and tinzaparin (a LMWH) were determined by dose-titration. Following pretreatment with heparin or tinzaparin in rats, tail-transection was performed, and the effect of rFVIIa and NN1731 on the bleeding was observed. Results:  rFVIIa (5, 10 and 20 mg kg⁻¹) reduced bleeding time and blood loss caused by heparin- and tinzaparin-induced bleeding, using doses of 200 IU kg⁻¹ ( n  = 8) and 500 IU kg⁻¹ ( n  = 9), respectively. Similarly, 10 mg kg⁻¹ NN1731 significantly reduced both heparin- and tinzaparin-induced bleeding to the normal level. Following severe anticoagulation with 1800 IU kg⁻¹ tinzaparin, 10 mg kg⁻¹ NN1731 reduced and normalized the bleeding, while the effect of 20 mg kg⁻¹ rFVIIa failed to reach statistical significance. These data are consistent with the hypothesis that rFVIIa/NN1731 are capable of generating sufficient thrombin locally on the surface of activated platelets to induce hemostasis in the presence of heparin/LMWH. Conclusions:  This study suggests that rFVIIa and NN1731 may have the potential to control bleedings caused by heparin or LMWH.     

Nicotinamide inhibits endotoxin-induced monocyte tissue factor expression

Summary.  Background : Tissue factor (TF) is the main initiator of blood coagulation in vivo . Its increased expression on activated monocytes is associated with thrombotic complications and mortality in conditions such as sepsis, disseminated intravascular coagulation and coronary artery disease. Objective : The effect of the vitamin B derivative nicotinamide on endotoxin-induced monocyte TF and CD11b expression, soluble interleukin(IL)-6, and clotting onset time (COT) was studied. Methods : Experiments were conducted in human peripheral blood leukocyte suspensions and in whole blood from eight healthy volunteers. Free oscillating rheometry (measuring COT) and flow cytometry were applied to evaluate the effect of endotoxin on TF, CD11b, IL-6 and the overall coagulation response of plasma supplemented with activated autologous leukocytes. Results : In response to endotoxin, there was an increase in IL-6, TF and CD11b expression and a procoagulant shift of COT. At 4 mmol L⁻¹ nicotinamide, inhibition of TF expression and IL-6 and a normalization of COT were seen. At 16 mmol L⁻¹ nicotinamide, CD11b decreased also. The level of monocyte TF expression correlated with the COT readings, and the endotoxin-induced procoagulant shift of COT could be totally inhibited by blocking TF with an inhibitory antibody. Conclusions : These results demonstrate the ability of nicotinamide to inhibit the activation of coagulation associated with endotoxemia. We have previously shown that nicotinamide exerts strong anti-inflammatory effects. Evidence is accumulating for nicotinamide to have a therapeutic potential in modulating disease states in which there is a profound activation of coagulation and inflammation, such as in sepsis and disseminated intravascular coagulation.     

Cisplatin-induced renal effects and thromboxane A2 receptor blockade

The aim of this study was to evaluate the renal protective effect of linotroban, a thromboxane A₂ receptor antagonist, in 25 patients with malignant tumours scheduled for cisplatin therapy. Cisplatin was administered 1 h after the start of a 24-h continuous infusion of linotroban or placebo. Glomerular filtration rate and effective renal plasma flow were measured. Infusions of cisplatin decreased glomerular filtration rate by 17 ± 25 mL min⁻¹ ( P  = 0.049 vs. baseline) and effective renal plasma flow by 94 ± 150 mL min⁻¹ ( P  = 0.049 vs. baseline) in the placebo group. In the linotroban group a decrease in glomerular filtration rate by 11 ± 18 mL min⁻¹ ( P  = 0.050 vs. baseline) and in effective renal plasma flow by 26 ± 63 mL min⁻¹ ( P  = 0.2 vs. baseline) was noted. However, no difference was noted between groups in response to treatment. Our findings indicate that linotroban may not be useful for prevention of cisplatin's acute nephrotoxic effects.     

The benefit-to-risk profile of melagatran is superior to that of hirudin in a rabbit arterial thrombosis prevention and bleeding model

Summary.  Although hirudin is better than heparin at preventing recurrent ischemia in patients with unstable angina, hirudin produces more bleeding. The purpose of this study was to use a rabbit arterial thrombosis prevention and ear bleeding model to determine whether for equivalent efficacy, melagatran, a synthetic direct thrombin inhibitor, is safer than hirudin. A combination of balloon injury and stasis was used to induce thrombosis in the distal aorta, and patency and blood flow were continuously monitored with ultrasonic flow probes. Rabbits were randomized to melagatran (in total doses of 78–313 nmol kg⁻¹), hirudin (in total doses of 18–107 nmol kg⁻¹), or saline over 90 min. To assess safety, blood loss from standardized ear incisions was measured. Both melagatran and hirudin produced dose-dependent increases in patency and blood flow. At doses that maintained the highest levels of patency, however, melagatran produced 2–3-fold less bleeding than hirudin. Thus, at maximally effective doses, melagatran causes less bleeding than hirudin in this model. These findings raise the possibility that some direct thrombin inhibitors are safer than others.     

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.     

Effects of low-dose l-arginine on insulin-mediated vasodilatation and insulin sensitivity

The present study was carried out to evaluate the effect of a low-dose intravenous supplementation of l -arginine on insulin-mediated vasodilatation and insulin sensitivity. The study was performed in healthy subjects ( n  = 7) and patients with obesity ( n  = 9) and non-insulin-dependent diabetes mellitus (NIDDM) ( n  = 9). Insulin-mediated vasodilatation was measured by venous occlusion plethysmography during the insulin suppression test, evaluating insulin sensitivity. Experiments were performed twice in each subject in the presence or absence of a concomitant infusion of l -arginine (0.52 mg kg⁻¹ min⁻¹). l -Arginine restored the impaired insulin-mediated vasodilatation observed in obesity (22.4 ± 4.1%, P  < 0.01 vs. without l -arginine) and NIDDM (20.3 ± 3.2%, P  < 0.01 vs. without l -arginine). In healthy subjects, no effect on insulin mediated-vasodilatation was observed (24.8 ± 3.1% vs. 21.4 ± 3.1%). Insulin sensitivity was improved significantly ( P  < 0.001) in all three groups by infusion of l -arginine. No effect of l -arginine was observed on insulin, insulin-like growth factor I (IGF-I), free fatty acids (FFAs) or C-peptide levels during the insulin suppression test. Our data indicate that defective insulin-mediated vasodilatation in obesity and NIDDM can be normalized by intravenous l -arginine. Furthermore, l -arginine improves insulin sensitivity in obese patients and NIDDM patients as well as in healthy subjects, indicating a possible mechanism that is different from the restoration of insulin-mediated vasodilatation.     

The hormone sensitive lipase gene in familial combined hyperlipidemia and insulin resistance

Backround Insulin resistance in the most common familial dyslipidemia, familial combined hyperlipidemia (FCHL), could be due to variations in the hormone sensitive lipase (HSL) gene.
Materials and methods The coding region of the HSL gene was screened with the single strand conformation polymorphism analysis in probands of 27 FCHL families with 228 members. In addition, the C-60G promoter substitution of the HSL gene was determined by the restriction fragment length polymorphism analysis in these subjects.
Results No variants in the coding region of the HSL gene were found and the allele frequencies of the C-60G promoter substitution and the silent variant (G3138A) in the 3' untranslated region did not differ between 110 control subjects and 27 probands with FCHL. However, in control women the C-60G substitution was associated with high body mass index [30·6 ± 0·9 kg m⁻² (mean ± SD) in subjects with the C/G genotype and 24·8 ± 4·6 in subjects with the C/C genotype, P  = 0·012], and in control men with high rates of insulin-stimulated whole body glucose uptake (70·1 ± 14·7 vs. 56·7 ± 14·2 µmol kg⁻¹ min⁻¹, P  = 0·014). In 228 FCHL family members this substitution was associated with high low-density lipoprotein cholesterol levels in men (4·51 ± 1·12 vs. 5·17 ± 1·28 mmol L⁻¹, P  = 0·049), but not in women.
Conclusions The HSL gene is not a major gene for FCHL. However, the − 60G allele of this gene may affect body weight, insulin sensitivity and serum cholesterol levels.     

Population pharmacokinetics of amikacin in intensive care unit patients studied by NPEM algorithm

Summary— The population pharmacokinetics of amikacin was studied in 40 intensive care unit patients (212 plasma concentrations) by NPEM algorithm using a one-compartment model. The population was best characterized by the following pharmacokinetic parameters: renal clearance relative to creatinine clearance (C_s = 0.96 ± 0.33), and either the total volume of distribution (V_d = 23.9 ± 7.0 I) or the volume of distribution relative to body weight (V_s = 0.36 ± 0.10 1·kg⁻¹. The volume of distribution was increased with respect to the usual value of 0.25 1·kg⁻¹. The statistical distribution of these pharmacokinetic parameters was approximately gaussian, with no significant correlation between volume of distribution and clearance. The medians and standard deviations of C_s and V_s were used as reference population values to estimate the pharmacokinetics of amikacin in a second group of 29 patients by the bayesian method, with two blood samples per patient. For each patient, the fitted parameters were able to predict the plasma concentrations of amikacin during the next 72 h with no significant bias and good precision (2.9 mg·1⁻¹ for peaks and 0.5 mg·1⁻¹ for troughs). This study confirms the ability of the NPEM algorithm to provide reference population values for use in bayesian monitoring of aminoglycoside therapy.     

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.